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Clinical outcomes following total hip arthroplasty for bony ankylosed hips: a propensity score-matched analysis | c28db74b-3f6e-470f-8fca-e42d3b278c6d | 11849327 | Surgical Procedures, Operative[mh] | Patients with spontaneous ankylosis due to hip arthritis or those who have undergone arthrodesis in adolescence for hip disease often achieve pain relief but experience difficulty with activities of daily living and disability of adjacent joints, such as the lumbar spine and knee . Therefore, pain due to adjacent joint disorders caused by ankylosed hips is an indication for total hip arthroplasty (THA) for ankylosed hips . THA is known to be one of the most useful treatment procedures for hip arthritis . However, the postoperative outcomes of THA in patients with bony ankylosed hips are nonconclusive. To our knowledge, no studies have compared clinical outcomes following THA in patients with bony ankylosed and non-ankylosed hips using propensity scores. In this study, we examined the postoperative results of THA for bony ankylosed hips using propensity score matching.
This retrospective cohort study used data obtained from the THA database of our institution. Data obtained included the Japanese Orthopaedic Association (JOA) hip score, laboratory data, postoperative complications, and computed tomography (CT) images. The study protocol was in accordance withSaga University Hospital (Reference number: 2020–06-R07). All patients provided written informed consent prior to participation. Patients Propensity score matching Surgical procedure Surgical indication Types of implants Outcomes Statistical analyses Statistical analyses were performed using JMP Pro software (version 15.2.0, SAS Institute Inc., Cary, NC, USA). Categorical variables were expressed as absolute and percentage values, and continuous variables were expressed as mean ± standard deviation. The Shapiro–Wilk test was conducted to evaluate the normality of the distribution of the continuous variables. A two-tailed F-test was used to evaluate variance. The Wilcoxon signed-rank test was used to compare the postoperative JOA hip score at the last follow-up and post-propensity score matching with the preoperative and pre-matching values, respectively, in the same group. Comparisons between the bony-ankylosed and control groups were performed as follows: a two-tailed Student’s t-test was used to compare the post-propensity score-matched height, weight, preoperative total JOA score, haemoglobin level, and alkaline phosphatase on the day prior to surgery and postoperative day 1. A two-tailed Welch’s t-test was used to compare the pre-propensity score-matched height, body mass index, postoperative blood loss, and creatine phosphokinase level on postoperative day 1. The Mann–Whitney U test was used to compare the preoperative JOA hip score, except for the total score, postoperative JOA hip score at the last follow-up, preoperative and postoperative use of walking aids, length of hospital stay, follow-up period, operating time, intraoperative blood loss, allogeneic blood transfusion, laboratory data except for the haemoglobin level, alkaline phosphatase on preoperative day and postoperative day 1, and creatine phosphokinase level on postoperative day 1, and postoperative CSA of the gluteus medius and CT values at that site. Post-hoc analyses were performed for the postoperative total JOA hip score at the last follow-up (effect size d = 1.13, two-sided alpha = 0.05, sample size = 40 and 40), CSA of the gluteus medius (effect size d = 1.88, two-sided alpha = 0.05, sample size = 11 and 8), and the CT value of the gluteus medius (effect size d = 1.39, two-sided alpha = 0.05, sample size = 11 and 8), resulting in calculated power values of 0.99, 0.96, and 0.79, respectively. Regarding CT imaging studies, intra-rater reliability was assessed by two measurements recorded by one orthopaedic surgeon 1 week apart, and inter-rater reliability was assessed by measurements recorded by two orthopaedic surgeons. The intra-rater intraclass correlation coefficient (ICC) and inter-rater ICC values were 0.9448 and 0.8133 for the postoperative CSA of gluteus medius and 0.9446 and 0.8512 for the postoperative CT values of gluteus medius at CSA, respectively, all of which were sufficiently reproducible to be classified in the first class by Wheeler’s Evaluating the Measurement Process method . Following Cochran’s rule, Fisher’s test was used for the number of hips that underwent revision surgery and time of postoperative CT imaging, where more than 20% of the squares had an expected number less than five . The chi-square test was used for to analyse sex, preoperative and postoperative use of walking aids, number of hips with allogeneic blood transfusions, number of hips with postoperative complications, where no squares had an expected number less than five . Survival analysis was performed using Kaplan–Meier and log-rank tests for the acetabular side, femoral side, and both sides of the prosthesis, with total hip arthroplasty revision as the endpoint. The relationship between intraoperative blood loss and other variables, including the type of ankylosis, was evaluated using Spearman’s correlation coefficient in the bony-ankylosed group. For all analyses, statistical significance was set at a p -value < 0.05.
Data were extracted for 3338 hips (2863 patients) that underwent primary unilateral THA at our institution between January 1999 and December 2011 (Fig. ). Cases with follow-up periods of less than 10 years and those with missing data were excluded. The hips included in the study were then temporarily divided into two groups: a bony-ankylosed group (40 hips, 38 patients) and a non-ankylosed (control) group (829 hips, 729 patients). Bony ankylosed hips were defined as cases with joint ossification on simple radiography and no range of motion (Fig. A). The remaining cases were defined as non-ankylosed hips.
To minimise confounding factors, propensity score matching was used to match bony ankylosed to non-ankylosed hips. Using logistic regression, propensity scores were calculated using the five variables of age, sex, height, weight, and body mass index, which were selected based on previous studies . Propensity score matching was then performed using nearest neighbour matching without replacement, with each bony ankylosed hip matched to a control hip . We used a calipre width of 0.2, which is the standard deviation of the logit of the propensity score . To check the balance of the matches, a standardised mean difference threshold of 0.1 was set to determine the residual imbalances .
THA was performed using the posterolateral approach under spinal anaesthesia with identical cementless implants. In the bony-ankylosed group, 33 of the 40 hips underwent an additional approach from the anterior edge of the gluteus medius.
The main surgical indication for the bony-ankylosed group was pain due to adjacent joint disorders caused by the ankylosed hips and for the control group it was a decline in activities of daily living due to pain and limited range of motion in the hip joints. Walking training within the allowable pain range was initiated without weight-bearing limitations after drain removal on day-2 postoperatively.
In both groups, proximal hydroxyapatite-coated cementless femoral components with a proximal porous coating consisting of pure titanium (PerFix-HA femoral component; Kyocera, Kyoto, Japan) and hydroxyapatite-coated cementless hemispherical acetabular shells with a porous coating consisting of pure titanium (AMS-hydroxyapatite acetabular shell; Kyocera, Kyoto, Japan) were implanted. An alumina or zirconia ball and ABS (alumina ceramic inlay mechanically fixed to a polyethylene liner) or AMS (polyethylene liner) liner were used for the bearing surface (Fig. B).
The outcomes of this study were the JOA hip score and use of walking aids, preoperatively and at the last postoperative follow-up; length of hospital stay; follow-up period; operating time; intraoperative blood loss; postoperative blood loss calculated from a drain tube; allogeneic blood transfusion; number of hips with allogeneic blood transfusions; number of hips with postoperative complications; number of hips that underwent revision surgery; laboratory data obtained preoperatively and at postoperative days 1 and 7. Postoperative haemoglobin included data for patients who received allogeneic blood transfusions. The JOA hip score consists of four subcategories and is calculated on a 100-point scale: pain, 40 points; range of motion, 20 points; gait ability, 20 points; and activities of daily living, 20 points. Laboratory data, including platelet count and creatine phosphokinase levels, were evaluated using routine blood tests. Additionally, data were missing after propensity score matching; however, postoperative CT images were used to evaluate the gluteus medius on the surgical side in the transverse section, and the image information unification system ShadeQuest/ViewR (version 1.26.10; Yokogawa, Tokyo, Japan) was used to take measurements. A complete-case analysis was used for the evaluation. The midpoint of the line connecting the superior anterior iliac spine and the apex of the greater trochanter was defined in the frontal simple radiograph image of the pelvis for positioning when the CT image was captured. The cross-sectional area (CSA) and CT value of the gluteus medius were measured at the midpoint level (Fig. A and ) .
Propensity score matching Clinical results Laboratory results Radiographic results Survival of acetabular and femoral components The cumulative survival rates of the cup, stem, and overall, with revision as the endpoint, were not significantly different between the bony-ankylosed and control groups ( p = 0.5620, p = 0.1753, and p = 0.3378, respectively; Fig. A–C).
The propensity score-matched population consisted of 40 bony ankylosed (38 patients) and 40 non-ankylosed (40 patients) hips as matched controls. The area under the receiver operating characteristic curve for fitting the propensity score was 0.68. This value is an appropriate value for calculating the propensity score as it ranges from 0.6–0.9 and is not extremely close to 0.5 or 1.0, which are considered inappropriate . The standardised mean differences in baseline demographics for the matched study population are shown in Table and Fig. : all variables achieved an appropriate balance (standardised mean difference < 0.1). In the bony-ankylosed group, the mean duration of ankylosis, as assessed by preoperative interviews with patients, was 36.1 ± 19.0 years, with 22 cases of spontaneous ankylosis and 18 of arthrodesis.
Intragroup comparison Comparison between the bony-ankylosed and control groups Preoperatively, the JOA hip scores for “total” and “pain” in the bony-ankylosed group were significantly higher than those in the control group ( p < 0.0001 and p < 0.0001, respectively), whereas the scores for “range of motion” and “percentage of use of walking aids” were significantly lower ( p < 0.0001 and p = 0.0368, respectively). No significant differences were observed between the two groups with regards the JOA hip scores for “gait ability” and “activities of daily living” ( p = 0.5654 and p = 0.0563, respectively). Regarding the postoperative values, the JOA hip scores at the last follow-upwere significantly lower in the bony-ankylosed group than in the control group ( p < 0.0001, p = 0.0382, p < 0.0001, p < 0.0001, and p = 0.0083, respectively). The percentage of patients who used walking aids postoperatively, length of hospital stay, operating time, intraoperative and postoperative blood loss, allogeneic blood transfusion, number of hips with allogeneic blood transfusions, and number of hips with postoperative complications were significantly higher in the bony-ankylosed group than in the control group ( p < 0.0001, p < 0.0001, p < 0.0001, p < 0.0001, p = 0.0003, p < 0.0001, p = 0.0015, and p = 0.0021, respectively; Table ). Complications in the bony-ankylosed group included eight cases of heterotopic ossification (HO), with four grade 1, three grade 2 and one grade 3 in Brooker's HO grade ; and loosening of the acetabular side prosthesis; destruction of the femoral side prosthesis; periprosthetic joint infection; and dislocation in one case each. Complications in the control group included periprosthetic joint infection and dislocation in one case each.
In both groups, the postoperative JOA hip scores at the last follow-up had significantly improved compared to the preoperative scores for all components of “total”, “pain”, “range of motion”, “gait ability”, and “activities of daily living” (bony-ankylosed group: p < 0.0001, p < 0.0001, p < 0.0001, p = 0.0038, and p = 0.0001, respectively; control group: p < 0.0001, p < 0.0001, p < 0.0001, p < 0.0001, and p < 0.0001, respectively; Table ).
the bony-ankylosed group on postoperative day 1, the platelet and creatinine phosphokinase levels were significantly lower and higher, respectively, than those in the control group ( p < 0.0209 and p < 0.0034, respectively; Table ). A positive correlation was found between operative time and intraoperative blood loss; a negative correlation was found between age at primary THA and intraoperative blood loss, and between the duration of ankylosis before primary THA and intraoperative blood loss ( p = 0.0001, p < 0.0080, and p < 0.0289, respectively; Table ).
To study the additional outcomes, CT was performed in 11 and eight patients in the bony-ankylosed and control groups, respectively (19 cases). The mean postoperative CSA of the gluteus medius in the bony-ankylosed group was 1363.3.9 ± 495.1 mm 2 , which was significantly lower than that in the control group (2377.5 ± 580.2 mm 2 ; p = 0.0030). The mean postoperative CT values of the gluteus medius at the CSA in the bony-ankylosed group was −4.4 ± 28.6 Hounsfield unit, which was significantly lower than that in the control group (31.8 ± 23.0 mm 2 ; p = 0.0039). The mean time of postoperative CT imaging in the bony-ankylosed and control groups were 7.6 ± 3.5 and 10.1 ± 2.6 years, respectively, which were not significantly different ( p = 0.2437).
is study revealed two important clinical findings. First, except for pain, patients with bony ankylosed hips had improved JOA hip scores after THA; however, the pain scores were worse. Furthermore, the postoperative JOA hip scores in all subcategories and the rate at which the use of walking aids was not required in the bony-ankylosed group were significantly lower than those in the control group. Second, tThe results of the current study showed that although the total JOA hip score of the bony-ankylosed group improved, the pain score worsened: this disadvantage indicated some discomfort and fatigue in the hip that was originally pain-free. Compared to the control group, JOA hip scores were lower in the bony-ankylosed group in all categories. More than half the patients (52.5%, 21/40) with bony ankylosed hips used a walking aid postoperatively (odds ratio, 7.7) compared to 12.5% (5/40) of those in the control group. These results suggest that THA for patients with bony ankylosed hips can somewhat improve the JOA hip score, but not to the same extent as THA for patients without ankylosed hips. This should be preoperatively communicated to patients with bony ankylosed hips who undergo THA. Herein, a greater number of hips in the bony-ankylosed group had postoperative complications than those in the control group and the complications were characterised by HO. HO may be a consequence of the large number of bone fragments produced during osteotomy . Another possible reason is soft tissue damage during surgery. Soft tissue damage due to surgery provides an environment for osteoblasts to develop from mesenchymal cells, causing HO . We observed that blood creatine phosphokinase levels on the first postoperative day in the bony-ankylosed group were higher than those in the control group; creatine phosphokinase levels increase with soft tissue—including skeletal muscle—damage . We hypothesise that damage to the soft tissue during surgery was greater in the bony-ankylosed group than in the control group because of the significant adhesions and anatomical variations around the hip joint, which required a more extensive approach than in normal THA procedures. THA for an ankylosed hip requires a higher level of surgical skill for osteotomy and soft tissue approach and a longer operative time than normal THA . In the current study, intraoperative blood loss, operative time, and blood transfusion were higher in the bony-ankylosed group than in the control group, and the platelet count on postoperative day 1 was lower. Additionally, intraoperative blood loss and operative time were positively correlated. Age at the time of THA and the duration of ankylosis also showed negative correlations with intraoperative blood loss. Skeletal muscle is a vascular-rich tissue; however, muscle mass and vascularity decrease with age and prolonged lack of use . We believe that this negative correlation occurred because the amount of muscle approached at the time of surgery differed depending on the age and duration of ankylosis. Therefore, adequate blood transfusion preparation should be ensured prior to THA for ankylosed hips, especially in young patients or patients with short duration ankylosis. The gluteus medius is one of the major muscles of the hip abductor group and an important determinant of hip function: it ensures hip stability and controls pelvic posture during standing and walking . Decreased gluteus medius volume is the main cause of limp gait in patients with hip joint osteoarthritis . A decrease in the CT values of the gluteus medius represents an atrophic change in the nature of the muscle and muscle weakness in patients with gait disturbance . Herein, the bony-ankylosed group had significantly lower CSA and CT values of the gluteus medius than the control group. Thus, gluteus medius atrophy may have contributed to lower JOA hip scores and longer hospital stays in the bony-ankylosed group. Several studies have reported implant survival rates for THA of ankylosed hips. Hamadouche et al. reported an 8-year survival rate of 96.7% and Joshi et al. reported a 10-year survival rate of 96.1% . Paxton et al. conducted an international comparison of implant survival rates of primary THA for osteoarthritis and reported survival rates of approximately 93–95% . In the current study, the 10-year survival rate of implants in the bony-ankylosed group was 95%, which is comparable with the three aforementioned reports and the current study’s control group. In this study, the THAs performed on the patients with bony ankylosed hips had high survival rates. In contrast, Richards and Duncan reported a 10-year survival rate of 74.2% for conversion of hip arthrodesis to THA . They reported lower survival rates than those of our study. Although a statistical examination was not possible, the mean age of the patients in their study (50 years) was lower than that in our study (60.2 years). The relatively older age of our patients at the time of surgery and the different generations of implants may have affected the survival rate of the implants. This study had four limitations. First, this was a retrospective, single-centre study. However, we used propensity score matching to minimise confounding factors. Second, due to inadequate data recording, we could not assess the outcomes of adjacent joint disorders. Third, only a small number of patients underwent postoperative CT. However, the statistical power of the outcomes was sufficient. Fourth, many patients were excluded due to follow-up periods of less than 10 years. The reason for this is that many patients came to the hospital from far away and the follow-up may have been carried out by the patient's neighbouring doctor, making the follow-up untraceable. In summary, THA for patients with bony ankylosed hips achieved positive results, such as increased JOA hip scores for all items except pain; however, these scores were inferior to those observed in patients with non-ankylosed hips. The number of hips with postoperative complications was significantly higher in the bony-ankylosed group than in the control group.
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Nationwide survey on the usage of ovulation-induction agents among obstetricians and gynecologists in China | 43be6b14-9a18-4666-bf31-ae61a8925766 | 6831081 | Gynaecology[mh] | The authors are grateful to the Chinese obstetrician and gynecologists for participating in the study. They also thank the Gynecological Endocrinology Committee of the Chinese Maternal and Child Health Association for helping to carry out the survey.
This study was supported by a grant from The National Natural Science Foundation of China (No. 11471024).
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Association of sociodemographic factors with the prescription pattern of opioids for dental patients: A systematic review protocol | 54793020-244d-44e2-b651-401f098ed1ab | 8341526 | Dental[mh] | Opioids have long since been used for pain relief, with a key role in modern anesthesia, as well as in postoperative, palliative, and emergency care [ – ]. The analgesia produced by opioids is derived by its complex interaction with receptors of the Central Nervous System . Though effective and largely used worldwide [ – ], opioids are associated with a wide variety of pernicious side effects . When administered at higher doses, the probability of addiction and abuse increases over the long-term . The growth in opioid use has led to a health crisis in some countries . In 2017, the United States (US) declared the opioid epidemic a public health emergency. Opioid misuse has been recognized as a national health issue in Australia . The roots of this epidemic rely on the overall recognition that pain has been underestimated by healthcare providers in the past. In 1990, Max stated that pain was being poorly managed and suggested that authorities should encourage the therapeutic use of opioids. In addition, in 1995, pain was described as the fifth vital sign . Associated with pharmaceutical companies’ aggressive marketing of new opioid formulations and other initiatives that came thereafter, healthcare providers became more sensitive in the treatment of pain . Consequently, opioid sales skyrocketed . The health and social burdens of an opioid epidemic are significant. From 2005 to 2015, there was a 22.3% increase in disorders associated with opioid use worldwide . In the US, data from 2016 estimated 42,245 deaths provoked by opioid overdose (a mean of 118 deaths daily) . Young adults between 25 and 34 years were the most affected (n = 11,552; 20%), and among individuals within this age range, one in five deaths were associated with opioids . In this scenario, deliberate attempts to mitigate the number of prescriptions of opioids by healthcare providers, including oral health practitioners, have been made [ , , ]. Oral health practitioners accounted for 8.6% of all providers who prescribe opioids in the US . The prescription patterns of these drugs in Dentistry seems to be influenced by many factors, including the type of procedure performed, the patient’s threshold for pain, and sociodemographic characteristics [ – ]. It seems that women are more likely to receive an opioid prescription . However, one systematic review reported that an individual’s sex was not identified as an associated factor for opioid prescriptions after surgery or trauma . Compared to white patients, African-Americans are more likely to receive an opioid prescription provided by a dentist . Conversely, another study reported that race was not a predictor for opioid prescriptions in Dentistry . In Brazil, the more privileged the area, the higher the opioid sales . However, in Australia, living in lower socioeconomic conditions was associated with a higher risk of receiving an opioid prescription . Regarding dental insurance, the association with opioid use is still unclear [ , , , ]. In view of the opioid epidemic, awareness of the underlying risk factors for opioid prescriptions could contribute to the rational use of this medication . Recently, protocols to aid health professionals in opioid prescriptions, such as the Centers for Disease Control and Prevention (CDC) guideline , have been provided, but there is still a consensus that a multi-faceted approach is needed to address this issue . Hence, a better knowledge of sociodemographic determinants, associated with the prescription pattern of opioids, may be helpful in the development of interventions to tackle the problem and in the reduction of the perpetuated disparities in the use of pain medications between privileged and underprivileged groups . Primary studies have assessed the possible influence of sociodemographic factors on the prescription pattern of opioids in Dentistry; however, the evidence produced by such studies seems controversial [ , , , , ]. A preliminary search was conducted in the PROSPERO, MEDLINE (PubMed), and Web of Science databases, and no systematic review on this issue was identified. Therefore, the objective of this systematic review is to identify if patients’ sociodemographic factors are in fact associated with the prescription pattern of opioids in Dentistry.
The proposed systematic review was registered in the International Prospective Register Of Systematic Reviews (PROSPERO): CRD42020211226. The Preferred Reporting Items for Systematic Review and Meta-Analysis Protocols (PRISMA-P) checklist is presented ( ). This study is supported by the Coordination for the Improvement of Higher Education Personnel (CAPES, in Portuguese). This funding agency played no role in the study design, decision to publish, or preparation of the manuscript. Review question Inclusion criteria Search strategy Study selection Data extraction Assessment of methodological quality Data synthesis Certainty of evidence If there is sufficient evidence to make recommendations, the Grading of Recommendations, Assessment, Development and Evaluation (GRADE) will be used to rank the certainty of evidence. The GRADEpro (McMaster University, ON, Canada) will be used to construct a Summary of Findings. GRADE is based on five domains: the risk of bias within individual studies, the study design, the indirectness of the evidence, the inconsistency, the imprecision of the effect size estimates, and the risk of publication bias. The certainty of the evidence for each outcome may be ‘very low’, ‘low’, ‘moderate’, or ‘high’ .
Are sociodemographic factors associated with the prescription pattern of opioids for dental patients?
Participants Exposure Outcome Types of studies This systematic review will consider studies with individuals at any age, in which the influence of patients’ sociodemographic factors on receiving an opioid prescription provided by an oral health practitioner was assessed. Studies investigating opioid prescriptions for dental conditions delivered by healthcare providers other than oral health practitioners (e.g., physicians, emergency room [ED] doctors) will be excluded.
In this systematic review, the exposure will consist of dental patients’ sociodemographic characteristics, including sex, age, race, income, educational level, living environment, and dental insurance. Data sources for the assessment of sociodemographic factors will include validated questionnaires, dental charts/records, and oral healthcare system databases.
This systematic review will consider the prescription pattern of opioids provided by oral health practitioners as an outcome.
Observational studies: cross-sectional, case-control, and cohort studies will be included. Letters to the editor, editorials, ecological studies, case reports, case series, and literature reviews will be excluded.
For the definition of the search strategy for this systematic review, three steps were taken. First, a preliminary search was performed in MEDLINE (PubMed) to identify articles that meet the inclusion criteria. Thereafter, the titles and abstracts of these studies were used to identify keywords and indexing terms to develop the final search strategy. Second, with the assistance of a librarian, the final search strategy was tailored for MEDLINE (PubMed) using MeSH terms, entry terms, and synonyms linked with Boolean operators ( ). The search was adapted for each of the other databases, using controlled vocabulary (MeSH, EMTREE, and others), as well as entry terms. Finally, the list of references of all studies included in the systematic review will be screened. Before final analysis, the searches will be updated. No restrictions will be applied to language and year of publication, or geographic limits. Information sources Searches will be conducted in the following electronic databases: MEDLINE (PubMed), EMBASE, Scopus, Web of Science, LILACS and SciELO. A search in Google Scholar, limited to the first 200 most relevant studies , will also be conducted. Grey literature will be searched in OpenGrey.
All identified citations will be uploaded into EndNote 20 (Clarivate Analytics, PA, USA) and duplicates removed. Two reviewers will select the studies independently. They will begin by assessing the titles/abstracts. Those that meet the inclusion criteria will be selected. If the title/abstract does not provide sufficient information for a decision, the full text of the reference will be evaluated. References whose full text fulfills the eligibility criteria will be included. Any disagreements arising during the study selection will be discussed and resolved by consensus. If an agreement is not reached, a third reviewer will decide. Interrater reliability will be estimated with the kappa test.
Two reviewers will extract all data from the included studies independently and in duplicate. If divergences between reviewers occur, a discussion will be set in place until a consensus is reached. Data extracted will include study details (author, year, journal), study methods (design, setting, sample, recruitment process, exposure), prescription of opioids (frequency, dose, duration of prescribed opioid), and the results of evaluation of the association of exposures (sociodemographic factors: sex, age, race, income, educational level, living environment, and dental insurance) with opioid prescriptions. The form developed for data extraction is displayed as supporting information ( ).
Two reviewers will assess the methodological quality of the included studies independently. Disagreements arising during this process will be discussed and resolved by consensus. In the cases in which agreement is unattainable, a third reviewer will be consulted. Depending on each study design, the following JBI’s critical appraisal tools will be deployed: Checklist for Analytical Cross-Sectional Studies, Checklist for Case Control Studies, and Checklist for Cohort Studies will be used, depending on each study design . In the cross-sectional studies, eight items will be assessed: definition of the inclusion criteria; depicted information of participants, study´s setting, and time period; use of valid and reliable methods for the assessment of the exposure; if objective and standard criteria were employed to assess the condition; awareness of confounders; reliable measurement of the outcome; and use of adequate statistical analysis . In the case control studies, 11 items will be assessed: similarity between groups, presence of disease in cases or the absence of disease in controls; matching of cases and controls; adoption of the same criteria to identify cases and controls; adoption of a valid and reliable strategy for the measurement of the exposure; same measurement of the exposure for cases and controls; awareness of confounders; strategies to handle confounders; adoption of a valid and reliable method for the measurement of the outcome for cases and controls; period of interest of the exposure, if this period was long enough to be significant; and adoption of adequate statistical analysis . In the cohort studies, 11 items will also be assessed: recruitment of participants of the two groups from the same population; similar measurement of the exposure to assign participants to both exposed and unexposed groups; use of a valid and reliable instrument for the measurement of the exposure; identification of confounders; strategies to deal with confounders; groups of individuals without the outcome at the study’s onset; adoption of a valid and reliable strategy for the measurement of the outcome; report of the follow-up period and whether this period is long enough for the occurrence of the outcome; information whether follow-up was complete or at least a statement on the reasons for follow-up losses; strategies to handle incomplete follow-up; and adoption of adequate statistical analysis . In each study, three ratings may be assigned to the items: ‘low risk of bias’ (if the answer to the question is ‘Yes’), ‘high risk of bias’ (if the answer to the question is ‘No’), and ‘unclear risk of bias’ . All studies will be submitted to data extraction and synthesis. The impact of risk of bias will be considered when developing conclusions and recommendations (if feasible).
In the first step of data synthesis, we will present the results of the study selection process using the PRISMA statement flowchart. The interrater reliability (agreement between the two reviewers) estimate will be given with the kappa test. In the second step of data synthesis, homogeneous data will be aggregated into meta-analyses. Effect measures will be expressed as either odds ratio/risk ratio (for dichotomous data), or mean differences (for continuous data). In the case of meta-analyses of continuous outcomes, for which included studies used different scales, the standardized mean difference will be determined. The 95% confidence interval (CI) will be calculated as well. The possibility of combining studies of continuous and dichotomous outcomes through transformations will be assessed. Heterogeneity will be assessed using chi-square and the I 2 statistic . The confidence interval of the I 2 will be determined as well. The following formula will be used: exp (ln I 2 ±1.96× SE [ ln ( I 2 )]) . The random effect model will be deployed in the meta-analysis . We will also check whether the results of the meta-analyses remain even if undetected heterogeneity is assumed. The random effect model will be implemented through the DerSimonian and Laird inverse variance . Meta-analyses will be developed at RevMan 5.4 (Copenhagen, The Nordic Cochrane Centre, Cochrane). Sensitivity analysis might be conducted with the removal of studies one by one in an attempt to reduce heterogeneity. If possible, subgroup analyses will be performed according to the different sociodemographic factors assessed (sex, age, race, income, educational level, living environment, and dental insurance). The design of the included studies will also be considered during the analysis of subgroups. The results of meta-analysis will be presented as forest plots. Publication bias will be addressed by developing a funnel plot . The RevMan 5.4 (Copenhagen, The Nordic Cochrane Centre, Cochrane) will be used. Considering that the visual evaluation of the funnel plot might be subjective, we will run a test for funnel plot asymmetry, so long as at least ten studies are incorporated into the meta-analysis . The regression method for the detection of funnel plot asymmetry proposed by Harbord and colleagues (2006) will be used for dichotomous outcomes. For the meta-analysis of continuous outcomes, the assessment of funnel plot asymmetry will be performed with the Egger test . Moreover, if less than ten studies are incorporated into the meta-analysis, no funnel plot will be constructed, and this will be reported and discussed as a limitation of the systematic review. Bias assessment, considering the year of publication of the articles, will also be performed, as publication bias in systematic reviews of more recent studies is lower .
S1 Checklist PRISMA-P 2015 checklist: Recommended items to address in a systematic review protocol. (PDF) Click here for additional data file. S1 File Search conducted in MEDLINE (PubMed) on September 28th, 2020. (PDF) Click here for additional data file. S2 File Form to extract data of included studies. (PDF) Click here for additional data file.
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Correlation between marginal bone loss around dental implants and various systemic diseases: a cross-sectional study | 9a15e94e-c66d-4be8-b4c4-47d6536aa3e3 | 11502616 | Dentistry[mh] | Dental Implants are a widely utilized option for patients suffering from tooth loss that can help restore both function and esthetics. The long-term survival of dental implant therapy is reported widely in various studies . A dental implant failure can occur in the early stages due to a lack of osseointegration or in the late stage due to peri-implantitis . Marginal Bone Loss [MBL]There is a bidirectional relationship between systemic and oral health, which may compromise the survival of the dental implant . Hypertension is defined as a systolic value over 140 mmHg and/or a diastolic reading of over 90 mmHg and is considered the main cause of premature diseases and death in the world . Blood pressure readings over time are valuable predictors of an individual’s risk of developing cardiovascular disease (CVD). Not only does it impose a risk of CVD, it can lead to comorbidities such as chronic renal failure and stroke. Bone abnormalities have also been linked to hypertension including a decrease in the regeneration, density, and quality of the alveolar bone due to impaired calcium metabolism and delayed healing . Hyperlipidemia occurs as a result of abnormal lipid levels within the blood. This is characterized by increased levels of total cholesterol, triglycerides and low-density lipoproteins (LDL) as well as a decrease in the levels of “good cholesterol” known as high density lipoproteins (HDL) . Currently, nearly 86 million adults in the United States 20 years and older have total cholesterol levels above 200 mg/dL . A high level of cholesterol is a major controllable risk factor for other health complications including heart disease, myocardial infarction, and cerebrovascular accidents. Several studies have investigated the effects of hyperlipidemia on bone and have found that elevated amounts of plasma lipoproteins may increase the number of osteoclasts in the alveolar bone . This phenomenon will lead to the inhibition of osteoblastic activity needed for osseointegration of the dental implant . Diabetes mellitus is one of the most common chronic health issues within the world and is directly correlated with impaired wound healing due to an overactive immune response to pathogens. Inflammatory mediators (IL-1, IL-6, IL-8) and tumor necrosis factor (TNF-ɑ) associated with diabetes are released into the oral tissues. These factors can lead to increased inflammation and a reduction in collagen synthesis which ultimately affects formation of bone and the healing capability of oral tissues . Hyperglycemic conditions over the long-term may degrade the vascularity within the oral cavity and that supplying the alveolar bone . Just as in hyperlipidemia, the differentiation of osteoclasts is promoted and osteoblast formation is inhibited. Due to these risk factors, dental implants may not be considered on these individuals. While complications are evident among diabetic individuals, they have been seen to diminish in those with properly controlled diabetes mellitus . In 2016, a systematic review narrated a significant delay in the osseointegration of dental implants in poorly controlled diabetic patients . A recent meta-analysis indicated a direct association between hyperglycemia and the risk of peri‐implant diseases, and there is a high risk for MBL of dental implants for type 2 diabetes mellitus (T2DM) control patients . It has been suggested that successful implants must present ≤ 2 mm of MBL during the first year after placement, followed by ≤ 0.2 mm per subsequent year . It was indicated that there is an increased risk of implant failure if the MBL was 0.44 mm, at six months post-loading . In 2018, Neves et al. concluded that rheumatologic and cardiovascular disorders are associated with an increased risk of peri-implant pathology . A cross-sectional biochemical study comparing the inflammatory and lipid profiles of patients with and without peri-implantitis suggested that even healthy individuals with peri-implantitis exhibited a low-grade systemic inflammatory state, evidenced by elevated circulating white blood cell levels, as well as dyslipidemia, characterized by increased LDL cholesterol and total cholesterol levels . A recent umbrella review emphasized the need for further studies to assess the long-term effects of cardiovascular disease, neurological disorders, and the use of certain medications on dental implant survival rates . Another systematic review also highlighted that more research is required before drawing definitive conclusions about the association between cardiovascular disease and peri-implantitis, as the current body of literature contains too few studies to establish a clear link . The present study aimed to evaluate marginal bone loss (MBL) around dental implants in patients with hypertension, hyperlipidemia, and diabetes mellitus attending the University of Nevada, Las Vegas (UNLV) dental clinics. The null hypothesis proposed that there would be no significant difference in MBL among patients with hypertension, hyperlipidemia, and diabetes.
Study design and population Search strategy Inclusion and exclusion criteria Data extraction Ethical approval Radiographic analysis This study is a cross-sectional study of patients who attended the University of Nevada, Las Vegas dental clinics.
Clinical notes from patients at the University of Nevada, Las Vegas (UNLV) dental clinics were analyzed using AxiUm™ software. AxiUm™ is a comprehensive dental practice management software for educational institutions, particularly dental schools. It helps streamline various administrative, clinical, and financial operations by integrating tools and features that manage patient records, treatment planning, billing, scheduling, and academic workflows. A search was conducted using keywords such as ‘systemic disease,’ ‘marginal bone loss,’ ‘dental implant,’ ‘high cholesterol,’ ‘hypertension,’ and ‘diabetes’ within the system (Fig. ).
The study included patients with dental implants diagnosed with hypertension, diabetes mellitus, and hyperlipidemia who attended the UNLV School of Dental Medicine clinics from 2012 to 2022. Exclusions were made for patients with acquired immune deficiency syndrome and those with a limited number of radiographs.
All patients are informed during their intake to the UNLV dental clinics that they are being treated at an educational institution and their information may be used for research purposes. Their information is protected according to regulations dictated by the Federal Health Insurance Portability and Accountability Act of 1996 (HIPAA). Socio-demographic data including the patient’s age, gender, ethnicity, smoking habits (yes/no and type), and alcohol consumption (yes/no) were recorded. Dental and medical histories were collected from the existing chart records and evaluated. Data collected from these forms included systemic health issues and prescribed medications. Data were extracted by AC and NHA.
As no supplementary radiographs or examinations were conducted specifically for the study, and the data collected were analyzed and presented anonymously, the study was granted exempt status by the Institutional Review Board (IRB) of the University of Nevada at Las Vegas (UNLV; #UNLV-2022-256). The study adhered to the principles of good clinical practice in accordance with the World Medical Association (WMA) Declaration of Helsinki (1975), revised in 2013.
All radiographs were obtained using a long-cone technique at 70 kV and exposure time of 0.16 s. Only periapical radiographs were considered for the study, no CBCT imaging was included. All periapical radiographs were standardized using sensor holders and the parallel technique. Additionally, experienced dental technicians supervised all radiographic images taken at the radiology clinic to ensure quality and consistency. If the implant was placed at the UNLV dental clinic, peri-implant bone measurements were obtained from the most recent radiographs and radiographs at the time of placement. If the patient presented to the UNLV clinics with an existing implant, new radiographs taken over the course of at least 6 months were included in the data. The marginal bone level is defined as the vertical distance from the tip of the implant body to the coronal edge of the first bone-to-implant contact . Marginal bone level was evaluated based on this principle as well as methods used in previous studies. Measurements were taken from the implant-abutment junction to the crest of the bone at both mesial and distal sides of each implant following the methodology described by Shi et al. (Fig. ). MBL was confirmed by comparing subsequent bone-to-implant contact levels to the initial radiographs. The MiPACS (Medical Imaging Picture Archiving and Communication System) is a comprehensive digital imaging system fully integrated with the axiUm™ dental software, and it was used for all radiographic measurements. MiPACS is the primary imaging module for capturing, viewing, and managing dental radiographs such as intraoral radiographs, panoramic images, and CBCT scans. MiPACS enhances clinical diagnostics with advanced image enhancement tools, including zoom, contrast adjustment, annotations, and measurement tools. One investigator (first author), conducted the measurement in order to eliminate inter-examiner variation. Using Cohen’s kappa in SPSS (version 28), the intra-examiner reliability was calculated to be 85%, indicating a strong level of agreement between repeated measurements.
After screening the 1,310 potentially eligible electronic Axium records, 57 patients with 165 implants met the selected inclusion criteria (Fig. ). Table shows the descriptive statistics of the sample. The majority of the patients were aged 65 and older (79%) followed by those between 55 and 64 years of age (18%). Social factors such as smoking and alcohol consumption accounted for < 50% of the sample (Table ). The more systemic health disease a subject presented with, the more likely they had bone loss surrounding an implant as 45.6% of patients reported having more than four systemic diseases and 67% were taking four or more prescription medications (Fig. ). A decrease in the marginal bone surrounding dental implants in this sample was strongly correlated with patients diagnosed with hypertension (78.95%) or hyperlipidemia (73.68%) compared to those with diabetes mellitus (40.35%). Patients diagnosed with both hypertension and hyperlipidemia comprised 29.82% of the sample data and held statistical significance ( p < 0.05) (Fig. ). Overall, varying combinations of these systemic diseases and comorbidities were more closely associated with peri-implant MBL than patients with a single systemic disease diagnosis. Prescribed medications to combat these health issues, such as statins and antihypertensive, also showed the same trends and corresponded to a higher prevalence of MBL (Table ). Over 90% of patients in each category reported taking the daily doses of medications on a regular basis.
The aim of the present cross-sectional study was to investigate the correlation between hypertension, diabetes mellitus and hyperlipidemia, on the marginal bone loss (MBL) surrounding dental implants among patients attending the University of Nevada, Las Vegas dental clinics. After analyzing the results, it was found that there was a statistically significant difference in MBL between patients with cardiovascular diseases (HTN, hyperlipidemia) and diabetic patients. This study has successfully demonstrated that implant failure and peri-implant bone loss occur more in individuals that present with cardiovascular disease. Therefore, the null hypothesis was rejected. Several studies have been published that report a direct relationship between diabetes and peri-implantitis. They concluded that diabetic patients, particularly those diagnosed with type 1, have a greater estimated MBL over time . This has been attributed to the reduced angiogenesis due to hypercoagulation as well as a hindrance of bone formation markers . For Diabetic patients, it is important to note that the type and stability of diabetes is a determinant in the overall success of the implant. Deeper pocket depths, bleeding on probing and increased MBL were more commonly found among those with poor metabolic control compared to those with ‘well controlled’ diabetes . This data correlates with the present study as diabetic patients presented with Type 2 diabetes had varying level of glycemic controls. Bone metabolism is affected by hyperlipidemia through both osteoclasts and osteoblasts which may promote bone loss and inhibited osseointegration of dental implants . In an experimental study on rats, Teken and Toker found that the rats that were fed a high cholesterol diet showed significantly lower bone-to-implant contact values than the control group . The findings of their study give support to the hypothesis that hyperlipidemia can lead to a decrease in implant osseointegration and implant stability. Statin medications used to treat hyperlipidemia were also correlated to peri-implant bone loss. Behrami et al. conducted a retrospective study on the influence of statin use on the severity of peri-implantitis and the incidence of peri-implant bone loss . However, several studies reported an increase in osseointegration of the implant body into the alveolar bone when coupled with Simvastatin. Implants were either coated with the Simvastatin or a gel containing the medication was placed into the alveolar socket at the time of placement . These findings illustrate the need for further investigation into the effects of hyperlipidemia and statins on endosteal implants. Hypertension has a positive association with moderate to severe periodontitis as bleeding on probing, CAL, and pocket depths were poorer on hypertensive patients . Singh et al. found that hypertension led to a 20.8% increase in the failure of dental implants. This phenomenon may be due to impaired calcium metabolism and delayed healing associated with higher arterial pressure . In a cohort study by Wu et al., of the 1,449 implants, a failure rate of 0.6% was observed in people using antihypertensive drugs and 4.1% for nonusers . The results of these two studies show a possible failure of compliance in hypertensive patients taking their medications. This should be accounted for in the present study as the data relies on self-reporting from patients. Future studies should also evaluate the influence of time and follow-up periods on MBL which was not considered in this study. There are several limitations to the present study. First, this is a cross-sectional dental-record based study that relied on the accuracy of the examination and documentation. Incomplete documentation was anticipated as patients are seen by a variety of students at University Dental Clinics. Second, there was a lack of quantifiable measurements of MBL radiographically and also no CBCTs were used in the investigation. The correlation was related to the main and mostly reported systemic diseases and not all systemic diseases. The measurements were performed as a visual representation of the effects associated with specific health issues and medications rather than a diagnostic tool. Third, oral hygiene and parafunctional habits could not be properly assessed due to the nature of this study; therefore the oral environment and the forces applied to the prosthesis might have an impact on the clinical outcomes. Lastly, a limited number of subjects, implants and restoration types were identified within the study.
Within the limitations of the present investigation, patients diagnosed with hyperlipidemia and hypertension were more likely to exhibit MBL surrounding dental implants. Further investigation is required to correlate the presence of MBL around dental implants with antihypertensive and statin medication use.
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SOCIAL DETERMINANTS OF MORTALITY FROM COVID-19, A RETROSPECTIVE STUDY OF 6,000 PATIENTS | c718c3a0-ec83-4251-860b-f83f7d1fb422 | 8972426 | Internal Medicine[mh] | Health disparities and Coronavirus disease 2019 (COVID-19) mortality is an evolving topic. This study sought to explore the relationship between patient ethnicity, annual household income, and COVID-19 mortality.
A chart review was conducted of 6,000 hospitalized patients with positive COVID-19 polymerase chain reaction (PCR) tests from March 2020 to June 2021 at Methodist Health System in Dallas, Texas. Patient age, gender, ethnicity, and zip code were collected. The sample included 3,114 males and 2,886 females with a mean age of 61.6±17.1 years. Ethnicity selected by the patient was used. Median annual income by zip code was obtained from the 2020 U.S. Census Data. A Chi-square test was used to calculate p-values.
No statistically significant difference in mortality based on ethnicity or median annual income by zip code was found. Asian American patients had the lowest mortality rate, while Hispanic, Latino, or Spanish origin had the highest mortality rate with p(0.92), independent of other factors. Patients living in a zip code with a median household annual income of $40-80K had the lowest mortality rate while those with ≥$80K had the highest mortality rate with p(0.12), independent of other factors as shown in Table 1.
Our study suggested that neither ethnicity nor income predicted mortality in admitted COVID-19 patients, independent of age and gender.
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Low Skeletal Muscle Mass: A Strong Predictive Factor for Surgical Complications After Free Forearm Flap Reconstruction in Oral Cancer Patients | cb29d944-aaa9-4b76-8a51-f4f4831188f2 | 11907681 | Musculoskeletal System[mh] | Introduction Free forearm flaps (FFAF), that is, the free radial forearm flap (FRFF) and free ulnar forearm flap (FUFF), are frequently used free vascularised flaps for reconstruction of large head and neck soft tissue defects following ablative cancer surgery. A number of preoperative risk factors are known to be associated with postoperative complications after free tissue transfer, which include age, gender, tobacco use, diabetes, hypertension, body mass index (BMI) and prior radiotherapy . Sarcopenia, a condition characterized by loss of skeletal muscle mass (SMM) and low muscle strength, has been found to influence both treatment outcomes and survival in head and neck cancer (HNC) patients . Low SMM alone has been associated with a higher incidence of postoperative complications, chemotherapy related toxicity, longer hospital stays and diminished disease‐free and overall survival in HNC patients . Patients with HNC are more likely to develop sarcopenia because of swallowing disorders caused by the localization of the primary tumor, decreased nutritional intake and cancer‐induced catabolism . Some studies found that sarcopenia is a significant independent risk factor for free flap complications, surgical site infection and other postoperative complications in patients with HNC [ , , , ]. The aim of this article is to analyze the association of low SMM with free flap related and other postoperative complications in a subgroup of HNC patients who underwent an FFAF reconstruction following resection of an oral cancer. Furthermore, the relationship between low SMM and the duration of hospital stay is investigated.
Material and Methods 2.1 2.3 2.4 Statistical Analysis The data analyses were performed using IBM SPSS Statistics version 25.0 (IBM Corp., Armonk, NY, USA). The baseline characteristics were presented as frequencies and percentages. Correlation analysis was performed by use of Pearson's correlation analysis for variables with a normal distribution and Spearman's correlation analysis was used for non‐normally distributed variables. Logistic regression was used for univariate and multivariate analysis of surgical complications. Covariates used in the multivariate analysis were selected based on clinical significance or selected based on statistical significance ( p < 0.20) in univariate analysis. A test of normality (One sample Kolgomov–Smirnov) was performed for the duration of hospital stay. If hospital stay was normally distributed, an independent sample t ‐test or a linear regression model was performed if the assumptions of linear regression were met. Statistical significance in this analysis was evaluated at the p < 0.05 level using two‐sided tests.
Ethical Approval The design of this study was approved by the Medical Ethical Research Committee of the University Medical Center Utrecht (approval ID 17‐365/C). All proceduresPatients and Study Design A retrospective study was performed of consecutive patients who underwent reconstruction of oral cavity defects with FFAF after resection of a malignancy between 2003 and 2020 at the Departments of Oral and Maxillofacial Surgery, Otorhinolaryngology and Head and Neck Surgery and Head and Neck Surgical Oncology of the University Medical Center Utrecht, Utrecht, the Netherlands. All surgical procedures were performed by experienced microvascular head and neck surgeons. The choice of an ulnar or radial forearm flap was based on the surgeon's preference and experience with raising the specific flap. Patients were included if they had a recent (less than 1 month before surgery) CT or MRI scan of the head and neck. Clinical and demographic data were collected from the medical records. Data collected included age at surgery, gender, BMI, alcohol consumption (defined as drinking more than 2 units alcohol per day), smoking history (categorized as current smoker or having stopped more than 12 months), diagnosis, TNM stage (pathological), localization of the defect, comorbidity as expressed by the Adult Comorbidity Evaluation‐27 (ACE‐27) score, duration of hospital stay and occurrence of postoperative complications Patients with prior chemoradiation therapy to the head and neck were not included. Neither were patients with recurrences or preoperative nasogastric tube insertion included. All postoperative complications were scored according to the Clavien–Dindo classification of surgical complications . Patients with multiple complications were scored according to their highest grade of complication. Complications with a Clavien–Dindo grade III–V were graded as severe complications. Postoperative complications specifically related to the free flap were also analyzed and scored. These were categorized as congestion or thrombosis, partial skin paddle necrosis or dehiscence, donor site morbidity and flap failure.
Body Composition Measurement SMM was measured as muscle cross‐sectional area (CSA) on pre‐treatment CT or MRI imaging of the head and neck area at the level of the third cervical vertebrae (C3). The first axial slide of the imaging when scrolling from cranially to caudally, which showed both transverse processes and the entire vertebral arc, was selected for segmentation of muscle tissue. For CT imaging, muscle area was defined as the pixel area between the radiodensity range of −29 and +150 Hounsfield Units (HU), which is specific for muscle tissue. For MRI, muscle area was manually segmented, and fatty tissue was manually excluded. The CSA was calculated as the sum of the delineated areas of the paravertebral muscles and both sternocleidomastoid muscles. Segmentation of muscle tissue was manually performed using the commercially available software package SliceOmatic (Tomovision, Canada) by a single researcher (NCM) who was blinded for patient outcomes. An example of segmentation at the level of C3 is shown in Figure . CSA at the level of C3 was converted to CSA at the level of L3 using a previously published formula 1 (23). The lumbar skeletal muscle index (LSMI) was calculated by correcting SMM at the level of L3 for squared height as shown in formula 2. Low SMM was defined as a LSMI below 43.2 cm 2 /m 2 , a cutoff value which was determined in a separate cohort of head and neck cancer patients . Formula 1 : CSA at L3 cm 2 = 27.304 + 1.363 * CSA at C3 cm 2 – 0.67 1 * Age years + 0.640 * Weight kg + 26.44 2 * Sex Sex = 1 for female and 2 for male Formula 2 : Lumbar SMI cm 2 / m 2 = CSA at L 3 / length m 2
Results 3.1 3.2 3.3 3.4 Patient Characteristics Descriptive data are presented in Table . In total, 174 patients were included. Low SMM was identified in 115 (66.1%) patients. One hundred and thirty‐eight (79.3%) patients underwent a FRFF and 36 (20.7%) patients underwent a FUFF.
All Complications Table shows the non‐flap complications. Clavien–Dindo scores of all postoperative complications are described in Table . In total, 117 (67.2%) patients had any postoperative complication, of whom 77 (65.8%) had low SMM. Forty‐one patients (23.6%) had severe complications (Clavien–Dindo III–V), of whom 27 (65.9%) had low SMM. Four patients (2.3%), all with low SMM, died in hospital within 1 month due to a complication. The results of the univariate analyses on potential risk factors for any postoperative complications are shown in Table . Age, alcohol use, smoking, BMI, ACE‐27 score and SMM were included in the multivariate logistic analysis. The results of the multivariate analysis are shown in Table . Low SMM was not associated with any postoperative complications (OR 1.18; 95% CI 0.58–2.57, p = 0.64). Furthermore low SMM was significantly associated with severe postoperative complications (Clavien–Dindo III–V) (OR 1.46; 95% CI 1.20–2.09, p = 0.02).
FFAF Related Complications FFAF‐related complications are described in Table . Complications related to the FFAF occurred in 47 (27.0%) patients, of which 25 (53.2%) occurred in patients with low SMM. Five (3%) patients needed flap revision due to venous thrombosis or arterial occlusion. In 3 (1.7%) patients, the complete flap was lost. In the logistic regression, low SMM was associated with flap related complications (OR 2.14; 95% CI 1.02–4.39, p = 0.029).
Duration of Hospital Stay Median length of hospital stay was 14.3 days (95% CI 12.99–15.58, SD 8.54). Mean number of days in hospital was similar for patients with or without low SMM (14.40 days, SD 7.23 and 14.23 days, SD 9.05, respectively).
Discussion This study assessed risk factors for the occurrence of postoperative complications in patients who underwent reconstruction of head and neck defects with FFAF after resection of a malignant oral cavity tumor. We compared several potential perioperative predictive factors, of which low SMM was significantly likely to cause more postoperative FFAF related complications such as flap dehiscence, flap necrosis, thrombosis and flap failure. Postoperative complications of any type were not significantly higher in patients with low SMM. However low SMM was predictive for the occurrence of severe complications (Clavien–Dindo III–V). The finding of the current study is in line with previous studies. A systematic review with meta‐analysis demonstrated that low SMM was associated with the occurrence of severe postoperative complications in patients with head and neck squamous cell carcinoma (OR 4.79, 95% CI: 2.52–9.11) . Previously we evaluated postoperative complications among patients who had undergone reconstruction of segmental mandibular defects with free fibula flaps . In another article low SMM in FRFF was found to be a predictor for postoperative complications (OR 2.0, 95% CI 1.1–3.8, p = 0.03) . We observed that low SMM was a negative predictive factor for postoperative flap complications. A retrospective case–control study by Alwani et al. evaluated complications among 168 HNC patients who received autologous free vascularized tissue reconstruction. Patients with low SMM had higher rates of complications, including pneumonia, venous thromboembolism, longer mechanical ventilation times, delirium, wound disruptions/fistula, and intensive care unit stays. Overall, these patients had higher rates of any postoperative complications and also flap‐specific complications . In another study of 239 HNC patients who underwent free flap reconstruction, low SMM was a predictor of perioperative blood transfusion requirements . In a cohort of 206 HNC patients 30.1% were discharged after free flap reconstruction to post–acute care facilities, including skilled nursing facilities, in‐patient rehabilitation facilities, and long‐term care hospitals, for extended support and recuperation beyond the immediate postoperative setting. Low SMM was found to be independently associated with discharge to the above mentioned post–acute care facilities . In this cohort flap, dehiscence was particularly present in the low SMM group 44.7% versus 31.9% in the normal SMM group. In line with this finding is a study of patients undergoing total laryngectomy, where sarcopenia was found to be the sole predictive factor of any wound complication (OR, 7.54; 95% CI, 1.56–36.4) . The pathophysiological mechanisms underlying the association between preoperative sarcopenia and the risk of postoperative complications have not been elucidated. SMM depletion is associated the production of anti‐inflammatory cytokines and adiponectin decreases and the production of pro‐inflammatory molecules, such as leptin, chemerin, resistin, tumor necrosis factor‐α, interleukin‐1 and ‐6 increases . Based on this mechanism, patients with sarcopenia are considered to be in a pro‐inflammatory state. The pro‐inflammatory state leads to a weakening of the immune system and poor wound healing after surgery, thereby exerting an impact on the risk of postoperative complications . Postoperative complications can have devastating consequences for both functional and cosmetic outcomes and can have a serious psychological impact on patients. Assessment of SMM is an objective measure that can provide valuable information in the clinical setting. It can be used to predict postoperative outcomes and consequently aid in surgical decision making. For instance, as in whether or not to opt for a microvascular reconstruction or a reconstruction with a local flap, which entails less risk of wound dehiscence and shorter operative time. Selecting patients with low SMM for a local flap reconstruction can potentially reduce postoperative complications. Several methods to increase skeletal muscle mass and to decrease systemic inflammation have been reported in the literature . In a double‐blind, randomized placebo‐controlled trial by Rooks et al. bimagrumab treatment, a monoclonal antibody that blocks activin type II receptor (ActRII) to inhibit myostatin signaling and stimulate protein anabolism was added to optimized standard of care in community‐dwelling older adults with sarcopenia . The results showed no difference in the improvement of physical function between bimagrumab versus placebo, although participants who received bimagrumab had an increased lean body mass and reduced fat mass versus participants who received placebo. Physical activity is also effective at mitigating sarcopenia. Several studies have demonstrated that prehabilitation in patients undergoing major abdominal surgery, particularly with exercise programs, results in lower rates of postoperative morbidity . However, these studies did not stratify for sarcopenia. Optimizing nutritional status may be valuable in patients prior to surgery because this can potentially decrease systemic inflammation and promote better wound healing. An example is the area of immunonutrition supplements that contain arginine, omega‐3 fatty acids, and dietary nucleotides that modulate prostaglandin E2 production, decrease IL‐6 production, and promote T‐cell differentiation. Mueller et al. demonstrated that the use of immunonutrition for 5 days prior to salvage surgery among patients with HNC compared to a control group, caused a significant reduction in overall complications (35% vs. 58%, p = 0.027) and in overall length of hospital stay (median 6 vs. 17 days, p = < 0.001) . A study by Aeberhard et al. found that the use of immunonutrition for 5 days prior to surgery was associated with a significant decrease in the occurrence of wound abscesses and orocutaneous or pharyngocutaneous fistulas compared to the control group (7.4% vs. 15.3%, OR = 0.30, p = 0.006) . The combination of nutritional intervention and exercise has also been reported. A recent meta‐analysis of 42 RCTs compared multiple exercise intervention arms in 3728 older people with sarcopenia . This analysis found that adding nutritional interventions to exercise had a larger effect on handgrip strength than exercise alone while showing a similar effect on other physical function measures to exercise alone. Our study was limited due to its single‐institution and retrospective nature and therefore limited by the available medical documentation. Heterogeneity in terms of types of flaps, that is, only FRFF and FUFF, was limited (particularly when compared to other studies), but may have introduced bias with respect to flap complications. Although we found an association between low SMM and severe complications and also flap complications, we could not determine the odds of specific complications such as pneumonia, wound infections, delirium and flap failure due to the limited number of events. SMM was assessed on cross‐sectional imaging at the level of C3 because of the availability of routinely performed head and neck CT and MRI and its high correlation with the most often used SMM assessment at the third lumbar vertebra (L3) [ , , ]. Changes in SMM occur over time since cancer‐related skeletal muscle depletion is a continuous process. This study is limited to preoperative SMM at a single point in time, although the degree and effect of eventual perioperative change of SMM is unknown. In conclusion, low preoperative SMM has a negative impact on the occurrence of FFAF‐related complications in patients undergoing an FAFF reconstruction for oral cavity cancer. It is also a negative impact factor for severe (CD ≥ III) postoperative complications. Identification of high‐risk patients by SMM assessment allows for alternative surgical treatment planning, for example, less extensive surgery and less complex reconstructions, pre‐ and perioperative management and counseling.
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A feasibility study of DNA ploidy analysis, HPV, and TCT for screening of cervical cancer: A retrospective study | 3e00b45f-17b7-4de7-9d1c-87b5329e3452 | 11630942 | Biopsy[mh] | Cervical cancer (CC) is the second most prevalent malignant tumor in females ; it is the only disease with a clear pathogenetic mechanism and can be prevented by vaccination and screening. However, because of outdated screening technology and a lack of awareness regarding prevention, there is still high morbidity and mortality due to CC in some areas. According to a recent estimation in 2018, the number of new global cases of CC and the number of deaths due to CC were 569,847 and 311,365, respectively. Precancerous and early invasive CC can be detected by CC screening, which effectively reduces the associated morbidity and mortality. Presently, the “three-step” diagnostic method is widely used to diagnose cervical diseases; this method includes cytology and/or human papillomavirus (HPV-DNA) test, colposcopy, and histopathological diagnosis. HPV-DNA testing is a pivotal element in CC screening, focusing on the identification of high-risk HPV types that are strongly linked to the development of CC. This method has significantly augmented the capability of screening programs to predict and prevent CC by targeting the primary cause of the disease before significant cellular changes occur. Concurrently, the thin-prep cytologic test (TCT) has refined the traditional Pap smear approach by employing liquid-based cytology. This advancement enhances the quality of cell samples and the accuracy of the results, allowing for better detection of cellular abnormalities. TCT categorizes cells more precisely, which significantly decreases the rate of false negatives and increases the sensitivity of detecting precancerous states. However, the efficacy of TCT largely depends on the subjective assessment of cytologists, which can introduce variability in diagnosis. In recent years, many new CC screening methods have emerged. Deoxyribonucleic acid (DNA) ploidy analysis for cervical epithelial cells is a new method of cellular DNA quantitative diagnosis, and it is used to estimate the physiological status and pathological changes in cells by detecting the ploidy in the cell nucleus. An advantage of this method is that it can be combined with HPV tests to enhance the accuracy of CC screening. Despite the effectiveness of HPV testing and TCT, there are inherent limitations that affect their diagnostic precision and ability to detect all cases of precancer and early invasive CC. Variability in human interpretation of TCT results, and the limited ability of HPV testing alone to predict which infections might progress to cancer, highlight the need for improved screening methods. This study proposes an innovative approach by integrating DNA ploidy analysis with HPV testing and TCT. This combination aims to leverage the strengths of each method to enhance the overall accuracy and predictive capability of CC screening. Therefore, in the present study, random samples were collected from 14,019 patients who underwent both TCT and HPV-DNA test in Shandong Provincial Hospital, Liaocheng People’s Hospital, and First People’s Hospital of Tancheng County from June 2021 to June 2022. The results of DNA ploidy analysis were compared with those of TCT, HPV-DNA, and biopsy, and samples with inconsistent results were rechecked to investigate the application of DNA ploidy analysis in the preliminary screening of HPV (+) CC.
2.1. Sample source 2.2. HPV-DNA test 2.3. Thin-prep cytologic test 2.4. DNA ploidy analysis 2.5. Evaluation criterion for biopsy 2.6. Inclusion and exclusion criteria 2.7. Statistical analysis The study included 14,019 patients who underwent both TCT and HPV-DNA tests from June 2021 to June 2022 (10,199 patients from Shandong Provincial Hospital, 2450 patients from First People’s Hospital of Tancheng County, and 1370 patients from Liaocheng People’s Hospital). Before conducting DNA ploidy analysis, informed consent was obtained from all patients. This study was approved by the Ethics Committee of Shandong Province Hospital.
Cells were collected from the cervical transformation zones of the patients by using a special cervical brush and stored in a bottle containing a special storage solution. HPV type 16 and 18 and other 12 high-risk subtypes (type 31, 33, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68) were detected by PCR and relevant reagents within 3 days of sample collection. In accordance with the method protocol, the hybridization test showed a clear bluish-violet spot that indicated a positive result. The results of typing were determined according to the HPV scattergram (a clear bluish-violet spot in the hybridization test indicated a positive result for the genotype being tested, while no clear bluish-violet spot indicated a negative result).
By using a sterile cotton swab, the patient’s cervical surface mucus secretion was cleaned, and the cells were collected from the cervical transformation zones of the patient by using a special cervical brush. The collected cells were immediately rinsed in a bottle containing a special cell preservation solution, and thin-layer slide staining was subsequently performed. All TCT slides were diagnosed by professional pathologists by using an optical microscope. TCT results were classified according to TBS classification standards 2001 recommended by “The International Agency for Research on Cancer” as follows: negative for intraepithelial lesion or malignancy (NILM), atypical squamous cells-cannot exclude high-grade squamous intraepithelial lesion (ASC-H), atypical squamous cells of undetermined significance (ASC-US), atypical glandular cells (AGC, and squamous cell carcinoma (SCC). Non-NILM was considered a positive result in TCT classification.
The automatic cellular DNA image analysis system, abbreviated as TAD system (Registration No.: Lu Xie registration 20192220050), is controlled by a fully automated scanning platform; it includes a high-definition digital camera with software to automatically read and quantitatively analyze the exfoliated cell smear stained with Feulgen under a microscope. Based on the DNA index, a single cell with DI = 1 to 2 (i.e., 2C–4C) is considered a normal cell, while those with DI ≥ 2.5 (i.e., 5C) are considered cells with abnormal ploidy. If no abnormal ploidy cells were observed in the cell smear, no visible aneuploid cell peak was observed, or the number of proliferating cells accounted for <5%, the result was considered negative. If 1 to 2 cells with abnormal ploidy were observed or the number of proliferating cells accounted for 5% to 10%, the result was considered suspiciously positive. If 3 or more cells with abnormal ploidy were observed, if a visible aneuploid cell peak appeared, or if the number of proliferating cells accounted for more than 10%, the result was considered positive.
According to the pathological diagnosis results, the patients were assigned to the nonlesion group (NILM, including chronic cervicitis with or without squamous hyperplasia, condyloma-like lesions, papillary erosion, and polypoid hyperplasia) and the lesion group (LSIL and other high-grade lesions).
The inclusion criteria were as follows: patients who consulted a doctor because of vaginal bleeding, abnormal leucorrhea, and other symptoms; and patients who were willing to undergo DNA ploidy analysis, TCT, and HPV-DNA test. The exclusion criteria were as follows: patients with dysfunction of the heart, liver, and kidney or other severe underlying diseases; patients with malignant tumors; and patients with psychological diseases.
Statistical analysis was performed by SPSS 26.0 statistical software. Enumeration data were represented by [case (%)]. The χ 2 test was used for group comparison, the Kruskal–Wallis test was used for ordinal data, and Kappa test was used for consistency analysis; the biopsy result was considered as the golden standard. In the Kappa test, a score of <0.4 indicated low consistency, 0.4 to 0.6 indicated medium consistency, and >0.6 indicated high consistency. The significance level was set at .05 ( P < .05).
3.1. Features of lesions in DNA ploidy analysis and TCT cytodiagnosis 3.2. Status of infection in different grades of cervical lesions (TCT cytodiagnosis) 3.3. Distribution and correlation of DNA ploidy analysis in HPV-DNA (+) patients 3.4. Analysis of different diagnostic methods and biopsy results 3.5. Comparison of the diagnostic value of TCT, HPV test, DNA ploidy analysis, and biopsy The DNA ploidy analysis showed the highest sensitivity and significant differences ( P < .05) in diagnosing LSIL (+) and HSIL (+) patients. Tables and show the results of the combination of TCT and HPV-DNA test, TCT and DNA ploidy analysis, and HPV-DNA test and DNA ploidy analysis. The combination of the HPV-DNA test and DNA ploidy analysis exhibited the highest sensitivity. A significant difference was observed in sensitivity between the TCT + HPV-DNA test and HPV-DNA + DNA ploidy analysis ( P < .05). In contrast, the results of HPV-DNA + DNA ploidy analysis and biopsy showed the best agreement.
First, the features of lesion in DNA ploidy analysis and TCT cytodiagnosis were analyzed. The results (Table ) showed that in the DNA ploidy test, positive, suspected, and negative results accounted for 9.22%, 19.3%, and 71.48%, respectively. Positive results for DNA ploidy were 4.27% in NILM, 41.08% in ASC/AGC, 80.33% in LSIL, 88.89% in HSIL, and 100% in SCC. With the increase in TBS grading, the positive rate of DNA ploidy also gradually increased. A Kappa value of 0.230 ( P < .05) was achieved in the Kappa test. The χ 2 value was 3874.973 ( P < .01).
Next, we analyzed the features of lesions in the HPV-DNA test and TCT cytodiagnosis. The results (Table ) showed that the HPV-DNA infection rates in HPV16/18 type and other types were 4.90% and 14.85%, respectively, and the negative infection rate was 80.25%. For HSIL and SCC, the HPV-DNA infection rate in HPV16/18 type was higher than that for other types. However, for NILM, ASC/AGC, and LSIL, the HPV-DNA infection rate in HPV16/18 type was lower than that for other types. With the increase in TBS grading, the positive rate of HPV16/18 type also gradually increased [Kappa = 0.263, P < .05; Kruskal–Wallis test (χ 2 = 2122.202, P < .01)].
A correlation analysis was performed for the results of the HPV-DNA test and DNA ploidy analysis. As shown in Table , among HPV-DNA (−) patients, the results of the DNA ploidy analysis were negative, suspected positive, and positive for 77.24%, 18.15%, and 4.60% patients, respectively. Similarly, for HPV-DNA (+) patients, the results of DNA ploidy analysis were negative, suspected positive, and positive for 48.08%, 23.91%, and 28.01% patients, respectively (Kappa = 0.173).
A total of 979 patients underwent cervical biopsy examination; of these, 25, 181, 234, and 539 patients were diagnosed to have SCC, HSIL, LSIL, and cervicitis, respectively. Among the 979 patients, 408 had normal TCT results, while 571 had abnormal results. A total of 277 patients had negative results for high-risk HPV detection, while 702 patients showed positive results (including 203 patients with HPV16/18 positivity). The DNA ploidy analysis showed normal results for 235 patients and abnormal results for 744 patients (including 458 patients with DNA ≥ 3). The details are provided in Table .
In China, approximately 131,500 new cases of CC are recorded each year, which accounts for 26.3% of the global cases ; this situation reflects the inadequate screening and detection of CC in China. According to previous studies, only 1-fifth of Chinese women undergo the Pap smear screening test for CC. In recent years, the incidence and mortality of CC have been steadily increasing, and CC is being increasingly detected in young patients. A reduction in the incidence and mortality of CC largely depends on the CC screening test. Following the introduction of CC screening in Europe and America in the mid-20th century, the incidence and mortality of CC have significantly decreased. In China, CC screening started late, and it was not until 2009 that the government organized a CC screening project in rural areas. According to previous research in 2010, the coverage of CC screening in China was only 20%, while the coverage in urban areas did not exceed 30%. This is also 1 of the main reasons for the continuous increase in the incidence of CC in China. For a long time, TCT has been used as the primary screening method for CC in China, as this test is easy to conduct and is easily accepted by patients. TCT has remarkably enhanced the quality of smears and adopts the TBS system (a scientifically rigorous system) for classification. This has led to a decrease in the false negative rate and an increase in the positive rate. This technique, however, has a limitation because the diagnostic criteria are mainly based on the differences in cellular morphology, and the outcome mainly depends on the experience and subjective judgment of the laboratory personnel. DNA ploidy analysis is a computer-based automated method that can offset the disadvantage of TCT. DNA ploidy analysis has been widely used as an auxiliary method for CC screening overseas, particularly in North America and Europe, where it has been used as 1 of the routine clinical examinations. DNA ploidy analysis began to be used in China in the year 2000. This test detects the genetic material in the patient’s cell nucleus and determines the extent of changes in nuclear DNA content that precede morphological changes in cells. During the carcinogenesis process, most cells in the malignant solid tumor are aneuploid cells. If this type of cell is detected, it indicates early malignant lesions. DNA ploidy analysis can detect early precancerous cells and cancer cells. It may play a role in triaging patients to determine whether they require further examination by colposcopy or biopsy. Thus, DNA ploidy analysis can also detect lesions. DNA ploidy analysis is used to evaluate the physiological status and pathological changes in cells by detecting the DNA content or ploidy in the cell nucleus. Some studies have suggested that the accuracy of results can be increased by combining DNA ploidy analysis and TCT for screening CC and precancerous lesions. In our present study, we found that the positive rate of DNA ploidy analysis was 37.14%, and the probability of having abnormal ploidy in patients with positive results for squamous intraepithelial lesion (ASC/AGC, LSIL, HSIL, and SCC) in TCT was higher than that for no abnormal ploidy. With the increase in DNA ploidy, the incidence of squamous intraepithelial lesions also increased; this finding indicates a correlation between the number of cells with abnormal DNA ploidy and the degree of squamous intraepithelial lesion. In a previous study, DNA ploidy analysis was combined with the HPV test, and the results showed that the detection rate of cervical precancerous lesions and CC positivity was consistent with cervical biopsy results. In the present study, DNA ploidy analysis showed a positive correlation with the results of the HPV-DNA test. In patients with positive DNA ploidy results, the proportion of HPV (+) was significantly higher than that for HPV (-); this finding indicates that HPV-DNA plays an important role in auxiliary diagnosis in CC screening. The HPV-DNA and TCT combination test is the primary method for CC screening. In the present study, we analyzed the correlation of HPV infection with TCT. The results showed that among LSIL and other high-grade lesions evaluated by TCT, the number of patients with HPV (+) was more than that for HPV (−), thus, indicating that HPV infection is 1 of the causes of squamous intraepithelial lesions. In patients with HSIL and SCC, the HPV infection rate in HPV16/18 type was higher than that in other types; however, an opposite result was noted for patients with NILM, ASC/AGC, and LSIL. With the increase in TBS grading, the positive rate of HPV16/18 also gradually increased; this finding suggests that HPV16 and 18 types are the key factors that lead to the occurrence of CC. A total of 979 patients underwent cervical biopsy examination, and the results showed that there were 539, 234, 181, and 25 patients with chronic inflammation, LSIL, HSIL, and SCC, respectively. The results of TCT showed that there were 440, 384, 151, 36, and 187 patients with chronic inflammation, ASC/AGC, LSIL, HSIL, and LSIL, and high-grade lesions, respectively. The detection rate of TCT was significantly lower than that of pathological screening. A total of 702 patients were positive in HPV-DNA screening, with a positivity rate of 71.71%. Furthermore, a total of 744 patients were positive in DNA ploidy screening, with a positivity rate of 76.00%. A single screening test has its own advantages and disadvantages. Regarding the screening of high-grade cervical lesions (pathological results showing HSIL (+)), the sensitivity of the results shows as ASC-US and higher-grade lesions, high-risk HPV positive and DNA ploidy suspected is 66.99%, 79.13%, and 81.55%, respectively. T86%, 90.91%, and 97.95%, respectively. For screening high-grade cervical lesions (pathological results show HSIL (+)), the sensitivity of the TCT and HPV-DNA test combination, TCT and DNA ploidy analysis combination, and HPV-DNA test and DNA ploidy analysis combination was 88.35%, 85.92%, and 98.06%, respectively. The positive and negative predictive values of the HPV-DNA test and DNA ploidy analysis combination were relatively high, and the comprehensive detection showed a similar outcome as that for a histopathology examination. In this study, DNA ploidy analysis was employed alongside traditional methods such as the HPV-DNA test and TCT to improve the predictive accuracy for precancerous cervical lesions. We consider patients to be eligible for DNA ploidy analysis in the following situations. Firstly, patients showing positive results for high-risk HPV types, especially HPV16 and HPV18, also tended to have abnormal DNA ploidy outcomes. This correlation suggests that DNA ploidy analysis could be particularly insightful in cases of high-risk HPV positivity, providing a stronger basis for the predictive assessment of cervical lesions. Additionally, abnormal TCT results, especially those classified as ASC-H, HSIL, or greater, indicate another scenario where DNA ploidy analysis adds significant diagnostic value. Lastly, in instances of discrepancy between TCT and HPV-DNA test results, implementing DNA ploidy analysis offers a critical triage tool. It aids in resolving diagnostic uncertainties and directs the clinical pathway towards further investigation through colposcopy or biopsy where necessary.
The findings of this study underscore the significant clinical utility of DNA ploidy analysis as a complementary diagnostic tool in the mass screening for CC. By integrating DNA ploidy analysis with the HPV-DNA test and TCT, we observed a marked improvement in the diagnostic accuracy for detecting precancerous lesions. This was particularly evident in patients who tested positive for high-risk HPV types and those presenting with abnormal TCT results, where DNA ploidy analysis contributed to a more definitive interpretation of the potential for disease progression.
Conceptualization: Jin Li, Qing-Feng Bu, Ming-Li Zuo, Jia Wang. Data curation: Jin Li, Qing-Feng Bu, Ming-Li Zuo, Jia Wang. Formal analysis: Xin-Yi Bi, Zheng-Wu Pan, Shu-Lan Liu, Xiao-Ming Chen. Investigation: Wen-Ping Sun, Yan Zhang, Wei Liu, Fei Wang, Chang-Zhong Li. Methodology: Xin-Yi Bi, Zheng-Wu Pan, Shu-Lan Liu, Xiao-Ming Chen. Resources: Jin Li, Qing-Feng Bu, Ming-Li Zuo, Jia Wang. Software: Wen-Ping Sun, Yan Zhang, Wei Liu. Supervision: Wen-Ping Sun, Yan Zhang, Fei Wang, Chang-Zhong Li. Writing – original draft: Xin-Yi Bi, Zheng-Wu Pan, Shu-Lan Liu, Xiao-Ming Chen. Writing – review & editing: Wei Liu, Fei Wang, Chang-Zhong Li.
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Association of self-reported periodontal disease and inequities with long haul COVID-19 | c64736dc-e46e-485b-9bde-23a990bb18c9 | 11469594 | Dentistry[mh] | In 2000, the Surgeon General’s Report on Oral Health clearly stated that oral health is connected to overall health and well-being . The most prevalent oral diseases are dental caries and periodontal diseases, which are largely preventable. Populations at higher risk for developing medical conditions are the same as populations at higher risk for developing oral diseases . Socioeconomic status refers to the absolute or relative levels of economic resources, power, and prestige closely associated with wealth of an individual, community, or country . Populations of lower socioeconomic status have increased prevalence of comorbid conditions, generally poorer oral health, and more limited access to health care services, all summing up to health inequities . While the COVID-19 pandemic had an impact that could be felt worldwide, populations that most experience oral health inequities disproportionately felt its effects . This is due to many risk factors, a number of which were heightened by the COVID-19 pandemic: stress, alcohol use, tobacco use, poor diet, domestic violence issues, behavioral health problems, and poverty . Among patients hospitalized due to COVID-19, two of the most prevalent comorbidities reported are diabetes and cardiovascular disease . There is enough literature to support the association of these two conditions with periodontal disease . According to the Institute of Medicine and National Research Council, “poor and minority children are substantially less likely to have access to oral health care than their nonpoor and nonminority peers .” These populations are also more likely to lack dental insurance or depend on Medicaid. The Centers for Disease Control and Prevention (CDC) notes that “non-Hispanic Band the same populations have been found to have significantly higher incidence of COVID-19–related infection and death . A meta-analysis by Magesh et al. concluded that members of racial and ethnic minority groups had higher risks of testing positive for COVID-19 and of having a more severe disease course. They also determined that socioeconomic determinants were strongly associated with COVID-19 outcomes in racial and ethnic minority populations . For example, African American and Hispanic individuals were most likely to test positive for COVID-19. Asian American individuals had the highest risk of intensive care unit admission. Decreased access to clinical care was positively associated with COVID-19 positivity in Hispanic and African American individuals . Early in the COVID-19 pandemic, the American Dental Association recommended postponement of elective dental procedures, with the provision of only urgent or emergency care if necessary. In March 2020, 95% of surveyed dental offices were either entirely closed, or closed and seeing emergency patients only. This also resulted in patients’ lack of prioritization of oral health care, and delay in obtaining care . These changes caused an exacerbation of disparities already present amongst communities already at elevated risk . While several systemic issues exist to prevent marginalized communities from receiving equal access to care, reduced emphasis on the importance of dental care during the COVID-19 pandemic resulted in deeper oral health disparities within the population . Additionally, with shrinking of public health insurance benefits like the Medicaid in several states, suspension of preventive dental care programs such as free clinics and school sealant programs in areas with a shortage of dental providers, thousands if not millions of adults and children were left without the care they may otherwise not have access to . In 2020, Northridge et al. reported that “in response to fiscal challenges, many states have reduced or eliminated Medicaid dental coverage over the past decade, with a concurrent 10% decline in oral health care utilization among low-income adults” . Regarding at-risk populations who have dental benefits under Medicaid, they further report that there is often “difficulty finding Medicaid-contracted dental providers, because only 20% of dentists nationwide accept Medicaid .” Literature notes, in respiratory conditions such as COVID-19, potential mechanisms of pathogenesis include aspiration of oral pathogens into the lungs, alteration of respiratory tract mucosal surfaces to favor adhesion of pathogens, and secretion of hydrolytic enzymes from pathogens that inhibit the innate immune response within the respiratory tract . Additionally, several studies have demonstrated a connection between poor oral hygiene and conditions such as pneumonia, or good oral hygiene and reduced incidence of respiratory disease . There is also evidence that the virus may reside and replicate within periodontal pockets . The symbiotic relationships between microorganisms in the oral cavity are disrupted by poor oral hygiene and periodontal disease. Bacteria in a disturbed biofilm further stimulates cytokine release, in addition to those triggered by the condition of periodontitis alone. These cytokines, upon aspiration, may induce infection and inflammation in the lungs. In early stages of infection, throat is a key area of replication of the virus . It has been shown that within the first week of infection, patients infected with SARS-CoV-2 had elevated concentrations of viral RNA in oropharyngeal swabs, indicating active replication in the region . Severity of infection with SARS-CoV-2 appears to be amplified as a result of comorbidities such as diabetes, hypertension, and cardiovascular disease. These comorbid conditions also have a connection with periodontal disease . While a direct causal relationship cannot be established, it is possible that periodontal disease can intensify the severity of a COVID-19 infection through mechanisms such as enhancing inflammatory responses, causing microbial dysbiosis, and immune system overstimulation . A study performed by Larvin et al. investigated a potential impact of periodontal disease on hospital admission and mortality associated with COVID-19 . While the study could not conclusively link periodontal disease with an increased risk of infection, it was found that within the sample population of patients infected with COVID-19, there was significantly higher mortality amongst participants with periodontal disease . The primary objective of this study is to assess the disproportionate impact of the COVID-19 pandemic (long haul COVID) on populations from lower socioeconomic status in the state of Indiana. The study also aims to assess the association of self-reported periodontal disease and COVID-19 disease course and severity. Considering all the above factors, it is hypothesized that the COVID-19 pandemic did in fact heighten oral health disparities.
The Indiana Clinical and Translational Sciences Institute (CTSI) was contacted to identify a sample population with the inclusion criteria of 1) resident of the state of Indiana and 2) positive diagnosis of SARS-CoV-2 in the past year. The sociodemographic distribution (age, gender, race/ethnicity) and information about the social determinants of health for this cohort (income, zip code/neighborhoods and education levels) were also requested. Information about the research study was shared with the cohort, and participants were given a study information sheet detailing its purpose, procedures, risks, and benefits. After reviewing the information sheet, participants were asked to indicate their consent electronically before proceeding to the survey. This method ensured that consent was documented and stored securely within the REDCap platform. This study was IRB exempt. The IRB number associated with the research is #15239. Upon completion of this portion of the project, a questionnaire was sent to the cohort via the CTSI inquiring whether they are experiencing any symptoms of “long haul COVID.” The survey tool was developed from validated questionnaire available from literature . Questions relevant to long haul COVID included inquiring about symptoms such as cough, muscle pain, and loss of taste/smell after the resolution of the infection. The survey tool also inquired about the course of COVID-19 disease progression (mild/moderate/severe ‐ requiring hospitalization), symptoms of gum disease, oral hygiene habits, and presence of co-morbid chronic conditions (such as diabetes and cardiovascular issues). Example questions are, “ How do you feel about your sense of taste compared with the status prior to COVID-19 ?” to determine if a participant is still experiencing symptoms after a positive COVID diagnosis, and “ Have you visited an oral healthcare center (dental clinic) for the following in the past 12 months for treatment for gum disease such as scaling and deep cleaning ?” to determine if the participant has a history of periodontal disease and received any treatment for it previously. The survey can be found in , and was administered via REDCap, a secure virtual platform. Once completed surveys were returned, the data was consolidated and analyzed to best align it with the research question. Associations of patient characteristics and self-reported periodontal-related survey items with COVID-related survey items were evaluated using chi-square tests, considering the ordinal response categories when appropriate. Spearman correlation coefficients also were calculated when both sets of response categories were ordinal. A two-sided 5% significance level was used for all tests. Analyses were performed using SAS version 9.4 (SAS Institute, Inc., Cary, NC, USA).
The results of the study underscore the intricate link between periodontal disease, socioeconomic factors, and long-term effects of COVID-19. The percentages represent the proportion of respondents within each category who reported a specific outcome, while ’n’ values denote the actual number of individuals within those percentages. When reporting multiple categories, the proportion was calculated out of our total sample (209) respondents. The comprehensive analysis included a cohort of 209 individuals from Indiana, who in the past year, tested positive for COVID-19. The demographic data, from “ ”, reveal a predominant representation of females (n = 154; 74%), while male respondents accounted for a smaller percentage (n = 55; 26%). The age distribution showcased a relatively balanced spread across the different age brackets, with a marginally higher prevalence in the 30–39 years (n = 48; 23%) and 50–64 years (n = 52; 25%) age groups. In terms of racial demographics, most participants were Whites (n = 18; 87%), followed by other racial categories comprising remaining 13% of the participant population. The educational background of the participants was notably high, with most of the participants (n = 122; 59%) holding a college or a postgraduate degree (n = 73; 35%). Employment status predominantly included employed for wages (n = 135; 65%) individuals. The income distribution indicated that a significant portion of the cohort, (n = 93; 44%), had an annual income of $75,000 or more. COVID vaccine uptake among the surveyed individuals shows that the majority (n = 131; 63%), reported having received the initial vaccine series plus one booster shot. “ ” reveals a significant correlation between past COVID-19 hospitalization and oral health practices; Past COVID-19 hospitalization was associated with more frequent dental floss use (p = 0.049), more frequent rinsing mouth (p = 0.041), and treatment of gum diseases within the past 12 months (p<0.001). It must be noted, our sample only had 11 participants (5%) who had reported hospitalization due to COVID-19. Among participants who reported a history of hospitalization, a higher percentage used dental floss once or more in a day (n = 9; 4%). Similarly, most participants with past hospitalization rinsed their mouth once or more in a day (n = 10; 5%). And lastly, many participants who reported hospitalization, visited a clinic in the past 12 months for the treatment of gum disease (n = 7; 22%). There was no statistically significant association between self-reported periodontal disease factors (detected loss of bone around teeth, permanent teeth lost due to gum disease, bleeding gums and previous diagnosis of gum disease), socio-demographic factors, or oral hygiene variables, with past COVID-19 hospitalization. “ ” shows the association of sense of smell with socio-demographic and oral factors. Reports of a worsened sense of smell were associated with lower education levels (p = 0.121). A greater proportion of individuals with Grade 12 or GED level of education reported worsened sense of smell (n = 6, 43%) than individuals with higher levels of education such as post-graduate (n = 19, 26%). In addition, lowered sense of smell was significantly more common amongst participants who were unemployed or disabled (p = 0.008). The survey results indicated that a greater percentage of unemployed individuals (n = 12; 75%) had a worsened sense of smell post COVID-19 infection compared to their employed counterparts. Furthermore, there was a significant association observed between the participants having a worse general health status (p<0.001), bleeding gums (p = 0.031), history of toothaches (p = 0.000) and a lower oral health rating (p = 0.002). Among the respondents, a significant number of those who reported a total loss of or sense of smell worse than before (n = 18; 9%), also reported having fair to poor general health status. Among the same group, a significant number also reported having bleeding gums (n = 33, 16%) and a history of toothaches (n = 38, 18%). A majority of participants with a self-assessed oral health rating of ’poor,’ (n = 4; 67%) reported a worsened sense of smell post-COVID when compared with those who had an oral health rating of ‘very good’ (n = 16; 27%) or ‘excellent’ (n = 8; 24%). Lastly, a significant association was also noted between COVID-19 vaccination status and sense of smell (p = 0.011). A greater percentage of participants reported a worsened sense of smell if they had not received the vaccine (n = 7, 64%). A significantly lower percentage of participants reported worsened sense of smell if they had received a booster (n = 41, 31% and n = 7, 24%). “ ” shows association between sense of taste post COVID-19 and socio-demographic and oral factors. Lower sense of taste ratings were associated with older age (p = 0.018). Participants within the age group of 40–49 years reported a worsened sense of taste at a higher frequency than others (n = 19; 46%); followed by the >65 years age group (n = 12; 39%). The least affected group was those under the age of thirty (n = 7; 19%). Lower sense of taste was also associated with worse overall health status (p<0.001), mobile or loose teeth (p = 0.010), tooth loss (p = 0.013), bleeding gums (p<0.001), possibility of gum disease (p<0.001), permanent teeth lost due to gum disease (p = 0.006), toothache (p<0.001), and lower oral health ratings (p = 0.001). Among the respondents, a significant number of those who reported a total loss of or sense of tastemobile or loose teeth (n = 12, 6%) a history of tooth loss (n = 11, 5%), bleeding gums (n = 35, 17%), possibility of gum disease (n = 26, 12%), permanent tooth loss due to gum disease (n = 10, 5%), and a history of toothaches (n = 34, 16%). Mosttaste post-COVID when compared with those who had an oral health rating of ‘very good’ (n = 14; 23%) or ‘excellent’ (n = 10; 29%). And lastly, a significant association was noted between COVID-19 vaccine and sense of taste (p = 0.001). A greater percentage of participants reported a worsened sense of taste post COVID-19 if they had not received the vaccine (n = 8, 73%). A significantly lower percentage of participants reported worsened sense of taste if they had received the vaccine and booster(s) (n = 35, 27% and n = 9, 31%).
The study’s primary focus was to investigate the impact of COVID-19 on lower socioeconomic populations and its association with self-reported periodontal disease. It has been demonstrated that the COVID-19 pandemic and all of the changes that came with it–dental offices closing temporarily, free health care programs being suspended, and patient lack of pursuit of dental treatment for a variety of reasons–largely stood in the way of patients obtaining dental care necessary for their well-being. As a result, the oral health status of marginalized populations deteriorated. Literature also notes the association between income loss during the COVID-19 pandemic and unmet dental care for children, emphasizing the financial barriers to dental care during the pandemic . Oral health is connected to overall health, and a worsening condition of the oral cavity, potentially combined with comorbid conditions, can impact the course of an individual’s COVID-19 infection, potentially increasing the severity and increasing the probability for symptoms remaining long term. While some results were statistically significant, with only eleven hospitalizations (5%) observed in the sample, some frequencies reported were higher in opposing categories. It is important to note that while these statistically significant associations between variables provide valuable insights, they may not necessarily indicate direct causal relationships due to the limitations of a small sample size and data scope. Therefore, it is also important to emphasize that the study’s results are not entirely generalizable. When considering demographic factors, age was the sole variable that displayed a significant association with changes in gustatory function following a COVID-19 infection. This is consistent with a recent study by Perlis et al. which found that long haul COVID was associated with older age and female sex . Per our study results, for changes in olfactory function, education and employment were the only socio-demographic factors that showed statistical significance in relation to changes in sense of smell compared to pre-COVID. Individuals with lower levels of education seemed to report a worsened sense of smell, along with individuals that are unemployed as opposed to those who are not. These results align with our study’s overarching theme of addressing disparities in oral health, supporting the notion that socioeconomic factors impacted access to dental care during the COVID-19 pandemic. This may be contrasting with a cross-sectional study by Mahmoodi et al. found that individuals with higher education levels and underlying comorbid conditions were at greater risk for having symptoms of long haul COVID . Despite this contrast between the two studies, one portion of Mahmoodi et al.’s findings does align with results found in our data; individuals with lower general health status were more likely to report having symptoms of long haul COVID such as worsened sense of smell or taste. Periodontal disease factors such as bleeding gums, previous diagnosis of gum disease, and permanent teeth lost due to gum disease exhibited significant association with a worsened sense of taste post COVID-19. As for worsened sense of smell post COVID-19, only bleeding gums of all the self-reported gum disease symptoms was found to be associated. Similar to our results, a case control study performed by Sari et al. concluded that poor periodontal health and higher incidence of periodontitis was observed in populations that was recently infected by COVID-19 . While our study cannot attribute a direct association between self-reported symptoms of periodontal disease and history of hospitalization, a case control study by Marouf et al. suggests periodontal disease was associated with COVID-19 complications including admission to the hospital, need for assisted ventilation, and even death . This is due to a possible mechanism of increased local and systemic inflammatory responses . Lastly, a study by Costa et al. determined that there was a positive association between oral health conditions such as periodontitis and severe COVID-19 outcomes such as hospitalization . This study focused on immediate consequences within a clinical setting, while our study uniquely explores post-COVID-19 sensory changes in a broader demographic context . This comparison highlights the complementary nature of the two studies, offering a more comprehensive understanding of the diverse impacts of COVID-19 on oral health outcomes. Interestingly, despite the expected potential benefits of oral hygiene practices, our study revealed that oral hygiene practices such as brushing teeth, using dental floss, and mouth rinsing were not significantly associated with reports of altered sense of taste or smell. In contrast to this, a study by Catton et al. indicates a link between flossing and rapid taste recovery, suggesting that oral hygiene practices may actually reduce COVID-19 viral entry and dissemination . As mentioned previouslyOur results in this category may not indicate direct causal relationships as a result of the limitations of a small sample size and data scope. With regards to vaccination status, individuals who had not been vaccinated for COVID-19 generally reported worsened levels of both sense of smell and taste. Additionally, those who had not received a COVID-19 booster also reported worsened levels of sense of smell and taste. A recent study by Perlis et al. found that the risk of long-term COVID was reduced in populations that received the primary vaccination series . This complements results from other studies–vaccination generally results in a less severe disease course. And a less severe disease course is associated with a lower incidence of long haul COVID symptoms, such as worsened smell or taste. Research suggests that vaccination may alleviate certain long-COVID symptoms. This potential improvement could be due to an enhanced immune response triggered by the vaccine. Conversely, per a recent study, those who received at least one dose of a COVID-19 vaccine were more likely to report prolonged long-COVID symptoms more than a year after infection . A study by Strain et al., noted that the Moderna mRNA-1273 vaccine showed the most significant improvement in long COVID symptoms (66% improvement vs. 12% deterioration), followed by Pfizer-BioNTech BNT162b2 (56% improvement vs. 18% deterioration) and Oxford-AstraZeneca ChAdOx1 nCoV-19 (58% improvement vs. 19% deterioration) . In contrast, some studies indicated that the overall impact of vaccine type on symptom changes was minimal, with no significant differences reported between the types of vaccines . However, many studies have had small sample sizes, lacked diverse representation, and didn’t account for pre-pandemic symptoms or include a never-infected comparison group, which could show similar nonspecific symptoms . Despite these mixed findings, vaccines are known to significantly reduce the severity of COVID-19, including the risk of hospitalization and death . The observed association between vaccination and prolonged symptoms does not diminish these protective benefits but underscores the need for further research. Specifically, future studies should explore how different vaccines might influence long-COVID persistence and impact long-term oral health outcomes. Understanding these interactions, especially in relation to pre-existing conditions like periodontal disease, could provide valuable insights into post-COVID recovery. Strengths and weaknesses Future implications Future research should aim at expanding on these findings with a larger, more diverse cohort and multivariable analysis to dissect the complex relationships between sociodemographic variables, oral health and COVID-19 outcomes. Additionally, further investigation into the role of oral health practices and their potential impact on the severity of COVID-19 symptoms could provide valuable insights for public health interventions. Identifying these disparities is crucial for informing policy changes aimed at improving access to oral health care for marginalized groups.
Our study provides valuable insights into the progression and long-term effects of COVID-19, especially in relation to oral health. This approach is complemented by our attention to diverse demographic and socioeconomic factors, enriching our understanding of the pandemic’s impact across different population groups. These findings shed light on the complex interplay between oral health, socio-demographic factors, and COVID-19 outcomes, highlighting the relevance of oral health in understanding the impact of the pandemic and inform future public health strategies and research directions. Conversely, there are several limitations present in our study. The study data was cross sectional, which does not confer causality. The reliance on self-reported data for periodontal disease diagnosis is a limitation, as it lacks the clinical validation that a comprehensive periodontal examination, including radiographs and clinical assessments, would provide. In addition, self-reported data is often masked by recall bias which again is a big limitation. The small sample size, particularly in subgroups such as those hospitalized due to COVID-19, limits the generalizability of our results. This is evident in the inverse frequency of certain outcomes, such as more hospitalizations among participants who followed oral hygiene practices more religiously, which may be attributed to the small sample size and may not accurately reflect a larger trend. The survey did not collect specific data on the types of COVID-19 vaccines received by participants, which may limit the understanding of the impact of different vaccine types on long-term COVID-19 symptoms. Additionally, because our study population was geographically limited to Indiana, our results may not be generalizable.
This study reveals complex links between periodontal disease, socioeconomic factors, and long-term COVID-19 impacts. While associations were identified, limitations such as a small sample size and geographical focus exist. The findings underscore the importance of oral health in the context of the pandemic, suggesting potential connections with COVID-19 outcomes. However, the study also highlights the need for more comprehensive research, emphasizing the intricate nature of these relationships. Addressing oral health disparities, particularly among marginalized groups, is crucial for informed policy changes and improved health outcomes in future public health strategies.
S1 Appendix Long COVID-19 oral health survey. (PDF) S1 File (XLSX)
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A 40-Year-Old With Prior Stem Cell Transplant for Chronic Myeloid Leukemia Presents With Dyspnea and Respiratory Failure | 5c463d25-a482-424f-bd78-d5399fe647ca | 11867892 | Surgical Procedures, Operative[mh] | On arrival at the ICU, his temperature was 36.3 °C, heart rate 82 beats/min, blood pressure 109/65 mm Hg, respiratory rate 19 breaths/min, and pulse oxygen saturation 98% on 8 L nasal cannula. The patient was somnolent but awoke easily to voice and followed simple commands. He was oriented to person and place. Pupils were equal and reactive to light. There was no jugular venous distension. The cardiovascular examination was notable for no murmurs, normal rate and rhythm, and normal pulses. Diffuse wheezes and crackles were present bilaterally. He had 1-2+ pitting edema of the lower extremities. He had a mild erythematous papular rash on the bilateral lower extremities below the knee.
WBC count was 13.6 × 10 9 /L (normal, 3.8-10.6), hemoglobin was 8.5 g/dL (normal, 12.9-16.9), platelets were 22 × 10 9 /L (normal, 156-369), absolute neutrophil count was 16.7 × 10 9 /L (normal, 2.24-7.68), blast forms were 4%, creatinine 1.3 mg/dL, brain natriuretic peptide 16 pg/mL. Other electrolyte levels were normal. Previous Hospitalization Studies Current Hospitalization Studies A respiratory viral panel from nasal swab did not detect respiratory viruses. Legionella urinary antigen was negative. Blood cultures remained no growth at day +5. Sputum culture obtained on admission was pending. Cryptococcal antigen was negative. Urine culture showed no growth. Methicillin-resistant Staphylococcus aureus nasal screen was negative. Chest radiography demonstrated increasing indistinct opacity in lungs, more diffuse on the left but somewhat confined to the inferomedial right ( A). A chest CT showed increasing ground-glass opacities and interstitial thickening, right middle lobe atelectasis, and ill-defined reticulonodular pattern in the bilateral lower lobes ( B). Repeat transthoracic echocardiogram showed normal structure and function of the right and left ventricle, with an estimated pulmonary arterial systolic pressure of 32 mm Hg. A bedside right heart catheterization showed a right atrial pressure of 7 mm Hg, pulmonary arterial pressures of 30/15 mm Hg, pulmonary capillary wedge pressure of 6 mm Hg, and cardiac output of 10.1 L/min by thermal technique. Lung biopsy with video-assisted thoracoscopic surgery was initially attempted, but the patient was unable to tolerate single-lung ventilation. The procedure was converted to open thoracotomy, with successful biopsy without complication. The biopsy showed intra-alveolar exudates ( A) with periodic acid-Schiff (PAS)-positive globules ( B). Infectious pathogen stains from the biopsy specimen were negative. What is the diagnosis? Diagnosis: Pulmonary alveolar proteinosis secondary to hematologic malignancy
BAL performed 6 weeks before admission showed no bacterial, fungal, nocardia, or acid-fast bacilli pathogen growth. Herpes simplex virus polymerase chain reaction was negative. Galactomannan was 0.08 (normal, < 0.5) from blood and 0.14 (normal, < 0.5) from alveolar lavage. Blood BD-glucan was 211 pg/mL (normal, < 60). Blood Epstein-Barr virus and cytomegalovirus polymerase chain reaction were negative.
In patients with hematologic malignancies and allogeneic or haploidentical hematopoietic cell transplant, a differential diagnosis for acute hypoxemic respiratory failure can be summarized in broad categories depending on whether the complication occurs within the first 100 days of transplantation, including (1) infectious (bacterial, viral, or fungal, depending on presence of neutropenia); (2) noninfectious, noninflammatory (pulmonary edema, alveolar hemorrhage from thrombocytopenia, renal dysfunction, direct involvement from hematologic malignancy); and (3) noninfectious, inflammatory (graft-vs-host disease, drug pneumonitis, radiation pneumonitis, idiopathic pneumonia syndrome, alveolar proteinosis, veno-occlusive disease). Pulmonary alveolar proteinosis (PAP) is a rare disease, defined by protein (surfactant) accumulation within alveoli impairing gas exchange. Prevalence is between 3.7 to 40 cases per million, and incidence is 0.2 cases per million. Although the most common cause of this rare disorder is an autoimmunity to granulocyte-macrophage colony-stimulating factor (GM-CSF), which accounts for 90% of all cases, PAP also may be attributable to secondary causes, such as infectious pathogens (eg, Pneumocystis ), hematological malignancies, occupational exposures, and lung transplantation. In neonates, congenital defects in surfactant protein also can cause PAP. This discussion focuses on secondary PAP caused by hematologic malignancy. Secondary PAP accounts for less than 10% of cases and is caused by a reduction in the number or function of alveolar macrophages, not because of GM-CSF antibodies seen in the autoimmune PAP. The resulting buildup of surfactant in alveoli therefore causes the typical symptoms of cough and dyspnea. Secondary PAP can be associated with hematopoietic cell transplantation, medications such as sirolimus and ruxolitinib, or opportunistic pathogens. Considering hematologic disorders, myelodysplastic syndrome and chronic myeloid leukemia are often associated with PAP. The mechanism of PAP development is poorly understood in hematologic malignancy but may have to do with monocytopenia or GATA2 deficiency. GATA2, a hematopoietic differentiation transcription factor, has been implicated in rare cases of PAP. GATA2 deficiency can be seen associated with hematologic malignancies such as chronic myeloid leukemia, as well as viral or fungal infections. Whether there are specific treatments to alter the course of PAP in this population is unknown. The diagnosis of secondary PAP is determined with radiographic findings, bronchoalveolar lavage cytology, or lung biopsy findings. Lactate dehydrogenase may be elevated but is nonspecific. Chest radiography findings include bilateral alveolar opacities, usually in a perihilar and basilar distribution. The “crazy paving” pattern is observable on CT scan and is attributable to a combination of intralobular thickening and diffuse ground-glass opacities. Only 33% of cases have this described pattern. Bronchoalveolar lavage or lung biopsy is the confirmatory diagnostic test(s) of choice. Cytology will reveal large foamy macrophages and PAS staining. In the absence of crazy paving, PAP can be misdiagnosed, and it also can have overlap with infectious causes, particularly in the setting of immunosuppression. Treatment for secondary PAP is reliant on the underlying condition. This patient had already had a bone marrow transplant in the setting of his leukemia, and this was a recurrence. Whole-lung lavage can be used as supportive treatment in addition to GM-CSF. Apart from symptomatic and supportive respiratory care and therapy directed toward the underlying hematologic malignancy, no specific therapy has been identified for the treatment of PAP associated with hematologic malignancy. Clinical Course Vancomycin and diuretics were administered initially in addition to his prior antimicrobials, including posaconazole, amphotericin, and acyclovir. Following the diagnosis of PAP, GM-CSF was administered pending GM-CSF antibody testing, which was not detected. The patient subsequently underwent whole lung lavage for 3 sequential days. Cloudy fluid, characteristic of PAP, was obtained during the lavage, as depicted in . The patient’s oxygenation briefly improved, but unfortunately, his respiratory failure persisted. In the context of his multiple comorbidities, multi-organ dysfunction, and refractory hematologic malignancy, he and his family transitioned to comfort measures only. The patient died days later while hospitalized.
1. The differential diagnosis for acute hypoxemic respiratory failure in hematopoietic cell transplant patients is vast, although it can be broadly categorized as infectious (viral, fungal, bacterial), inflammatory (drug pneumonitis, idiopathic pneumonia syndrome, graft-vs-host disease), and noninfectious, noninflammatory (pulmonary edema, renal dysfunction, congestive heart failure). 2. Autoimmunity to GM-CSF accounts for 90% of PAP cases in adults, although secondary cases occur from infection, hematologic malignancy, post-solid organ and hematopoietic cell transplantation, or occupational exposures. 3. Bronchoscopy and BAL plus or minus biopsy are the mainstay of diagnosing PAP, often with proteinaceous milky return and PAS-positive material on cytology or biopsy. 4. Treatment often includes GM-CSF administration if the cause is autoimmune, whereas whole lung lavage is supportive to improve oxygenation while pursuing treatment, depending on the cause. 5. GATA2 deficiency has been associated with PAP, although treatment remains unchanged for this rare entity.
None declared.
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Transformation of dental services from a governmental model to a revenue-generation model of operation in a tertiary care hospital: a health economics assessment | f2a4b2fb-77ce-4389-b9d9-6e16bfdd9501 | 9706714 | Dental[mh] | Healthcare in Saudi Arabia is undergoing a major transformation. King Faisal Specialist Hospital and Research Centre-Riyadh (KFSHRC-R) will transform its model of operation from a governmental hospital that delivers free service to its patients to a model where the service will be paid either by insurance or a governmental fund. It is hoped that this model of care will improve efficiency and productivity. The Department of Dentistry will consequently bill for its services and it is imperative to assess one's readiness for such a transformation in terms of efficiency, timely delivery of care and sustainable revenue streams. There is no precedent for this model in Saudi Arabia and KFSHRC-R is the first hospital to undergo such a transformation in the model of operation. The aim of this study was to identify how much the Department of Dentistry would generate in terms of revenue under such a model. Included in objectives are whether there might be any losses and to identify which specialties would incur such losses and the reasons behind such losses.
Anonymized data with regard to dental practice at KFSHRC-R were extracted from Cerner Millennium by Cerner, R4 Clinical+ v 4.7.0 by Carestream, Venus Billing System and Revenue Cycle Management System by Data Ocean for the period from January 2015 to December 2019. Visits to the emergency department were excluded from the analysis. The department employed on average 41 dentists as full-time employees and 8.6 hygienists on average as full-time employees. Each dental full-time employee is paid an average of 700 000 SAR (186 667 USD) per year. All visits for dentists and hygienists were included in the analysis. The demographics of the sample collected in terms of gender and age were analyzed. A descriptive analysis was carried out of the type of outpatient appointments and the category of current procedure terminology (CPT) code. Each CPT code had a billed rate as per the KFSHRC-R billing system. The billing rate was compared to a benchmark cost without profit that was provided through previous consulting work handled through a billing consulting company. If a CPT code was being billed at a rate less than the cost rate, then it was considered a CPT code that was incurring a negative margin of loss. If it was billed at a rate equal or higher than the cost rate then it was a CPT code that was incurring a positive margin of profit. Moreover, the billing rate was compared to an average calculated from three market leaders providing comparable services to that provided by KFSHRC-R to show if the billing rate was higher or lower than the prevailing market price for that CPT code. Data was collected from 835 individual data entries from 660 different CPT codes. Of these, 474 entries from 352 CPT codes had no volume in the year and thus were excluded from the analysis. In total 361 individual entries from 292 CPT codes were included for analysis. Data was collected detailing revenue for different types of dental work for a one-year period. The aim of the analysis was to detail the revenue for each type of work, and to categorize the amount of revenue in work with different profit levels. Revenue for each type of dental work was collected, with each type of work categorized into CPT codes. Most CPT codes consisted of one type of work only, but some codes had more than one individual type of work associated with them. For the purposes of analysis, one observation per CPT code was included in the analysis. Where there was more than one set of values per CPT code, the total revenue for the code as a whole was calculated. Where the margin and the difference with the benchmark price varied within a CPT code, a single value per CPT was calculated by weighting the results for each type of work by the revenue of the work. All analyses were descriptive in nature using the Dental Health Record C4, Kodak Dental Systems version 3.1.8 and analysis with IBM SPSS Statistics 29. Categorical variables were summarized by the number and percentage in each category. Continuous variables were summarized by the mean and standard deviation or median and data range. The main outcomes of interest were total revenue, and the percentage of all revenue for each type of work, or groups of work. Each type of work was categorized in three main ways: 1) based on whether the price of work was higher or lower than the ‘benchmark’ price, 2) whether there was a positive margin (profit) or a negative margin (loss), and 3) the speciality of the work. Summaries of revenue in each of these categories, or combinations of categories were quantified.
During the period of the study from 2015 to 2019, 11 214 (50.8%) and 10 840 (49.2%) female and male were treated, respectively ( ). They paid 179 554 (52.9%) and 159 858 (47.1%) outpatients visits, respectively, with an average 16 visits per female patient and 14.7 visits per male patient. Saudis constituted 89.4% of the patients and 93.7% of the visits. The number of patients treated and number of patients visits increased from 7294 and 51 505 to 9722 and 80 319 with an average annual increase of 486 (6.7%) and 5763 (11.2%), respectively, with a median number of visits per patient of 5 in 2015 to 6 in 2019, which equates to care delivered by fewer visits per patient since the number of patients increased from 2015 to 2019 ( ). Patients younger than 14 years of age and seniors older than 65 years comprised only 17.3% and 8.5% of all the visits and 18.6% and 8.95% of all the patients treated during the period 2015-2019, respectively. ( ). Distribution of visits showed that dental nursing clinic and hygienist clinic appointments constituted 23.5% and 7.2% of the total visits for the whole period of 2015-2019, respectively. During the same period, general dentistry, pedodontics, orthodontics, endodontics, oral and maxillofacial, prosthodontics, periodontics made up 28.1%, 12.5%, 7.1%, 7.0%, 6.9%, 4.4% and 3.3%, respectively, of the total number of visits ( ). On average each dentist and hygienist saw 2263 and 760 visits per year. The most common charge description master (CDM) codes are shown in for each year. Most were for x-rays or oral hygiene instructions. The revenue for all dental specialties for the period of the study was 51 922 240 Saudi Arabian Riyals (SAR) (13 864 597 USD) ( ). The revenue for the nursing and hygienist clinics was 7 167 530 SAR (1 911 341 USD). Thus, the total revenue for all the appointments was 59 089 770 SAR (15 757 272 USD). shows the revenue per year of study. Approximately 131 (44.9%) CPT codes incurred a negative margin of loss when compared to the benchmark cost of the CPT code while 161 (55.1%) incurred a positive margin compared to the benchmark cost of the CPT code. Eighty CPT codes (27.4%) incurred a positive profit margin and were higher in price than the average of the market leaders. Eighty-one (27.7%) CPT codes incurred a positive profit margin, but were lower in price than the average of the market leaders. On the other hand, 83 (28.4%) CPT codes incurred a negative margin of loss, but were higher in price than the average of the market leaders. Forty-eight (16.4%) CPT codes incurred a negative margin of loss and at the same time were lower in price than the average of the market leaders. shows the distribution of the CPT codes that incurred a negative margin of loss whether higher or lower than the average price of the market leaders among the different specialties. The estimated loss of revenue due to pricing that was lower than the actual cost was estimated at 29 922 900 SAR (7 979 440 USD).
There is a debate worldwide about who should cover dental care: the patient alone, the government alone, or by the patient with subsidization from the government. - This debate is ongoing in Saudi Arabia, which currently has a national health service covered by the government. Other countries have already started to reform. This research attempted to quantify the cost of care and the expected revenue along with suggestions to improve efficiency of dental services in an academic tertiary care hospital. During a period of 5 years, each patient paid 11 visits excluding hygienists and nursing appointments. However, the number increased from a median of 5 visits in 2015 to 6 visits in 2019 ( ). The study could not be extended to encompass the period 2020-2021 due to the reduction in delivery of dental services because of the COVID-19 pandemic. During that period, only emergency and necessary care was provided). The high number of visits probably reflects the nature of patients, many of whom were being treated for tertiary complex conditions like cancer and organ transplantation that required complex medical consultations prior to any dental intervention. But, more importantly, the increasing number of visits (27.7% in number of visits per patient per year), would have also meant a delay in execution of care, which would reduce patient satisfaction in a private model of operation. The average number of patient visits per year per dentist in the US was reported at 3566.4 in 2018 and has remained stable over the period 2015-2018 in a national study of private dental clinics. The average benchmark in the US fluctuates between 1600 and 2300 patient visits per year per dentist in contracted salary-based practice. The average number of outpatient visits per year per dentist at KFSHRC-R was 1256 in 2015 and reached 1959 in 2019 which is comparable to the benchmarks for a salary-based practice. This indicates that dentists at KFSHRC-R are performing on par with their colleagues in the US, but are underperforming by 45% in comparison to those performing in a feefor-service practice. The benchmark from the US for the hygienist should be 7.5-8.5 patients per an 8-hour day. This amounts to 1600 visits per year. Hygienists at KFSHRC-R see only 530 per year which means they are underperforming by 67%. As expected, nursing clinic and general dentistry clinic visits comprised 51.6% of the visits. The initial encounter for any patient is usually through these clinics, where any need for more specialized dental care is determined. Pedodontic visits comprised only 17.3% of all visits, which is comparable to a national study from Canada were pedodontic visits reached 18% of all visits. Total revenue for all the clinic appointments was 59 089 770 SAR (15 757 272 USD). There was an average increase in revenue of more than 10% each year, but it dropped to only around 2% in 2019. That drop coincided with a plateau in the number of visits in the same year at 80 319. This indicated a limited capacity to expand in terms of appointments and may be related to issues pertaining to infrastructure like the lack of more dental chairs and probably longer turnaround times between patients. The average salary for a dentist in the department was 700 000 SAR (186 667 USD) per year. The sum of salaries for the same period 2015-2019 was 143 500 000 SAR (38 266 667 USD). The revenue generated was only 36.2% of the salaries paid (51 922 240 SAR; 13 845 931 USD), which means that the dentists were underperforming in relation to salaries paid. Productivity should be increased to reach a sustainable budget in the long run. It was not possible to identify the risks for low productivity in a statistical analysis, but a trend was noticed in the data: the sections with the lowest productivity had a higher portion of dentists who worked in private practice, were within 5 years of the retirement age of 60 years, have been in practice more than 20 years, were administrative leaders or past leaders or were not North American board certified. This indicated a few things: local board training probably led to less experienced dentists who tended to have difficulty in delivering care in a timely fashion. Dentists near retirement or who had been in practice for 20 years or more were probably less motivated to engage in higher output due to their high salaries. Those working in private practice were probably accruing more income than their salaries, and were thus less inclined to increase productivity. Individuals in administrative positions spent more time than required on administrative duties. A solution to the low productivity could be a movement to a fee-for-service model, for dentists in practice for 20 years or more or near retirement. A downside of a fee-for-service model is the possibility of providing unwarranted care and overbilling, a practice that should be monitored and discouraged. The pricing of the CPT codes showed that 131 codes (44.9%) were incurring a negative margin of loss compared to the calculated cost of the CPT code. In fact out of these 48 CPT codes (16.4%) were priced even lower than the average of the market leaders. This indicated two things. First, the CPT codes at KFSHRC-R must be re-priced as the current prices will create a huge loss for the hospital when it moves into a private sector type of billing for services. Second, 83 CPT codes (28.4%) were incurring a negative margin of loss, but were higher than the average price of the market leaders, indicating a huge gap for improvement in terms of cost efficiency with regard to these CPT codes. The hospital must revisit the practice of the dentists to see if there was wastage in terms of materials used in the treatment plan or overutilization of laboratory tests and x-rays. Incorrect use of CPT codes does not explain the negative margin of loss as this analysis compared the registered billing price of the CPT code and the actual cost of that code and the average price of the market leaders regardless of what was actually delivered to the patient. Therefore, additional training about CPT codes would probably not resolve this problem but may still be useful. The three specialties that really needed to revise the prices of their CPT codes were general dentistry, endodontics and orthodontics. The two specialties that needed to look into their practice and improve efficiency and show more cost containment were prosthodontics and oral and maxillofacial surgery. This cost-containment can be approached through a reduction in total number of visits to deliver the care and also by a more judicial use of supplies. The loss of revenue due to the use of a billing price lower than the actual cost was estimated at 13 315 970 SAR (3 550 925 USD), which amounted to an annual loss of revenue of 10.1%. A market concentrated with dental providers will likely lead to less reimbursement in the insurance market as evidenced from a national study from US. Saudi Arabia is in a similar situation and this will compound the loss for revenue for KFSHRC-R. In conclusion, the analysis identified some delay in delivery of treatment, which might be expected due to treating complex tertiary conditions. Moreover, there was limited capacity to expand and meet the demand as shown by the plateau in the number of appointments. The productivity was low as might be expected, from a salaried model of practice, which could be improved by switching to a fee-for-service model. There was underproductivity among the hygienists. Furthermore, certain specialties must revisit their practice and improve cost-containment through a reduction in number of visits and judicial use of supplies. Last, the CDM price list needs revision to cover the cost of the services delivered.
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Using strain-resolved analysis to identify contamination in metagenomics data | 9a5fb524-2cb4-434c-824b-e8716b950338 | 9979413 | Forensic Medicine[mh] | The advancement of sequencing technologies has enabled researchers to investigate microbial communities at high resolution and throughput. However, contamination poses challenges for data analysis. Contamination refers to DNA within a sample that did not originate from that specific biological sample. Recognizing contamination, followed by appropriate decontamination, should be a critical first step for all microbiome analyses. Skipping this step can easily result in confounded results and false conclusions. Contamination can originate outside of a study. Microbial DNA from the surrounding environment, native microbiomes of researchers, and microbial DNA present in DNA extraction and library preparation kits are all considered external contaminants [ – ]. Detection of such contamination is enabled by the addition of negative controls (i.e., blank reagent controls) during sample collection, preparation, and/or sequencing. To date, strategies have been devised to minimize, detect, and/or remove externally derived contamination in silico [ , – ]. Cross-sample contamination is less well studied . Contaminants that originate within a study can be introduced during DNA extraction when DNA from one sample spills over into another. It can also occur during sequencing as a result of index switching or sample bleeding . Since the contaminating DNA in this case originates from microorganisms present in samples from the study, one cannot decontaminate by removing “contaminant species” present in controls from the actual dataset. While strategies have been developed for solving index switching and sample bleeding arising during sequencing , much less is known about the well-to-well contamination occurring prior to sequencing in metagenomics data and how to detect it. In recent work that characterized early-life intestinal strain colonization, we collected a large clinical dataset consisting of over 400 fecal samples from infants and their mothers . Unfortunately, we detected clear evidence of cross-sample contamination that required us to exclude one-half of the samples from the study. This motivated an in-depth investigation of the signals used to detect contamination in this and a second clinical dataset of preterm infants. Our approach relied upon a strain-resolved workflow. Strain-level analyses have been increasingly applied in microbiome studies, for example, to study mother-infant gut microbiome transmission [ – ] and conduct epidemiological surveillance . The high specificity and sensitivity of strain tracking methods are powerful, yet conclusions from these methods can also be confounded by cross-sample contamination . Here, we provide a framework for the detection of cross-sample contamination using strain-based surveillance, and in two case studies, we show how such a framework can be used to detect contamination in large-scale metagenomics datasets.
Case study 1: longitudinal preterm and full-term infant fecal samples Case study 2: longitudinal preterm infant fecal, mouth, and skin samples Study overview Conclusions from case study 2 Study overview Conclusions from case study 1 The first case study consists of 402 fecal samples collected from 19 preterm and 23 full-term infants from birth to age 1 and their mothers around time of birth . This study was designed to investigate strain persistence within infants, strain sharing between infants and their mothers, and strain sharing among different infants. DNA extraction was primarily achieved using 96-well plates (“ ”), and there were a total of five extraction plates, labeled P1 to P5. There were five reagent-only-negative controls, one for each DNA extraction plate, and they were labeled by the plate number (i.e., NC1 refers to the negative control on P1). One ZymoBIOMICS Microbial Community Standard (catalog no. D6300; termed “Zymo”) was included as a DNA extraction-positive control on P5. Following DNA extraction, all samples including controls were subjected to metagenomics sequencing and de novo genome reconstruction. Dereplication of the genomes constructed from fecal samples resulted in 1005 representative genomes, as previously described . Reads from all samples were then mapped to this dereplicated genome set for organism detection (“ ”). To detect potential sources of contamination, we examined strain sharing among unrelated samples within and across extraction plates (“ ”). Samples are considered unrelated if they are from different infants that are not biologically related or if one sample is a negative control. Evaluation of extraction negative and positive controls Evaluation of cross-sample contamination: index hoping and sample bleeding Evaluation of cross-sample contamination: well-to-well contamination Evaluation of underlying biological signals after removing contaminants The identification of well-to-well contaminated samples allowed us to assess strain sharing among supposedly uncontaminated infant samples on originally discarded P3 and P4. On P3, samples from six preterm infants shared a total of four strains. These four are strains of Clostridioides difficile , Clostridium paraputrificum , Clostridium butyricum , and Lactobacillus rhamnosus (Fig. ). Samples that shared these four strains were not often adjacent to one another. Furthermore, for each of these four organisms, near-identical strains were also found among samples that were extracted on different plates. Notably, C. difficile , C. paraputrificum , and C. butyricum strains were shared among preterm infants only. The pattern of strain sharing on P4 was similar to that on P3, except that there were more shared strains that were shared by fewer samples (11 strains were shared among samples from four infant pairs). As for P3, we detected no strong signal for contamination via dispersal of strains into all or most surrounding wells from single-source wells. Given minimal evidence of well-to-well contamination and since all infants in this study were born in the same hospital, we speculate that most of the strains shared by the infants whose samples were extracted on P3 and P4 were probably hospital derived, a phenomenon that has been reported previously .
No organism was detected in negative controls NC1 and NC5. However, negative controls NC2, NC3, and NC4 had at least one read mapped to ≥ 50% of at least one of the 1005 representative genomes (this value served as our threshold for detection; Fig. ). No contaminants were detected in the Zymo-positive control. The only genome detected in NC2 was Cutibacterium acnes . This organism was also found in nine fecal samples from five infants. These samples were extracted on P1, P2, and P4. Strain-to-strain comparisons indicate that all strains are sample-specific and different from that in NC2. Therefore, the presence of C. acnes in NC2 was not considered to be due to cross-sample contamination. As C. acnes is a common skin commensal bacterium , and is often detected in laboratory reagents and kits , we suspect this organism is an externally derived contaminant. NC3 and NC4 each had ~60 genomes that were above our read-mapping detection threshold. The organisms represented by these genomes could be externally derived and/or originated within the study via index switching, sample bleeding, and/or well-to-well DNA contamination. If any of these genomes were derived from external sources (e.g., from DNA extraction kits), we would expect the strains to be in the majority of samples from the same plate (because they were processed simultaneously) and perhaps across extraction plates. However, no strain was shared among negative controls. Furthermore, the strains found in NC3 and NC4 were only shared by a maximum of 7.7% and 6.5% of the unrelated samples from PC3 and PC4, respectively. Thus, we conclude that the genomes in NC3 and NC4 were unlikely due to external contamination and likely originated from other samples from this study.
The majority of strains found in NC3 and NC4 were only shared with samples from the same extraction plate. This observation rules out index switching and sample bleeding, as these phenomena should lead to contamination of samples from other plates because DNA from different plates was pooled for sequencing (P1 and P2 were pooled and sequenced at a different time than samples from P3-P5). Both index switching and sample bleeding refer to the misassignment of reads to samples. However, index switching results from indices being similar in multiplexing sequencing and can be essentially prevented by using unique dual indexes, which was what we used for sequencing in this study. Sample bleeding, on the other hand, occurs due to the close proximity of sample read clusters on the flow cell during sequencing . We confirmed that this is not the main explanation for the contamination in NC3 and NC4 by resequencing these two controls and finding that the community compositions were essentially the same as the originally sequenced NC3 and NC4.
To test for the remaining possible explanation for the contamination in NC3 and NC4, well-to-well contamination, we visualized strain sharing patterns in the context of the extraction plates. Based on the observation made by Minich et al. that neighboring wells are more prone to well-to-well contamination than distant wells , we hypothesized that, if between-sample contamination occurred within an extraction plate, nearby unrelated sample pairs would more likely to share strains than those that were farther apart. Distance-correlated strain sharing was not seen on P1, P2, and P5 ( p = 0.18, 0.31, and 0.32, respectively; Wilcoxon rank-sum test). This finding is consistent with the plate-based strain sharing visualization, which shows that unrelated samples on P1, P2, and P5 rarely shared any strains, and for pairs that did share strains, the majority of them were not immediately adjacent to each other (Fig. ). On P3 and P4, however, nearby samples were significantly more likely to share strains than those that were farther apart ( p = 2.3e-3 and 4.7e-3, respectively; Wilcoxon rank-sum test). Plate-based strain sharing visualization indicates that a few samples including NC3 and NC4 in particular (pink circles in Fig. ; see also Fig. S ) exhibited location-specific sharing patterns, consistent with well-to-well contamination. For instance, on P3, NC3 located on column L primarily shared strains with samples from infants #82 and #83 that were loaded onto columns K and L for extraction. This suggests that the fecal samples from infants #82 and #83 were potential sources of the contamination seen in NC3. NC3 also shared strains with a sample from infant #83 that was loaded onto column C for extraction. As expected, samples from infant #83 share strains, so strains in NC3 could have come from any of the samples from infant #83. Likely, the contaminant strains were derived from the samples adjacent to the negative control. Thus, we do not attribute this instance of sharing to long distance well-to-well contamination. NC4 on P4 exhibited a similar proximity-based strain sharing pattern to NC3 (Figs. and S ), and we deduce that NC4 was primarily cross-contaminated by adjacent samples. In addition to NC3 and NC4, four preterm infant samples displayed plate location-specific strain sharing patterns (unlabeled four pink circles in Fig. ). The first instance involved a sample from infant #98 (termed #98D4). #98D4 was extracted on P4, and it shared strains with nearly half of the samples extracted from the same plate, including those on columns J and K that were nine and ten columns away (Fig. ). Shared strains were from six infants, including a pair of twins (#122 and #123). Strains shared by each of the twins and #98D4 overlapped completely, and these strains were not found in any other infants. The other four of these six infants also shared strains that were otherwise unique to them with #98D4. #98D4 did not share strains with samples extracted from different plates. We therefore deduce that #98D4 was contaminated by multiple wells on P4. Besides #98D4, three other samples, each derived from a different infant (#63, #114 and #128), primarily shared strains with neighboring samples, similar to the patterns seen in NC3 and NC4. We confirmed that these four infant samples were indeed cross-contaminated by re-extracting and sequencing three of the four samples (the original stool sample from #128 and its close-by-date replacement were unavailable). For the sample from infant #63, a close-by-date sample (day of life 6 instead of 9) was selected as the original stool sample was unavailable. For all three re-extracted samples, their DNA concentrations became ~0 (Table S ) and their location-based strain sharing patterns were not observed.
Using strain-resolved workflow, we identified well-to-well contamination to be the major source of contamination in this dataset. The six contaminated samples (two negative controls and four preterm infant samples) were all low in microbial biomass. If such contamination was not addressed, we would have falsely concluded that strain sharing among non-related infants was not rare, and that some non-related infant pairs could share as many strains as sibling pairs do.
We applied our strain-resolved workflow to a different clinical dataset consisting of 533 samples collected from the skin, mouth, and stool of 16 preterm infants. These preterm infants were born in the same hospital as the infants from case study 1. This dataset was part of a study designed to elucidate strain transmissions between the hospital environment and preterm infants. DNA extraction was primarily achieved using 96-well plates. One reagent-only-negative control was included in each extraction plate. There were six plates (labeled P1 to P6) and six negative controls, labeled NC1 to NC6. In addition, P4, P5, and P6 each had ~3 Zymo standards as DNA extraction-positive controls. Two-hundred thirty-six of the 533 samples (including 3 negative controls (NC1, NC3, and NC5) and 3 positive controls, one from P5 and two from P6; termed PC5, PC6_1, and PC6_2) were selected for metagenomics sequencing (colored circles except for those light blue ones in plate maps in Figs. , , 7). Before library preparation, DNA was transferred from the extraction plates to three new 96-well plates, one for each sample type (“ ”). Following sequencing, de novo genome reconstruction and dereplication yielded 152 representative genomes, which served as the reference genomes for read-mapping based organism detection for this dataset (“ ”). To detect potential sources of contamination, we examined strain sharing among all unrelated samples, as described in case study 1. Evaluation of extraction negative and positive controls Evaluation of Zymo contamination in infant samples Evaluation of additional contamination not present in negative controls Using the strain-resolved approach developed in case study 1, we evaluated strain sharing among infants after excluding Zymo and Burkholderia strains. Five bacterial strains were widely shared by samples from different infants. Specifically, for each of these five strains, at least half of the infants had one sample that shared such a strain with a sample from an unrelated infant. Of these five strains, two are Staphylococcus epidermidis strains A and B, and the other three are Staphylococcus M0480, Corynebacterium aurimucosum , and Cutibacterium acnes . All five are common members of the healthy skin microbiome . S. epidermidis strain A and the Staphylococcus M0480 strain were shared among all sample types (skin, mouth and stool), and S. epidermidis strain B, the Corynebacterium aurimucosum strain, and the C. acnes strain were shared among skin samples only. Additionally, a near-identical S. epidermidis strain A was found in fecal samples from 16 out of 42 infants from case study 1. It is uncommon to find a single strain of each of these organisms in the majority of infants of a single dataset; we therefore identify these five strains to be externally derived contaminants (e.g., from staff who handled the samples). We re-examined strain sharing among samples of this dataset after excluding all identified external contaminants (Zymo strains, a Burkholderia strain, and five skin-associated strains). While most of the extraction plates did not exhibit location-based strain sharing, samples from one infant pair on P4 did, suggesting that there might be well-to-well contamination (pink circle in Fig. S ). A sample from infant #12 shared up to 3 strains with neighboring skin and oral samples of infant #13. These 3 shared strains were not shared by infant #13 and any other infants. In addition, none of the other samples from infant #12 shared strains with infant #13. This suspected cross-contaminated infant #12 sample was collected from skin, and its strain sharing pattern was similar to those of well-to-well contaminated samples in case study 1.
Of the three sequenced negative controls, one genome was detected in NC1 and NC3, and 9 genomes were detected in NC5 (Fig. ). No contaminants were detected in the Zymo-positive controls. Burkholderia sp. was the only organism detected in NC1 and NC3. The Burkholderia strain in NC1 was not detected in any sequenced samples. However, a different Burkholderia strain was found in NC3 and 12 infant skin samples that were extracted from four extraction plates (Fig. ). No Burkholderia was found in fecal or mouth samples, both of which were higher in biomass than skin samples ( p = 5.38e-37 and 1.58e-40, respectively; Wilcoxon rank-sum test). The skin samples that contained the Burkholderia strain had a significantly lower biomass than the skin samples that did not ( p = 2.79e-24; Wilcoxon rank-sum test). Burkholderia is not part of the normal skin microbiome , but it has often been reported as a reagent contaminant [ , , , ]. Identifying one single Burkholderia strain among infant and negative samples suggests this strain is a result of external contamination. Interestingly, this strain is also in one low-biomass gut sample from case study 1. This contaminant was likely introduced prior to library preparation as it was not detected in NC1 and NC5, both of which were on the same library preparation plate as those Burkholderia -containing skin samples (Fig. C). For NC5, one organism, Klebsiella pneumoniae , was present at an extremely low abundance (< 0.1%) and was not detected in any other samples. We cannot determine if adjacent samples on the extraction plate were possible sources of this organism because those samples were not sequenced. The remaining 8 organisms in NC5 were all bacterial members of the Zymo community (Fig. ). NC5 was adjacent to PC5, a Zymo-positive control, on the extraction plate. Thus, Zymo organisms in NC5 could be attributed to well-to-well contamination.
To further evaluate contamination by the Zymo strains, we searched for these strains in infant-derived samples. During DNA extraction, six skin and oral samples were deliberately spiked with 75 μL of the Zymo standard, four of which were sequenced (“ ”). By examining strains shared between positive controls and biological samples, we found 12 additional infant samples containing at least the four most abundant Zymo members (Fig. ). All but one of these samples were from skin or mouth, which had lower biomass than gut samples ( p = 1.58e-40 and 5.57e-9, respectively; Wilcoxon rank-sum test). Of these 12 infant samples, 9 were adjacent to a Zymo-spiked infant sample or a positive control. Since the contaminated samples generally only shared Zymo strains with neighboring Zymo-spiked samples (and not the other organisms in those samples), we conclude that the observed Zymo contaminants were more likely to be introduced accidentally, possibly via aerosolization or mis-pipetting, rather than via well-to-well contamination.
ur strain-resolved workflow identified external contamination to be the major source of contamination in this dataset. Suspected contaminants include Burkholderia strains, Zymo DNA, and five skin-associated strains. Our approach also suggested one skin sample to be cross-contaminated by adjacent samples from the same extraction plate. Notably, most of these contaminants were found in skin samples, which had lower biomass than oral and gut samples.
Contamination is an insidious and potentially unavoidable problem in metagenomics-based microbiome research. If not appropriately addressed, extraneous DNA sequences can skew conclusions, resulting in potentially false statements. Here, we devised a workflow that utilizes strain resolution to detect contamination based on the unexpected sharing of essentially identical strains. We demonstrate its usefulness in two clinical metagenomics datasets. By examining strain sharing based on genotype distribution across samples, and considering sample proximity on DNA extraction plates, our work is the first to show how to identify and differentiate, using non-synthetic microbiome data, contamination that derived from external sources and that which originated within the sample set. In a recent review, it was noted that negative controls have been included in ~30% of prior microbiome studies, although only a subset of studies sequenced and analyzed data from the negative controls . Although negative controls can identify foreign DNA that does not belong to the study, they only offer a limited view of contamination and do not constrain the contamination source or the number of samples impacted. In our first case study, analysis of the pattern of shared strains in the context of sample location on DNA extraction plates indicated that the DNA in negative controls was mostly derived from neighboring biological samples. Detection of contamination motivated a more complete analysis of well-to-well contamination in all biological samples in our study. In four cases, genotypes found in many samples from one infant were shared by only one of the samples from another infant. In each such case, the contaminated sample was located adjacent to the putative contamination source on the DNA extraction plate. This conclusion was verified, as the strains were no longer shared when the DNA was re-extracted and resequenced. In our second case study, two externally derived contaminants in the negative controls were identified: a Burkholderia strain and DNA from the Zymo-positive control. We thus investigated strains that were apparently shared by samples from unrelated infants and identified DNA from five additional contaminants in samples from the majority of infants yet absent in the negative controls. It may be possible to find true biological signals if the signal from contamination can be removed. In case study 1, after removing the well-to-well contaminated samples, we identified three Clostridia strains that were shared only among preterm infant samples. Since preterm infants in our study spent their first 2–3 months in the hospital, they have a higher chance to acquire hospital-associated strains than full-term infants. Given that strains of these bacteria have been found previously in preterm infants that were born in the same hospital and in neonatal intensive care unit room microbiomes , we hypothesize that these three strains may be circulating among infants in the hospital. In cases where samples from the same subject or sample set are loaded onto the same DNA extraction plate, the possibility that random and independent well-to-well transfer events could introduce strains that are not recognizable as contaminants should be considered. In our case study 1, although only ~3.3% of samples on P3 and P4 were identified to be contaminated by well-to-well transfer, these two plates had a much higher number of within-plate strain sharing than the other three plates. This raises the possibility of undetected well-to-well transfer events that may have artificially inflated strain sharing among samples. Our study provides a detailed workflow for contamination identification. Based on our observations and previous contamination-related studies, we list several suggestions for minimizing contamination in metagenomics-based studies. First, we encourage others to minimize plate-based extraction if possible. If plate-based extraction is a must, one could consider taking additional precautions such as limiting the number of open wells when extracting, using individual caps for covering wells on the plate, and fully spinning down the samples before removing the caps to prevent well-to-well contamination from occurring. In addition, one should consider including the DNA extraction plate maps in their published work. Second, we urge others to randomize samples when extracting DNA. Specifically, one should avoid loading samples from the same individual or experimental group or biologically related individuals adjacent to one another for extraction. Third, sequencing of sampling negative controls (i.e., empty swab that is used during sample collection) is recommended in addition to extraction negative controls. This should identify contamination introduced during sampling, which is important because such contaminants will likely not display extraction-plate-based sharing patterns. Removal of strains seemingly shared but introduced during sampling will clarify strains truly shared among samples.
Contamination may be unavoidable in high-throughput sequencing, and our results suggest that it can be particularly problematic for low-biomass samples. Genotype-level surveillance has the advantage that it does not require additional expenditures related to library construction and sequencing. Our work emphasizes the importance of routinely assessing contamination prior to data analysis so as to avoid incorrect findings. Harder to detect than external contamination, well-to-well contamination can be a concern of the microbiome field. Random mixtures of samples from the same extraction plate can result in distorted community diversity metrics and inflated strain sharing rate. This is particularly alarming for microbiome-based clinical studies since well-to-well contamination can inflate differences between the treatment and the control groups. As microbiome-based analysis and diagnosis are becoming more popular, we conclude that the use of genotype-specific surveillance methods as well as negative controls are recommended to ensure the integrity and reproducibility of the results.
Sample collection DNA extraction Metagenomic sequencing Metagenomic assembly, de novo binning, and taxonomy assignment Genome dereplication Detection of subspecies and identification of strains using inStrain Statistical analysis Infant fecal samples from case study 1 were collected either at UPMC Magee-Womens Hospital by trained nurses or at home by parents provided with detailed collection instructions. For sample collection and storage details, see Lou et al. . For case study 2, all infant samples (skin, oral, and stool samples) were collected at UPMC Magee-Womens Hospital by trained nurses. Skin and oral samples were obtained by the charge nurse using a BD BBL CultureSwab EZ under supervision of study personnel. Skin and oral samples were collected in duplicate at each timepoint for each preterm infant in order to increase the biomass available for DNA extraction. For details of sample collection and storage, see Olm et al. .
DNA was extracted using either the Qiagen QIAamp PowerFecal Pro DNA Isolation Kit (single-tube extractions; used for 14 of 402 samples in case studies 1 and 7 of 533 samples in case study 2) or Qiagen DNeasy PowerSoil HTP 96 DNA Isolation Kit (96-well plate extractions; used in the majority of samples in both case studies) with modifications to the manufacturer’s protocol. To minimize cross-plate contamination, no plates were extracted at the same time. For each 96-well DNA extraction plate, a reagent-only negative control was included. ZymoBIOMICS Microbial Community Standard (catalog no. D6300) was included as a positive control on one extraction plate from case study 1 and three extraction plates from case study 2. When loading samples into the wells of DNA extraction plates, samples were not randomly distributed among the plates, and often, samples from the same infant were present next to each other sequentially along columns on the same extraction plate. For DNA extracted from stool using the single-tube format, the manufacturer’s protocol was followed except for a heating step at 65 °C for 10 min before the homogenization step. For DNA extracted from stool with the 96-well kit, fecal samples were added to individual wells of the bead plate and stored overnight at −80 °C. The following day, the Bead Solution and Solution C1 were added, and the plates were incubated at 65 °C for 10 min. The plates were shaken on a Retsch Oscillating Mill MM400 with 96-well plate adaptors for 10 min at speed 20. The plates were rotated 180° and shaken again for 10 min at speed 20. All remaining steps followed the manufacturer’s centrifugation protocol. All skin and oral samples from case study 2 were extracted using 96-well plates. Specifically, for skin and oral swab samples, the swab head was cut off directly into the5 min at speed 20. The plates were rotated 180° and shaken again for 5 min at speed 20. The Solution C2 and C3 steps were combined (200 μl of each added) to improve DNA yield. All remaining steps followed the manufacturer’s centrifugation protocol. For six selected skin and oral samples, 75 μl of ZymoBIOMICS Microbial Community Standard (catalog no. D6300) was added to the wells of these six samples prior to the heating step during DNA extraction. Extracted DNA was quantitated using the Quant-iT High-Sensitivity dsDNA Assay Kit (Thermo Fisher Scientific) in 96-well plates, and measurements were made on a SpectraMax M2 microplate reader. DNA yields were used as proxies for sample biomasses. All extracted samples from case study 1 were sequenced, whereas only about half of the extracted samples from case study 2 were sequenced. DNA extractions and quantifications were performed at the University of Pittsburgh School of Medicine, Pittsburgh, PA, USA. Once completed, the extracted DNA was shipped to the QB3 Vincent J. Coates Genomics Sequencing Laboratory at UC Berkeley for library preparation and sequencing. For case study 1, the extracted DNA was sent in the same plates in which the DNA was eluted in the final step of the DNA extraction protocol. For case study 2, the DNA from selected samples was transferred from the extraction plates to three new 96-well plates (one for skin samples, one for oral samples, and one for stool samples) before shipping to Berkeley. For each pair of the duplicated skin and oral samples, their extracted DNA was combined into a single volume on the new 96-well plates.
Samples from case studies 1 and 2 had separate library preparation and sequencing runs. However, the overall sequencing workflow is essentially identical. Metagenomic sequencing of all samples was performed in collaboration with the California Institute for Quantitative Biosciences at UC Berkeley (QB3-Berkeley). Library preparation on all samples from each study was performed as previously described . Final sequence ready libraries were visualized and quantified on the Advanced Analytical Fragment Analyzer. All libraries were then evenly pooled into a single pool and sequenced on individual Illumina NovaSeq 6000 150 paired-end sequencing lanes with 2% PhiX v3 spike-in controls. Post-sequencing bcl files were converted to demultiplexed fastq files per the original sample count with Illumina’s bcl2fastq v2.20 software.
Sequencing reads from case studies 1 and 2 were assembled separately. However, the overall workflow of metagenomics assembly, de novo binning, and taxonomy assignment was essentially identical. See Lou et al. for details on read assembly, de novo binning, and taxonomy assignment on resulting genome bins .
To generate a set of study-specific, high-quality, and nonredundant reference genomes, all de novo assembled genome bins were dereplicated at 98% whole-genome average nucleotide identity (gANI) via dRep (v2.6.2) , using a minimum completeness of 75%, maximum contamination of 10%, the ANImf algorithm, 98% secondary clustering threshold, and 25% minimum coverage overlap. Since biological samples from case study 2 were deliberately spiked with Zymo standard (catalog no. D6300), 10 publicly available Zymo genomes ( https://s3.amazonaws.com/zymo-files/BioPool/ZymoBIOMICS.STD.refseq.v2.zip ) were added to the original genome set of case study 2 before dereplication. Genomes with gANI ≥ 98% were classified as the same “subspecies,” and the genome with the highest score (as determined by dRep) was chosen as the representative genome from each subspecies.
Reads from each individual sample were mapped to study-specific representative subspecies (generated via dRep as described above) concatenated together using Bowtie2 under default settings. inStrain (v1.3.4) profile was run on all resulting mapping files using a minimum mapQ score of 0 and insert size of 160. Genomes with ≥ 0.5 breadth (meaning at least half of the nucleotides of the genome are covered by ≥ 1 read) in samples were considered to be present. inStrain compare was used under default settings to compare read mappings to the same genome in different pairs of samples. In case study 1, samples were considered to share the same strain of the examined genome if the compared region of the genome from samples shared ≥ 99.999% population-level ANI ( popANI ), whereas in case study 2, the popANI threshold was set to be 99.995%. Only genomic areas with at least 5× coverage in samples were compared, and sample pairs with less than 50% of comparable regions of the genome were often excluded (≥ 0.5 percent_genome_compared ). For edge cases, such as when popANI values were within 0.005% of the threshold or when percent_genome_compared values were within 0.2% of the threshold, inStrain compare results were manually assessed to determine whether the sample pairs shared the same or different strains.
Statistical significance for two-group univariate comparisons was calculated using Wilcoxon rank-sum test (as implemented using the SciPy module “scipy.stats.ranksums”) as reported in the main text. For instance, to assess whether strain sharing was correlated with sample pair distance on each DNA extraction plate, we compared within-plate Euclidean distances of sample pairs that did not share strains to those that shared at least one strain using Wilcoxon rank-sum test.
Additional file 1: Figure S1. Details of strain sharing on P3 and P4 from case study I. Rectangular areas represent plates (P3 and P4) and circles show sample placements within each plate. Infant samples are named by infant number and infant day of life (i.e., #63D9 refers to infant #63 and this sample was collected when the infant was 9-day-old). If it is a maternal sample, such a sample is named by the infant number with a letter “M” in the end (i.e., #40M refers to the maternal fecal sample collected from infant #40). A line was drawn between unrelated samples if they shared ≥1 strain. The more strains a sample pair shared, the thicker and brighter the line. If a sample did not share any strains with other unrelated samples, its corresponding circle is colorless. Pink circles represent samples that were likely cross-contaminated. Additional file 2: Figure S2. Detection of one cross-contaminated sample on P4 from case study II. Merged circles represent duplicated samples that were extracted adjacent to each other and were merged before being transferred to the library preparation plates. Infant samples are named by infant number, infant day of life and sample type (“M” refers to mouth samples, “S” refers to skin samples, and “G” refers to gut samples). If a sample pair from unrelated infants shared ≥1 strain, the corresponding samples circles were colored gray and a line was drawn between them. The more strains a sample pair shared, the thicker and brighter the line. Additional file 3: Table S1*: The re-extracted stool sample (#63D6) is not the same as the original one (#63D9) as the original stool sample is unavailable. This alternative stool sample was collected 3 days earlier than the original sample. The original and the re-extracted DNA concentrations of four cross-contaminated samples from case study I.
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Development and Implementation of a Family Presence Facilitator Curriculum for Interprofessional Use in Pediatric Medical Resuscitations | d00399a1-3f92-4005-90e9-957766cccce8 | 11458738 | Pediatrics[mh] | By the end of this training, learners will be able to: 1. Prelearning/didactics: Articulate the purpose and benefits of the Family Presence Facilitator (FPF) role. 2. Prelearning/didactics: List the four components of the FPF role and key behaviors for each. 3. If participating in simulated participant training: Demonstrate the key personality types in a role-play scenario. 4. If participating in a simulated component: Demonstrate behaviors of the FPF Simulation Assessment Tool (FPF-SAT; i.e., respect and value, information-sharing, nonverbal communication, summary and follow-up) in a simulated resuscitation. Pediatric medical resuscitations are low-frequency, high-stakes events that require the coordination of many factors, including emergent medical decision-making, technical skills, interprofessional teamwork, and communication with patients and families. Family presence during pediatric resuscitations is increasingly the standard of care, and benefits include allowing family members (including parents, caregivers, and/or loved ones) to comfort and advocate for patients, decreasing emotional trauma, and easing families’ grief in cases of patient death. – Both the ACGME and the Joint Commission on Accreditation of Healthcare Organizations cite communication with patients and families as a core competency, , supporting the premise that this is also a prescribed and normative need in health professions education. Moreover, there is substantial evidence that family presence during resuscitations does not interfere with, but rather benefits, patient care. – Although families wish to be offered a supported presence, there are significant differences in how family presence during pediatric resuscitations is perceived by health care providers, with acceptance ranging from 35% to 85%, without significant differences between physicians and nurses. Despite data showing that health care providers are more comfortable facilitating family presence during invasive procedures and resuscitations after implementing specific practice guidelines, guidance and evidence are limited on how best to teach or evaluate team members providing emotional support to patients and families. Facilitating family presence during resuscitations requires training, refinement, and thoughtful evaluation of our practices. For optimal impact, training in complex skills like patient and family communication during critical events should include both didactic and experiential components and, when possible, allow for practice in the team's resuscitation space. Interprofessional, in situ simulation provides an ideal setting in which to develop and teach such skills, as it enables learners to practice with authentic teams, resources, culture, leadership, and implementation climates. Structured debriefing, such as that described in the Promoting Excellence and Reflective Learning in Simulation (PEARLS) debriefing framework, allows for discussion of crucial learning objectives from a variety of fields. Indeed, the simulation literature suggests that practicing simulation in a so-called blended model—in which the pursuit of medical and technical learning objectives is accompanied by a focus on interprofessional teamwork, situational awareness, and patient- and family-centered communication—heightens emotional arousal and psychological fidelity, thereby enhancing learner engagement and retention of targeted knowledge and skills. , However, creating such curricula in a manner that promotes psychological fidelity and experiential learning is resource intensive. Therefore, it is critical to create curricular materials that can be adapted and implemented across a variety of educational settings. In this Educational Summary Report (ESR), we describe the development, implementation, and evaluation of a curriculum to teach and promote the role of Family Presence Facilitator (FPF) during pediatric resuscitations and provide resources for clinician-educators to implement this curriculum for interprofessional learners. This curriculum was developed based on generalized and targeted needs assessments, including (a) a comprehensive literature review; (b) an appraisal of communication domains from existing patient communication tools , – ; and (c) focus groups with families and youth who have had the experience of being patients and/or family members in our health care system. We developed and used a modified Delphi process to refine a novel FPF Simulation Assessment Tool (FPF-SAT) in parallel to curriculum development. The FPF curriculum was revised in an iterative fashion, applying feedback from learners and FPF-SAT development. Our curriculum was designed with a broad and interprofessional audience in mind, including physicians (attendings, fellows, residents), medical students, nurses, advanced practice providers (APPs), child life specialists, chaplains, and social workers. The overall goal of the FPF curriculum is to empower all individuals on the health care team to provide psychosocial support to patients and families, and to improve humanistic patient- and family-centered communication during pediatric medical resuscitations. DevelopmentImplementationDebriefingAssessmentData Collection and AnalysisCurriculum designEquipment/environmentPersonnelWe used Kern's six-step approach, consisting of problem identification, general and targeted needs assessments, formulation of goals and objectives, creation of educational strategies, implementation of the curriculum, and evaluation and feedback from learners, to develop our curriculum. A literature review of existing curricula within medicine and allied professions demonstrated a paucity of structured curricula for the FPF role. Our local needs assessment identified existing resources, including a brief guide for communicating with patients and families during resuscitations, previously developed by members of our team. Additionally, our institution benefits from support and partnership with The Sala Institute for Child and Family Centered Care (i.e., “The Sala Institute”), a dedicated institute whose mission is to design, promote, and fund vital services in child and family support, resilience, safety, quality, and family partnership at NYU Langone Health/Hassenfeld Children's Hospital. The targeted needs assessment included key stakeholder meetings with health care providers and focus groups with The Sala Institute's family and youth advisory councils to identify behavioral domains essential to the FPF role. Family advisory council members consisted of parents of critically ill children and youth advisory council members composed of teenage patients who had experienced acute illness. We conducted two focus groups comprising six members each; because of the COVID-19 pandemic, focus groups were conducted using a virtual platform. We queried participants regarding key domains adapted from The Sala Institute's Patient and Family Faculty Program's Patient and Family Centered Competency Scorecard, which is a checklist previously developed to assess health care professionals in key patient- and family-centered communication skills. Domains comprised in this scorecard include role clarity (e.g., how patients and family members would like to be addressed and introduced), information-sharing practices, choice and autonomy (e.g., values and preferences, including deescalation techniques), culturally sensitive communication, nonverbal communication, and communication with conscious patients. These results were applied to determine curricular goals, objectives, and methodology. Our focus group discussions were not originally designed to address communication and management of patient death. While creating our curriculum, we noticed that most models of delivering bad news and communicating patient death assume that families are not present at the time of patient death. As such, based on iterative learner feedback, we modified our curriculum to provide recommendations in cases of patient death in the family's presence, recognizing that guidelines for detailed summary communication between physicians and families are beyond the scope of this curriculum. Our final curriculum is multimodal, with teaching materials of various lengths and formats that can be adapted to the learning needs, resources, time constraints, and preferences across various educational settings. Specifically, we created one set of materials designed to be delivered in didactic/workshop format, including (a) a 30-minute PowerPoint didactic to be delivered by an instructor , along with a prerecorded version of the PowerPoint for self-directed learning and (b) a 90-minute workshop that pairs the didactic with an interactive session based on a role-play demonstration performed by instructors and learners in the group (scripts in and ; worksheets in and ). All learners were asked to complete a posttraining survey after partaking in the workshop. No prerequisite knowledge was required to complete the curriculum. Additionally, we created materials that facilitated experiential teaching and learning of the FPF role using interprofessional, in situ simulation as a curricular framework. We encouraged learners to experience the PowerPoint (as a didactic, workshop, or self-directed learning) prior to participating in simulation-based practice and learning the FPF skills. Because simulated participants (SPs) were crucial for the simulation-based portion of our curriculum, we also developed a 90-minute SP curriculum to train SPs to portray family members during simulated pediatric resuscitations. A PowerPoint and simulation scenario for SP training were created in collaboration with the New York Simulation Center for the Health Sciences (NYSIM). The SP training introduced the FPF curriculum and described three core personality types (agitated, anxious, and quiet) to be portrayed by SPs. We also briefed SPs regarding their emotional starting points for the cases (on a scale of 1–10) and included sample conditional statements to adjust behavioral intensity based on the performance of the FPF. SP didactic instruction was supplemented with SP role-play and feedback, and SPs were trained to complete our novel assessment tool to rate learners’ performance in these domains (FPF-SAT; ). Training sessions were led by authors Ellen Duncan and Selin Sagalowsky in conjunction with NYSIM's SP Program Educator. Our SPs were recruited with help from the SP Program Educator as well, but SPs participating in this training do not need any prerequisite knowledge prior to the training. Both the FPF curriculum and the SP training curriculum began with a content advisory and provided resources regarding trauma-informed care. We recognize the sensitive nature of the curricula and endeavored to create an environment of psychological safety for all participants. The 30-minute, standalone didactic and SP training required only PowerPoint capabilities and audiovisual (AV) equipment (i.e., a computer and a projector). The 90-minute expanded workshop required PowerPoint and AV capabilities, along with simulation equipment (if desired) to carry out the two role-play demonstrations performed by the educator(s). These demonstrations were designed to be low fidelity and adaptable to different settings based on available resources; basic needs included a table, a low-fidelity infant manikin (though even a simple infant-sized doll can be used with mimicking of procedures) and, if available, airway equipment (including an infant bag-valve-mask, endotracheal tube, and laryngoscope blade and handle), IV equipment (with sharps removed for safety), two 3–5cc syringes to simulate medications, such as antibiotics, and a small blanket to use as a shoulder roll. Our implementation of the didactic curriculum was accompanied by a longitudinal, in situ simulation curriculum for a subset of learners. However, we recognize that this learning modality may not be available at all institutions and have thus developed a multimodal curriculum that does not rely on this simulation-based education component. For institutions that do have the resources for recurring in situ simulations, we recommend that either an SP or member of the team portray the family member role. Because the focus of this report is to discuss the FPF curriculum and how it may be generalized across practice settings, the specific needs for the in situ curriculum will not be elaborated upon. The 30-minute didactic and SP training curriculum may both be taught by a single educator. The expanded workshop required at least five participants for the role-play demonstration: team leader, airway provider, primary nurse, parent, and FPF. Any of these roles may be played by the primary educator. The in situ simulation was more labor intensive and required interprofessional participation. Programs with an existing in situ curriculum may integrate the FPF curriculum into their existing simulation by employing an SP or team member to portray the family member and ensuring that there is a team member who can fill the FPF role. Curriculum deliverySP recruitment and trainingIn situ simulationWe trained providers from different professions in the FPF role using the brief didactic, including institutionally (division of pediatric emergency medicine, emergency medicine residency training program, pediatric residency training program, and neonatal intensive care fellowship training program) and nationally at the 2023 Society for Academic Emergency Medicine conference. The didactic was presented via a computer and projector, as detailed above. We also conducted the longer workshop institutionally for the third-year medical student class as part of the existing Integrated Clinical Skills course curriculum and nationally at the 2023 Pediatric Academic Societies conference. For the workshop, we utilized a computer and projector to present the curriculum. We then ran the first role-play demonstration (FPF Role-Play Script Without FPF, ) using the equipment listed above and subsequently divided learners into small groups (four to five members per group) to discuss and complete the accompanying participant and instructor worksheets ( and ). The first scenario (without the FPF) is intended to facilitate active engagement such that learners could identify the performance gap and brainstorm when and how the FPF could provide support to the family member during the worksheet portion of the workshop. After the large-group report-out, we ran the second role-play demonstration , followed by small-group discussion and large-group report-out. This allowed learners to contrast the two scenarios and witness the ideas they generated during the worksheet portion in action during the second resuscitation scenario. Didactic material from this workshop was uploaded to our institution's educational website and sent to our physician, nursing, chaplaincy, and social work colleagues via hospital-wide listservs for asynchronous viewing. To conduct the simulation portion of this curriculum, we trained four SPs in performing the FPF role, using the curriculum described below and provided in and . The SP training for the FPF role was approximately 1 hour in length, comprising PowerPoint didactics and simulated role-play. We recruited SPs with the help of the NYSIM SP Program Educator from a dedicated group of SPs who worked with our institution's simulation center. Due to limitations associated with the COVID-19 pandemic, we conducted our SP training virtually for a group of four SPs; however, training may be conducted in person if feasible and/or desired. In our SP training, we first presented the SP Training PowerPoint , which summarized the purpose of the curriculum and the three main personality types. SPs then read through the SP Training Script and took turns role-playing those personality types. We also introduced the SPs to the FPF-SAT and together practiced completing the assessment tool to ensure that SPs felt comfortable with the checklist. This assessment tool was developed using existing assessment tools in OSCE work that were employed by our simulation center and with which our SPs had great familiarity. In this regard, we were fortunate that our SPs were very comfortable in the role of assessing learners. However, the SP training curriculum was designed to introduce SPs to the tool so that even those who have not used such a rating before are comfortable doing so. We conducted only one training session per group and provided detailed SP notes prior to each in situ simulation. All SP training sessions included a content advisory about the sensitive nature of these cases, the importance of psychological safety, and reiteration of the voluntary nature of SP participation. We also included a slide on the importance of deroling to encourage SPs to separate themselves from the person they portrayed in the simulation. Interprofessional members of our division of pediatric emergency medicine experienced the simulation-based component of FPF training as part of a longitudinal curriculum. In situ simulation sessions were conducted monthly in our pediatric emergency department and were led by a PEM faculty/fellow dyad. Simulation participants included PEM attending physicians and fellows; pediatric and emergency medicine residents; pediatric and general emergency medicine nurses; pharmacists and pharmacy residents; respiratory therapists; and, where applicable, consultant physicians (including teams from the neonatal ICU, neurosurgery, and obstetrics and gynecology). Each simulation followed a standardized format, with a 5-minute prebrief, 10-minute simulation, and 40-minute debrief. Each prebrief began with a statement about the importance of psychological safety in this learning environment. Starting in September 2022, after the initial delivery of our FPF didactics and workshops, we began integrating SPs into our simulations to portray the family member. In each simulation, the resuscitation team leader was responsible for assigning an FPF to communicate with and provide support to the patient and/or family. In the debrief, the facilitator discussed not only medical and teamwork domains but also patient- and family-centered communication. Portions of the curriculum that employed simulation-based instruction were debriefed. In the interactive workshop, both the small-group worksheet discussion and large-group report-out served as a mechanism for workshop participants to engage in focused facilitation with group leaders to reflect on the witnessed role-play demonstration. Our in situ simulation program comprised a 10-minute simulation, followed by a 40-minute scripted debriefing that utilized the PEARLS debriefing format and used a combination of direct feedback, Plus/Delta, and focused facilitation methodologies. We conducted a retrospective pre/post assessment using the Kirkpatrick New World model of program evaluation to query self-reported reactions and learning to evaluate the didactic curriculum, using 4-point Likert-like scales to dichotomize results . The retrospective pre/post design allowed for data collection from a broad audience, mitigated response-shift bias, and had been demonstrated to be effective and comparable to traditional pre/post self-assessments in health professions education. The same survey tool was used, irrespective of what version of the curriculum learners received (i.e., 30-minute didactic, 90-minute workshop, or asynchronous curriculum), with an option for learners who completed the experiential workshop to provide additional qualitative feedback. Due to the heterogeneous settings in which this curriculum was delivered, we did not record the total number of people who attended all sessions; however, an estimated <5% of participants declined to complete the evaluation. We did not separately assess learner evaluation of the longitudinal in situ simulation curriculum. The FPF-SAT was developed by the authors to provide feedback to the medical team on key behaviors identified in the FPF curriculum. After each simulation, the SP completed the FPF-SAT to evaluate FPF. The SP used their completed FPF-SAT to guide verbal feedback during the debrief, and the completed tool was also provided to the FPF and medical team as written formative feedback. These data, along with qualitative feedback, were incorporated into revisions of the curriculum and assessment tool. Data were collected via the Qualtrics platform. Posttraining survey results for the FPF curriculum were dichotomized to compare agreement ( agree / strongly agree ) with disagreement ( disagree / strongly disagree ), using chi-squared testing (IBM SPSS Statistics version 29). An estimated 160 learners completed the curriculum, of which 153 learners responded to the curriculum evaluation. Of these respondents, 26% experienced the brief didactic session; 67% experienced the expanded workshop; and 7% experienced the longitudinal simulation curriculum. None of the respondents experienced the asynchronous curriculum only. Results of the evaluation are shown in , with strong agreement ranging from 68% to 83% for each question. A demonstrative sample of qualitative feedback is provided in . Overall, posttraining surveys indicated that this curriculum was enjoyable, engaging, and relevant, with 95% to 100% of respondents reporting agreement ( agree / strongly agree ) with curriculum evaluation outcomes ( p < .001 across all domains). Although patient-oriented outcomes are the gold standard of medical education scholarship, our curriculum used proxy measures because pediatric resuscitations are rare and emotionally sensitive events. Notably, only 33/152 (22% vs. 78%, p < .001) of participants had received similar prior training. The workshop component of the training was particularly well received (100% of respondents reported it to be useful), with one participant noting, “The hands-on component was the most useful portion of the whole experience.” Given the highly skewed nature of our responses, we did not separate self-reported results by learning modality. In contrast to the majority of respondents, seven of 153 (5%) reported they would not change their practice based on this curriculum. Of these respondents, four of seven (57%) were attending physicians, two of seven (29%) were medical students, and one of seven (14%) were advanced practice providers. Of respondents, five of 152 (3%) reported that the curriculum was not relevant to their practice; these respondents comprised four of the five (80%) medical students and, interestingly, one of the five (20%) were child life specialists. Nevertheless, all these respondents reported the curriculum to be enjoyable, engaging, and resulting in improved knowledge and skills. One of these learners commented: “I will be more aware of designating someone to fill that role, I think it is important.” Constructive feedback suggested expanding the curriculum beyond resuscitation; including perspectives from a broader range of religions, ethnicities, and cultures; and highlighting the challenges of the FPF role. Results from the FPF-SAT, as rated by SPs in our in situ simulations , showed that, on average, FPFs performed well (>50% were rated well done ) on nine of 15 behaviors. Feedback on tailoring of information quantity was more variable: although 53% of SPs felt that this was well done, 33% reported that information quantity was not tailored to their preferences and needs. Qualitative feedback highlighted the importance of providing culturally competent care with appropriate language interpretation services (“There was an immediate disconnect the moment they found out the parent only spoke Spanish. This caused a lot of distress throughout the encounter, until the translator was ‘brought’”) as well as nonverbal communication (“The lack of eye contact or knowledge of myself made me feel extremely desperate and clueless, even before they realized I didn't speak English. I don't remember more than 1 team member looking into my eyes to try to understand me and help me.”) These data from the FPF-SAT represented a small number due to the resource-intensive nature of simulation, especially that with an SP component; however, we found the results were valuable with respect to general feedback as well as specific, domain-centered feedback. The FPF-SAT rated participant performance in the FPF role, in contrast to the posttraining survey, which evaluated learner self-reported reactions and knowledge acquisition. Because both tools were anonymous, we were not able to perform a retrospective analysis to evaluate whether self-reported learning outcomes correlated with objective performance in the FPF role. Facilitating family presence during pediatric resuscitations has myriad benefits and is an essential skill for interprofessional teams that provide care to pediatric patients. A prior study found that implementation of practice guidelines and training increased provider comfort with family presence during both invasive procedures and resuscitations. However, little research has been conducted to determine best practices for facilitating family presence during acute resuscitations, a crucial component of patient- and family-centered care that requires sensitive, dedicated training. In this ESR, we described the development, implementation, and evaluation of a curriculum designed to instruct all members of the health care team on how to provide this care. Through a literature review, appraisal of existing resources, and focus groups, we identified four main categories of behavior: respect and value; information-sharing; nonverbal communication; and summary and follow-up. Our curriculum provides essential training in applying these behaviors to provide emotional support to patients and families, in various formats adaptable to the needs of learners and educators (i.e., brief didactic; expanded workshop; asynchronous curriculum; and longitudinal simulation-based experiential learning). Our results indicate that the curriculum is enjoyable, engaging, and relevant. Dissenting results (i.e., those who did not find the curriculum relevant or think it would change their practice) may be explained in part by the narrower scope of pediatric emergency department-based resuscitations on which we focused, and on participants having had prior similar training in curricular components. These results were also balanced with learners concurrently endorsing the curriculum to be enjoyable and engaging: 95% agreed that they would change their practice because of the curriculum, and 100% agreed that doing so would improve the patient care experience. Feedback from SPs regarding FPF performance highlights the importance of providing culturally competent care with strong receptive listening skills. The PowerPoint materials are a core part of this curriculum and are intended to be consumed as prelearning, either in didactic or self-directed learning format prior to the simulation component. The first two learning objectives, namely articulating the purpose and benefits of the FPF role and listing the four components of the role and key behaviors for each, are level one objectives for knowledge acquisition on Krathwohl's revised Bloom's Taxonomy ; however, we strongly believe that when simulation-based health professions education includes novel, high-stakes, and/or emotionally charged concepts, conducting prelearning is necessary prior to entering the concrete experiences stage of the experiential learning cycle. Learning objectives three and four reflect the simulated participant and simulation-based components and necessarily build on the didactic materials. This curriculum was initially designed for use in emergency department-based pediatric medical resuscitations, and we acknowledge that there are likely differences in traumatic resuscitations, those taking place outside of the emergency department, and those involving adult patients; we are currently adapting the curriculum for use in these areas. Unfortunately, we did not collect data regarding residency type (emergency medicine vs. pediatrics). Additionally, this study was conducted at an academic, quaternary care institution in which staffing models generally permit additional team members to provide family support; however, this may not be true for all settings, especially smaller community-based emergency departments. To address this, we have included guidance in the curriculum regarding how to initiate communication with family members when there is no one to fill the FPF role. Our focus groups comprised youth and family advisory council members from only one institution in the northeastern United States, and their input may not be generalizable to all populations. Other limitations include the use of self-reported, immediate outcomes. We did not assess objective or patient care-focused outcomes, nor did we assess retention of learned skills over time. Because our in situ simulations were conducted monthly and with only one SP, our overall number of responses for the FPF-SAT was small, and we did not perform psychometric assessment of tool performance. Lastly, the workshops and simulation-based components of the curriculum are labor and resource intensive; however, our inclusion of briefer and asynchronous curriculum materials hopefully improves generalizability. The development of this curriculum benefits from the partnership between clinicians and families, which may have myriad additional advantages. Obtaining the perspective of patients and families in developing educational curricula (particularly simulation-based curricula) may also: improve health outcomes and patient safety; advance key communication skills by learners; mitigate implicit biases and promote health equity; optimize the portrayal of patients and families in curricula and by SPs; and enhance the psychological fidelity of the learning exercise. – The key behaviors described in this curriculum are not specific to pediatrics, nor are they specific to emergency department-based medical resuscitations. We believe that family presence during resuscitations should be supported across a wide range of patient ages, medical presentations, and settings. Next steps for research and education include identifying key barriers and facilitators to supporting family presence more widely, linking evidence-based practice to the patient experience and objective health outcomes, and developing best practices for communication and management around patient death when it is experienced by the family and health care teams together. We hope this curriculum will serve as a foundation for clinician-educators in various settings to train interprofessional teams in the FPF role, thereby improving patient- and family-centered care across the spectrum. FPF Curriculum.pptx FPF Curriculum Recording.mp4 Role-Play Script Without FPF.docx Role-Play Script With FPF.docx FPF Participant Worksheet.docx FPF Instructor Worksheet.docx FPF Survey.docx SP Training.pptx Simulated Participant Training Case.docx FPF-SAT.docx All appendices are peer reviewed as integral parts of the Original Publication. |
“It kinda helped us to be there”: students’ perspectives on the use of virtual patient software in psychiatry posting | 2a403728-da17-4da5-be58-cb78e427e8d2 | 10636819 | Psychiatry[mh] | The COVID-19 pandemic and the ensuing lockdown measures highlight the need for the incorporation of online learning in medical education. Among other tools, virtual patient software can be useful in teaching medical students across various clinical disciplines. While the value of virtual patient software in undergraduate psychiatry education is gaining recognition , understanding of medical students’ experiences with their use is still limited. Such understanding may help in the full utilisation of this educational tool to provide valuable learning opportunities for students. The term “virtual patients” has been used to describe various applications of information technology for clinical teaching. A review shows that interactive patient scenarios have been the predominant type of virtual patient in medical education . This is in line with the American Association of Medical Colleges’ definition that virtual patients involves simulation of real-life clinical scenarios using computer based programs . Other common types of virtual patients are high fidelity software simulations and virtual standardised patients . Comparing to other forms of virtual patients that emphasise different competencies, such as procedural skills or communication skills, interactive patient scenarios gravitate towards supporting the development of student clinical reasoning skills . Earlier studies have favourably compared the use of virtual patient software with standardised patients and real patients in undergraduate medical education . Students find it helpful that virtual patients can demonstrate clinical abnormalities, and there is a high level of acceptance . It may also be useful for early medical students with less clinical experience, as they may encounter difficulty in communication and history-taking with real or standardised patients . A recent systematic review shows that the use of virtual patients helps improve students’ clinical reasoning skills, especially in data collection, diagnosis, and patient management . This agrees with the values of virtual patient simulation in improving skills and knowledge in wider health education, including other allied professions . Students’ perceptions of virtual patient software have also been assessed in some qualitative studies. Students experience interactions with virtual patients as integrating biomedical knowledge and clinical experience, which provide learning in a logical and structured process. Nonetheless, the virtual patients were also perceived as monotonous, lacking in emotional interactivity and complexity of real patients . Besides the issue of authenticity, the adequacy of feedback in virtual patient tools has also been questioned . Virtual patients are a comparatively novel approach in psychiatry education as opposed to medical education as a whole. Nonetheless, considerable quantitative evidence has been accumulated. In a systematic review and meta-analysis involving 27 randomised controlled trials of the use of simulation in psychiatry for medical doctors , significant benefits in the improvement of students’ attitudes, skills, knowledge, and behaviours have been demonstrated, indicating the effectiveness of simulation training in psychiatry education. Focusing on undergraduate psychiatry education, including medical students and other allied healthcare professionals, a recent systematic review identified 46 articles . Interactive virtual patient scenarios were among the common types of virtual patient interventions. All included controlled trials reported statistically significant positive outcomes in terms of knowledge, skills, and attitudes. Notably, there were only five qualitative studies that attempted to explore students’ learning experiences with virtual patients, indicating a gap in this research area. There were two studies involved nursing students learning using clinical simulation tools . Another mixed methods study of virtual patient simulations focused on Masters level behavioural science students . The remaining two studies examined Danish medical students’ experience with video-based learning in psychiatry using recordings of standardised patients . Hence, in the current literature there is still very limited qualitative research on undergraduate medical students’ psychiatry learning experience with virtual patient tools based on interactive patient scenarios. At the Faculty of Medicine, the National University of Malaysia, we pioneered the use of DxR Clinician, a virtual patient software program based on interactive patient scenarios, for psychiatry education of fourth-year undergraduate medical students. The students had a seven-week psychiatry posting, during which they were exposed to the discipline through various teaching and learning methods, including ward rounds and clinic sit-ins, online, in-person and hybrid lectures, team-based learning (TBL), and workshops. One of them was a TBL module on neurocognitive disorders, which covered dementia and delirium. The TBL module consisted of the following six steps: pre-class preparation, individual readiness assurance test (IRAT), team readiness assurance test (TRAT), immediate feedback, clinical problem-solving activity, and closing . It was originally designed as a face-to-face session except for the pre-class preparation step. An initiative was taken in the first year of the pandemic (2020) to use DxR Clinician for the teaching of this module. In the modified module, two main changes were made: (1) A virtual interactive case scenario of dementia in DxR Clinician was used for the clinical problem-solving activity that was conducted asynchronously; (2) Other steps that were previously run face-to-face were switched to a videoconferencing platform. As the subjective experience of students with virtual patients may greatly vary depending on the educational setup and user profile, it is crucial to gain qualitative understanding of students’ experiences within specific contexts. Insights gained from a particular program may in turn inform improvements to current and future efforts in this area. In this study we aimed to explore medical students’ experiences in using the DxR Clinician program for a modified TBL module for neurocognitive disorders during their psychiatry clinical posting at the National University of Malaysia. The SPIDER (sample-phenomenon of interest-design-evaluation-research type) tool was used to frame the research question of this study : “What are the medical students’ experiences of participating in the modified TBL module for neurocognitive disorders using DxR Clinician?” More specifically, we aimed to explore the students’ ideas about and expectations, perceived advantages, and disadvantages of, and perceived barriers to the virtual patient software program in helping them to achieve the objectives of the modified TBL module.
The reporting of this study is guided by the Consolidated criteria for reporting qualitative studies (COREQ): a 32-item checklist for interviews and focus groups . The research team and reflexivity The modified TBL module for neurocognitive disorders Study design Study population Sampling and sample size Data collection Data analysis Ethics approval This study received approval from the Research Ethics Committee of the National University of Malaysia (UKM PPI/111/8/JEP-2021-355) on 16 July 2021.
The research team consisted of three clinical academics (One female and two males) at the Faculty of Medicine of the National University of Malaysia who were involved in undergraduate medical education. Two researchers were psychiatrists (L.S.-C.W. and T.I.M.D.) and another was a general physician (S.F.T.). L.S.-C.W. was the medical lecturer responsible for the modified TBL module for neurocognitive disorders. Both S.F.T and T.I.M.D. had extensive previous experience in conducting qualitative studies. L.S.-C.W. and T.I.M.D. were involved in the clinical teaching of the medical students who took part in this study during their psychiatry posting, but they were not individual supervisors of these students. To avoid bias and undue influence, a PhD student who was not part of this research team was appointed as the facilitator for the data collection process.
DxR Clinician is a web-based patient simulation software that contains a collection of problem-based case studies for the teaching and evaluating of clinical reasoning skills of medical students. It allows the student to interview the virtual patient, conduct a simulated examination, and order lab tests . A wide range of interview questions (up to 250 questions) is included for history taking. The software is also able to simulate physical exams and provide interpretation of findings, besides allowing the ordering of laboratory tests and imaging studies. Based on the findings, the students can develop a list of working hypotheses and follow the evidence to decide the final diagnosis. Finally, a treatment plan can be developed. Immediately after the completion of the case study, performance feedback in the form of a composite score with a breakdown of different component scores is provided to the students to indicate their strengths and weaknesses. Using DxR Clinician, a modified TBL module for neurocognitive disorders was developed. The learning objectives were to: define the condition; describe and define relevant symptoms and signs; discuss relevant differential diagnoses; discuss relevant investigations; and discuss principles of management. A virtual case in DxR Clinician was prepared to present a clinical scenario with the required primary psychiatric condition and associated secondary features that would stimulate students’ critical thinking about differential diagnoses. To ensure the authenticity of the case scenario, the virtual case was adapted from an actual case write-up for a patient with dementia, but with alterations of clinical features and modifications of sociodemographic characteristics to ensure anonymity. The class was conducted in three phases. In Phase 1, pre-reading materials and video instructions for the use of DxR Clinician were provided to the students a few days before the class. In Phase 2, an online session using a video-conferencing platform was held. An online quiz was administered to the students individually, followed by answering the quiz in groups and discussing the quiz questions. This formed the individual and group readiness assurance tests, respectively, for TBL. The student groups were then instructed to begin their case study using DxR Clinician (Phase 3). This section of the learning process was done asynchronously, whereby the students worked through the case with their group members. Students were expected to arrange the learning activity at this phase with their peers, including the time, place, and device to be used. As the case study was conducted in groups, each student group was given a group account name and a password to access the web-based program and solve the case. A final debriefing session was held after that, during which the instructor provided feedback, comments, and further instructions regarding the students’ case-based learning activity.
A qualitative study design was chosen for this research. A qualitative design is suitable to answer the research question regarding study subjects’ perceptions and experiences, as it allows a more in-depth and holistic understanding of phenomena within the natural context of the subjects . In this study, we adopted the basic positions of critical realism. Ontologically, we affirmed the mind-independent existence of reality. At the same time, we accepted epistemological constructivism, that the understanding of reality is a function of our perspectives, and there could be multiple interpretations of reality. We assumed that social reality is driven by mechanisms influenced by the context . However, we did not clearly delineate the context, mechanisms, and outcomes we investigated in this study. While we attempted to provide theoretical explanations for our findings, drawn from other theories, on how virtual patients for psychiatry education of medical students works, we did not develop or test a program theory .
Medical students at the National University of Malaysia were enrolled in a five-year undergraduate program for the Doctor of Medicine degree (MD). The program consisted of a preclinical phase of basic medical sciences for two years, followed by a junior clinical clerkship in the third year and two more years of senior clerkship. Students enter clinical posting in psychiatry during the fourth year. The inclusion criteria for this study were: (a) Fourth-year medical students; and (b) Completed psychiatry posting with the new modified TBL module for neurocognitive disorders. The exclusion criteria were: (a) Dropped out from studies; (b) Unable to devote time for participation in data collection due to commitment to other activities, physical or mental poor health, or other issues. A total of 128 fourth-year medical students completed the modified TBL module with DxR Clinician at the time this study began.
Purposive sampling was used to select study participants who were most likely to provide relevant information that would deepen the understanding of the research question . A diverse range of study participants was sampled by including different batches of students who underwent their psychiatry posting at different times (which in turn affected their clinical exposure due to various levels of restrictions on clinical contact at different periods), and students with different levels of mastery of the study subject. A general idea of the mastery of the subject was given by the DxR case study performance scores achieved by the student groups when they underwent the module. A focus group size of five participants was chosen. Group size may range from three to 12 . We chose a small group to facilitate the elicitation of in-depth insights . As it was expected that a single focus group would be inadequate, we planned for at least two focus group discussions with a total of 10 participants initially. Once potential participants were identified, invitations were emailed to the students. The email invitations were delivered by a faculty administrative staff and not one of the researchers to avoid any undue influence. As the potential study subjects were medical students, emphasis was made on the fact that their choice to participate or not will not affect their teaching and learning activities as well as assessments. It was also made clear to them that they were not under any pressure to participate against their will. Students who were available and willing to participate provided their informed consent and were included in the study. The study participants’ age ranged between 23 and 24 years old. There were eight females and two males. Data collection and data analysis was conducted as an iterative process, and as data saturation was achieved after the initial focus group discussions , no further sampling was done.
The focus group discussions were conducted online using a videoconferencing platform. A postgraduate student independent of the research team, who had training and experience in running focus group discussions, was appointed as the facilitator. The role of the facilitator involved explaining ground rules, introducing the discussion topic, encouraging participation from all group members, and keeping the discussion on track and on time. The discussions were carried out in English. A questioning route was developed to guide the discussions (Supplementary Material). The online discussions were recorded with the permission of the participants. No third party was present during the discussions. The discussions lasted between about one to one-and-a-half hours. Verbatim transcripts of the conversations during the discussions were made. The transcripts were returned to the participants for review. No correction to the transcripts was requested.
The thematic analytic approach was used for data analysis . Thematic analysis is a qualitative research method that provides a well-structured approach to handling dataset and summarising its key features and is useful in the examination of different perspectives of research participants . The same analytic approach has also been used by other qualitative studies on the student experience of virtual patients in undergraduate psychiatry education . The analysis was conducted according to the six phases described by Braun and Clarke . First, the transcribed collected data was read and re-read by the first author (L.S.-C.W.) to familiarise himself with the entire data. Through this process, the researcher actively searched for meanings and patterns in the data within the context of the study. Initial ideas were noted down. Transcripts were labelled with unique codes, which also included the dates FGD was conducted . Next, initial codes were generated. The researcher systematically worked through the data to code interesting features in the data. Relevant data for each code was collated. Data coding and collation of extracts were performed using Microsoft Excel (Redmond, Washington, USA). After all the data had been coded and collated, the codes were examined and considered for the identification of potential themes. With the help of a second researcher (T.I.M.D.), the initial thematic map was developed in this phase to sort the different codes into main themes and subthemes. Subsequently, all research team members reviewed the themes together, evaluating both the coherence of collated data for each theme and the validity of individual themes in relation to the entire data set. Once this was done, we refined the definitions of the themes and finalised their labels. We deliberated on the ‘story’ each theme tells and decided its place in the overall ‘story’. The results were shared with the study participants, but no additional feedback was received regarding the findings. In the last phase, we produced a write-up of our findings in the form of an analytic narrative with a selection of compelling data extracts to demonstrate the points.
ree main themes were identified regarding the medical students’ experience of using the DxR Clinician program in the modified TBL format in their psychiatry posting: (1) fulfilling the desired pedagogy (2), realism of the clinical case, and (3) ease of use related to technical settings (Table ). Participants’ quotations are included in the following subsections to illustrate the themes. Each quotation is identified by a unique code. Desired pedagogy Realism of the clinical case Technical setting The pedagogy theme was mainly about the respondents’ perception of the methods of the learning process using DxR Clinician. The theme covered the whole process of teaching-learning, whether it suited the levels of entry for students, the authenticity of materials, the delivery, peer learning, and assessments in the following sub-themes: Level of entry for students Flexibility of content presentation Provision of learning guidance Collaboration with peers Provision of feedback Assessment of performance The DxR Clinician was viewed favourably as an assessment tool by the study participants. The assessment scores given at the end of the case study served as helpful feedback in evaluating performance. It was suggested that the software program could potentially be used effectively as an assessment tool in examinations. “For the current situation, I think DxR is, from what, from what I see lah, from what I can think, it’s more like, err… something like exam-based software. Because, you can use DxR as an exam-based software to replace OSCE lah, in case, err… student cannot go to the wards or anything, so this platform is very good to use as an exam, exam, err… platform.” FGD1-28/9/2021 . In keeping with this idea, some felt that virtual patients can assist students in preparing for assessments. “It’s a good backup or at least a good, err… revision I would say to read, at least know what kind of questions that you can ask, although it’s not necessary, that means they give you like a set of questions that you know like oh this kind of question can be asked in different cases.” FGD2-28/9/2021 . Some, however, believed that it was not sufficient to aid preparation for clinical examinations. “When you are too used to that kind of platform, discussions are always going around DxR, and then when it’s during the exams, err… they get too comfortable with you know, a certain format and then they can’t go over again.” FGD3-28/9/2021 .
This subtheme was about what could be the appropriate levels or stages of learning to utilize DxR. First, some participants observed that this software program is suitable for medical students in their preclinical years in exposing them to clinical skills that are required before they enter their clinical postings. It has been suggested that this program can be used in a clinical skills lab or a problem-based learning module for this category of students as a form of preparation for their later clinical years. “DxR Clinician is a good platform to begin for preclinical students, at least for them to know what set of questions they should ask…Or what kind of investigation or physical examinations that’s available.” FGD2-28/9/2021 . At the same time, the virtual patient software program was also viewed as an excellent tool to compensate for the lack of clinical contact with patients during clinical years. The COVID-19 pandemic was cited as a reason for reduced clinical exposure due to the lockdown measures and learning clinical cases through DxR Clinician to some extent mitigated the shortfalls. This was considered especially pertinent for the neurocognitive disorder module in psychiatry posting because of the smaller number of clinical cases the students could encounter in real person compared to other, more common psychiatric disorders. “So, since we were like in the pandemic and especially since my psychiatry posting was affected, I feel like it kinda helped us in order to be there even though we can’t be there physically in the ward, but then we can somehow be involved in the entire history taking and all that.” FGD6-4/10/2021 . It was further suggested that the virtual cases’ difficulty levels could be specified so that students could gauge their competency levels based on case difficulty. “I would appreciate it if there is, err… a way to, umm… help different levels of students… I would like it if there were a platform where it tells us what we should know, at our level, so that we are, we are more targeted? And more, umm…able to study better for our level, a bit.” FGD7-4/10/2021 .
Regarding the presentation of the learning content, explanatory texts and case summaries provided in the case study were considered helpful by some participants. The way information was presented was flexible to fit the user’s style of learning. They can repeatedly use the content suiting their convenience. “After we complete the DxR we are provided with, err… many explanations and all the solutions that, err… the cases that we learn. So, I think with this, umm… it actually helps us a lot because sometimes when we learn we don’t- we do not know what sources are correct, or what answers we should give.” FGD10-28/9/2021 . At the same time, it has been commented that the explanations given were not organised systematically enough, making it hard to digest the content. “It actually has tons of explanations but, umm… I don’t, I’m not sure if it is, err… it is not compiled in a very systematic way, I can say. It is quite messy actually like they just put a lot of important points in many paragraphs…” FGD10-28/9/2021 . Thus, some felt that the manner of information presented in the virtual case study was passive and that they were just ‘going through motion’ when they took part in the learning activity. “I think err… some of us might just do it just to get it done. So, err…we don’t try hard, we don’t work hard for it because umm… we just want to get it done because our lecturer wants it to be done. Yeah.” FGD5-28/9/2021 . Additionally, the issue of the time limit for questioning was also raised. The current format of delivery of content that did not have a time limit might allow more thorough and careful case exploration, but imposing a time limit might improve the efficiency of the learning process. “I would prefer a platform with a limitation of time than a limitation of questions because time would be more of an exam-based format and we are used to that. But giving us a limited list or a number of questions would be very hard for us.” FGD3-28/9/2021 . Lastly, it was also suggested that virtual patients could be used for the development of other clinical skills, such as case presentations to lecturers based on the virtual patient’s history.
In this regard, the opinions of the participants were again varied. Concerning the comprehensive list of questions to select for history taking, some participants thought that it was a helpful resource in guiding their reasoning in actual clinical situations. “I would like to add about the variety of questions that can be chosen inside one of the particular cases. There are a lot of questions that we are asking, we will be asking during history taking and this DxR actually, err… includes all kinds of questions, so it provides a very real situation for us to, err… experience it.” FGD9-4/10/2021 . However, the cues could confuse them if the response did not go in line with their thinking. “It really affects the thinking process because rather than you thinking the questions out for yourself, like from the differential diagnosis or anything that you wanna come out with questions, it’s already displayed there then you just select… certain questions where you phrase it another way and you expect another answer, but it came out with a different answer.” FGD2-28/9/2021 . Yet some other participants viewed the long list of available questions to ask as a drawback. Sometimes the questions could be redundant; sometimes they were too broad or too specific in scope, making it hard for the students to decide if they were relevant to the case. “Although I can see there are still, there are a few err… improvements that still to be made lah. I mean like, for… for example for certain diagnosis err… or a certain situation, sometimes err…students want to ask, err… a more specific question or more broad question…” FGD1-28/9/2021 . The participants also observed that as they explored the case, they were not initially aware that the number and relevance of questions they asked would affect their overall performance score later. They would prefer such limitations to be communicated more clearly from the outset.
One aspect of pedagogy in this learning module using DxR Clinician was the team-based learning format. This format of learning, which involved collaboration with peers, was seen as beneficial in eliciting performance from the students. The students had the chance to work in a team and discuss the case together. Through such discussions, different perspectives emerged and contributed to the solving of the case. “And err, for err, for this psychiatry posting, we actually did this in a group. So, when I was doing it with my friends, I actually thought that I was discussing it with them, so… It’s somehow a good way for me err… to learn and study together. FGD5-28/9/2021 . The disadvantage of this learning format was that collaborative work could be more time-consuming. “My personal disappointment was because we were doing this as a group, and as a group we were only allowed to use, err… one device, instead of everybody using each device to put in their answers and all. So, I feel like because they were using only one device, it just, it, it keen to take a lot more time, because we had to wait for one person to type and the others to give answers, though it, err… makes our teamwork better but then I feel like it just takes some of our time which could be used for something else.” FGD6-4/10/2021 .
During the asynchronous learning phase using the virtual patient software, the students appreciated the instant, step-by-step feedback as they were working on the case. The feedback provided served as useful guidance to sharpen their clinical reasoning skills by indicating the level of importance of questioning. “And what I love the most, err… because DxR provides err… instant feedback, I can say. Err… we can know what we should ask, and what we should not ask, and yeah, the feedback is really good. FGD8-4/10/2021 . “But it does help you imprint questions, like when you see this patient, at least you know these questions, if you pick this you get lower marks in your clinic- (laughs) in your DxR so you’re not gonna do that again, and then you will pick a better question for that.” FGD3-28/9/2021 . An issue highlighted regarding feedback was the interaction with the instructor in the closing session. While instant feedback as assessment scores for the group learning activity was given, the participants also wanted more opportunities to have personal feedback and to ask questions directly. Indeed, such opportunities were provided in the closing session when the instructor went through the case again with the students, but the online session might not be very conducive for interactions. “I think some of us after, maybe after doing the whole exercise we might have questions that are more, for example, if it’s physical we could just ask the doctor any kind of questions and, umm… we’ll get their answer, so I’m not sure if this DxR could do the same as well, err… but this will probably require, err… more personal, err… a person to answer a more personalised questi-err… personalised question, yeah. So, I think if there is that function it will be… greater.” FGD7-4/10/2021 .
theme is about how closely the clinical case presented in the learning module in DxR Clinician resembled a real clinical encounter. There are two main aspects to this theme. Similarity to actual patient Cultural adaptation of the case Another subtheme was about the case scenario: how much the details presented in the scenario reflected real conditions in the local context. Some participants observed that some changes were made to localise the case history content for the learning module, such as the use of local-sounding personal names. Nonetheless, it was also pointed out that several terms used in the program were not consistent with the common terminologies applied in Malaysia. Furthermore, it has been suggested that having the virtual patient available for history taking in the Malay language (known as Bahasa Melayu ), the national language of Malaysia, would make the exercise more realistic as Malay is the language most used by students in their clinical encounters with patients, even though the use of English was also prevalent. “I’m just curious if this software is also available in Bahasa ? Yeah, I’m just curious about that because somehow, err, we are living in Malaysia so in our real setting, we’ll be interviewing err…our patient, and we are, maybe, most of our patients we are using Bahasa, err , Bahasa Melayu. If they wanna make it more real, I think maybe they can like you know… Innovate it into Bahasa. ” FGD4-28/9/2021 .
The first subtheme is related to the ‘feel’ of interacting with the virtual patient – how much it mimicked an actual patient. The opinion that the virtual clinical case was realistic was based on the features of the program that allow users to perform various clinical activities such as history taking, physical examination, and investigations consistent with what happens in real life. “It’s as of like you are really clerking a real patient, err… although you can’t get the feel of it, umm… but for example, there’s even the patient’s profile, the faces and it comes as a whole package, so I think it’s also easily accessible in that sense, where I can just, I just need to go into this website, and there is everything from the lab, everyth- investigations and err… the physical examinations, umm… so, I, I thought it’s very, err… err… wholesome in terms of it’s all in one package, and then there is also good visuals, so err… I like that, yeah.” FGD7-4/10/2021 . On the other hand, the main criticism of the lack of realism of the case was focused on the way history taking is conducted. The questions were pre-listed and the answers from the virtual patient were in written form, therefore differed considerably from real two-way communication between a clinician and a patient. “We don’t have really, umm… real feeling of asking patients, because if we are, umm… asking history from a real patient, we have a two-way communication like can hear their voices and all, but in here we just click the button and the, yeah the sentence will come out, the answer will come out like that, so we don’t have the real feeling of asking the patient.” FGD8-4/10/2021 .
technical setting theme revolved around two subthemes: access to the software program and the appearance of the user interface. Access User interface Regarding access, some participants remarked that in the current format, the DxR Clinician clinical case was only accessible to the students using a one-time account during the limited period given for the modified TBL activity. It was hoped that the clinical case could be made available to the students outside of that learning module so that they could explore the case on their own for learning and practice purposes. “Because if you want to access the DXR Clinician, you need to have the password of the particular case, but if it’s possible, or if it’s feasible maybe each of the students can have a particular code or particular account to it, and then when we access, we can access towards a different posting, because I believe that DxR Clinician is quite a good software to practise on. So, during our free time or anything I think we can use it for practise.” FGD2-28/9/2021 .
Several comments were made by the participants regarding the user interface (UI). On the positive side, the simple design of the program, which was readily accessible through an internet browser, made it easy to load without taking up too much internet bandwidth. “Umm, it is, it doesn’t use a lot of internet bandwidth…So, it is very quickly loaded. The page… (laughs) very simple, just click and then something appears, so it doesn’t have any complexity……on the website.” FGD3-28/9/2021 . Moreover, it did not require internal storage and was compatible with desktops, laptops, or handheld devices. On the other hand, the interface graphic design was deemed rather outdated and therefore unattractive. The case directory page was also be quite cluttered as all cases across various disciplines were listed together. The small button size for item selection could be hard to use, especially when navigating using a device with a small screen. Sometimes, the instructions in the software were too wordy or unclear. “All right, as for me I think it’s, it’s more about the UI lah. UI and the appearance of the website. The UI’s err… how to say ah, it’s, it’s like from, a very old computer program? I mean like the button, the appearance of the button, location and all… because there are a lot of err… choices and questions, right? Most of the choices are apparently all on the same pages, and so everything looks quite small lah.” FGD1-28/9/2021 .
In this qualitative study, we explored the experiences of undergraduate medical students participating in a team-based learning module in psychiatry using a virtual patient software program. In two focus group discussions, the students shared their perceptions of this mode of learning from their personal experiences. Following a thematic analysis of the transcribed content, we identified three main themes, namely, fulfilment of the desired pedagogy in this mode of learning, realism of the clinical case in the virtual patient program, and its ease of use concerning technical settings. The pedagogy main theme encompasses several subthemes, which are related to several events that occur in the teaching and learning process. The realism theme involves a personal aspect (how much the virtual patient mimics a real patient) as well as a wider sociocultural context (adaptation to the Malaysian context). Lastly, the technical setting theme consists of access issues and user interface appearance. The use of virtual patient software programs like DxR Clinician in medical education can be considered as a form of case-based learning. Its interactive nature lends itself well to the concept of active learning, which emphasises active and positive engagement of students in the learning process . It is student-centred instead of teacher-centred, where the teacher functions as a facilitator in the quest to acquire knowledge, rather than as the ultimate authoritative source of knowledge . Therefore, it has the potential to work well with the TBL format, which has a similar focus on fostering active learning . Moreover, as the online case study is done asynchronously between the facilitated sessions with ample time given, it also agrees with the flipped classroom approach, which gives students the freedom to engage themselves and review the learning materials at their own pace . In the current study, this is reflected by the participants’ positive experiences with how the program provides information and guidance based on users’ actions and informs their performance in a stepwise manner, and with the TBL format that allows collaboration with peers. Conversely, the lack of contextualised feedback and the logistics and technical difficulties when participating in this learning module were some of the cited issues that might have hindered a truly efficient and enriching active learning experience. As highlighted by some of the study participants, the learning module afforded them some sort of clinical exposure to neurocognitive disorders despite the severe restrictions during the pandemic lockdown periods, which greatly hampered their access to wards and clinics during psychiatry posting. This points out the value of learning by simulation in psychiatry education. Simulation allows a more ‘hands-on’ learning experience that is contained in a safe environment . A virtual simulated patient made it possible that our students could learn about the clinical case without the risk of COVID-19 infection at the height of the pandemic. Likewise, our study participants also indicated that such a virtual patient software can be very useful for students in preclinical years, precisely because junior students without adequate clinical skills and confidence can still safely learn a topic through a medium like this. Simulation education has been found to reduce anxiety and boost confidence among medical students, improving their readiness and clinical competency in various clinical scenarios . Further exploration of its potential in this aspect of psychiatry education should be considered. Virtual patients can be valuable pedagogical models serving as a scaffolding for students to prepare them for their clinical encounters with psychiatric patients . We think that the “desired pedagogy” theme could be viewed and understood from the perspective of the nine events of instruction as proposed by the American educational psychologist, Robert Mills Gagné (1916–2002). The events are: Gain attention, inform learners of objectives, stimulate recall of prior learning, present the content, provide learning guidance, elicit a response, provide feedback, assess performance, and enhance retention and transfer . This model is useful in improving instructional design and student performance in medical education . The identified subthemes in this study appear to correspond to some of the components of this model of instructional design. The flexibility of the content presentation subtheme is related to the event of presenting stimulus or content. As suggested by some of the participants, the learning content needs to be meaningfully organised and explained. Next, the provision of learning guidance was perceived as helpful when students were presented with new content, but it can be improved by making it more streamlined and clearer in boundaries. As students work on the virtual case to reach the diagnosis and produce the management plan, the instructional event of eliciting performance occurs. Working in small groups during the case study can help to produce better practice . Collaborative learning helps enhance medical students’ performance . The provision of feedback was seen as important by the study participants, and it highlights the importance of including timely, quality, and student-focused feedback as an essential element of the learning process . Finally, the design of the virtual patient software allows comprehensive assessment scores to be produced for the student group performance, which are directly tied to the learning objectives of the module. Some authors have also suggested that web-based learning modules in psychiatry can be suitably used for tracking learning outcomes . In their research of contextualised learning in the design of a multimedia learning environment, Herrington and Oliver have proposed an instructional design framework for authentic learning environments . Among the nine elements included in the framework, there are two that are closely mirrored by the “realism of clinical case” theme identified in this study. The first is the requirement that the learning experience provides an authentic context that reflects the way the knowledge will be used in real life. This is seen in the students’ desire that the case study demonstrates adaptation to the local Malaysian context in terms of language, culture, and terminology. Research also suggests that localisation of the technology and software for learning that involves cultural and linguistic adaptation is beneficial and may predict better learning outcomes . Furthermore, the extent the study subjects perceived interactions with the virtual patient mimicked actual interactions with a real patient also corresponds with the other element of the framework by Herrington et al., which is the provision of authentic activities during the learning experience. The activities involving the virtual patient should be well-defined and have real-world relevance. Authenticity is regarded as an important aspect of virtual patients, but technical limitations can be an issue . For instance, a software program like DxR Clinician, which employs a more traditional, textual approach to student-patient interaction, is unable to achieve as high a level of fidelity as tools that utilise more advanced technologies, such as immersive virtual reality (VR) . Medical students tend to have a positive attitude towards the use of electronic resources during their training, including psychiatry clerkship . At the same time, there is a concern about the technical quality of electronic educational resources in psychiatry . This technical aspect was given attention by our study participants. In their Conceptual Framework for E-Learning in Developing Countries, Andersson and Grönlund list individual characteristics, contextual factors, and technological challenges as relevant to the delivery of e-learning. Among the technical issues are access and interface design. It was mentioned by some of our study participants that the virtual patient software’s simple design was very helpful in ensuring easy and reliable access to the online platform. However, the settings of user access were something that the students would want to see improvement in. The challenge of the learning module designer is to find the optimal balance between creating greater flexibility in student access to maximise learning gain and ensuring adequate control and monitoring over its usage within the constraints of the software’s current design. Concerning the students’ impression of the software’s UI, a couple of principles drawn from the cognitive theory of multimedia learning may be applied here . The simple, direct UI used in the software could help minimise extraneous cognitive load caused by unnecessary details or ‘flashy’ features, following the Coherence Principle. Conversely, the monotonous appearance might be associated with a lack of cues that highlight salient material, in contravention of the Signaling Principle. Such understanding can be useful in improving the quality of the learning module. The present study uniquely explored medical students’ experience with virtual patient software based on an interactive clinical scenario in psychiatry education. Its qualitative study design allowed in-depth understanding of students’ perspectives. Several insights gained from this study may inform future research. Among others, strategies to effectively provide feedback to learners in an engaging manner using virtual patients should be further developed and tested. The potential of simulation with virtual patients in psychiatry education in relation to enhancement of clinical competency, especially soft skills, need to be explored. The use of web-based learning modules for the tracking of learning outcomes in psychiatry education also needs more research. Additionally, clearer guidelines are needed for the contextualisation of virtual patients, considering sociocultural differences, students’ technology savviness and available resources, as well as the unique features of the clinical practice of psychiatry in contrast to other medical disciplines. A few study limitations should be mentioned. We did not collect information from other students who were invited but declined to join the study, therefore we do not know their reasons and whether there was a difference in the characteristics of students who participated and those who did not. While we have tried to make the data collection process more neutral with an independent facilitator for the sessions, the pre-existing student-lecturer relationships between the participants and the researchers might still have influenced the way they responded and expressed their views during the discussions. The participants were in the same clinical year but had their psychiatry posting in several batches over the academic year, thus the time of their last exposure to the learning module varied and they might differ in their ability to recall and share their experiences.
Our findings suggest that the use of virtual patient software in psychiatry training for medical students has both advantages and disadvantages. From the pedagogical point of view, virtual patients the capability to offer a greater level of flexibility in catering to the needs of students at different levels of competency and with diverse learning objectives. At the same time, careful editing of the case study and related learning materials and instructional guides is required to ensure ease of navigation and use, as well as to maintain students’ focus and motivation to learn. A good match between the learning tool (virtual patient software) and the delivery format is crucial. Low technical specifications may improve learners’ access, but further refinement of the program’s features might be required to meet learners’ expectations of enhanced visual and interactive experience. The use of virtual patient software in undergraduate psychiatry education should be further explored.
Below is the link to the electronic supplementary material. Supplementary Material 1
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Dental Service and Resource Needs during COVID-19 among Underserved Populations | 059ee7d4-4192-4aee-b0bb-3cd2920a7267 | 9203663 | Dental[mh] | In Canada, dental care is primarily delivered through the private sector and effectively supports the oral health of the majority of Canadians, but inequalities in oral health and inequitable access to dental services disproportionally affect vulnerable and marginalized populations ( ). Approximately 6% of all dental care expenditures in Canada are publicly funded ( ), and only 5.5% of all Canadians have public dental benefits ( ) while 35.4% do not have dental insurance ( ). Although public dental programs are available for specific target underserved populations, including children living in low-income families, social assistance recipients, people with disabilities, and Aboriginal peoples, they are often limited and insufficient in meeting their needs and other underserved groups, such as low-income adults and older adults, are excluded ( ; ; ). In addition, those eligible for public dental benefits often face complicated insurance-related barriers to accessing dental care such as dental providers’ aversion to treating publicly funded patients due to dental providers’ being dissatisfied with the public plans ( ). Vulnerable populations are defined as having at least 1 interrelated dimension of vulnerability such as reduced individual capacities, lack of support networks, or their community lacking or having barriers to access essential services ( ). One of these services includes dental care. In Canada, these underserved populations who struggle to access dental services include people with low incomes, children in low-income families, people without dental insurance, older adults residing in institutions or with low incomes, Aboriginal peoples, refugees and newcomers, people with disabilities, and people living in rural regions ( ). People living with human immunodeficiency virus (HIV) or AIDS in particular have a stigmatizing, chronic condition that may lead to disability; hence, they are also identified as being vulnerable. Furthermore, the behavior model for vulnerable populations outlines factors that inhibit access to care such as mental health, substance use, competing needs, and incarceration history due to their social structure characteristics ( ). All these above underserved populations continue to face multiple barriers to accessing dental care such as cost, fear and dental phobia, denial of care, stigma, and discrimination that contribute to disproportionately higher rates of untreated dental caries, periodontal diseases, missing teeth, and oral pain, as well as greater dental care needs ( ; ; ; ; ). Substantial research supports that low-income Canadians face financial barriers to accessing dental care that contribute to lower dental care utilization as well as poorer oral health outcomes ( ; ). For people with severe mental illness, factors contributing to oral disease include amotivation, poor oral self-care, dental phobia, dental costs, difficulty accessing dental care, adverse effects of psychotic medications such as oral dryness, and stigma from dental professionals ( ; ). Barriers to accessing dental care among formerly incarcerated people include indifference toward oral health, stigma of incarceration, negative previous dental experiences, restrictions from parole or community supervision, and institutional conditioning ( ). Frequently reported challenges that impede access to dental care for people living with HIV/AIDS include cost, dental anxiety and fear, transportation issues, and dental provider discrimination and stigma ( ). Similarly, older adults face financial barriers, dental anxiety, ageism from dental professionals, reduced expectations of having their dental needs addressed, and transportation challenges ( ). Consequently, these adults with vulnerabilities experience greater oral health inequities when compared to the general Canadian population ( ). The coronavirus disease 2019 (COVID-19) global pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has raised concern among experts over oral health inequalities becoming exacerbated ( ) as dental services were restricted to urgent and emergent during the first wave of infections ( ). Vulnerable and marginalized populations with already limited access to needed dental services ( ; ; ; ; ) were at risk of being further affected by the curtailment of oral health care services. Previous studies have found that when access to oral health care services is limited, individuals may rely on hospital emergency rooms and substance use to cope with or address their oral problems ( ). In the United States, dental service utilization among insured patients decreased during the first wave of SARS-CoV-2 infections and reached the lowest point during the week of April 6 when there was a 94.5% reduction in service utilization compared to the previous year ( ). reported 66% fewer weekly dental visits in the week of April 12, 2020, across the United States. Similarly, a poll of US dentists revealed that in the week of April 20, 2020, 86.0% of respondents reported that their total patient volume was significantly reduced to less than 5% compared to their typical patient volume before the COVID-19 pandemic ( ). After restrictions were eased, dental service utilization began rebounding in the private sector but remained lower within the publicly insured population ( ; ). The full impact of the COVID-19 pandemic on oral health service utilization by underserved populations remains unexplored in British Columbia. This study aimed to: 1); and 2) identify their coping mechanisms employed during the curtailment of oral health care services. These 2 aims are addressed under the following research question: how did the COVID-19 pandemic affect dental services access and needs for underserved populations in British Columbia, Canada, during the first 6 mo of the pandemic?
A transformative research paradigm ( ) under a social justice framework was employed in this qualitative study that involved those who benefited from the research since its inception. This framework provides guidance for research projects collaborating with historically marginalized populations in methodological decision-making and conducting research to promote social justice. Over the past decade, the Faculty of Dentistry at the University of British Columbia has been collaborating with 6 community-based organizations that serve a variety of underserved members of our society to increase access to oral health care professionals within their communities. These populations historically experience structural vulnerabilities that result in reduced access to dental care and higher levels of oral disease ( ). These 6 organizations serve men and women with a history of incarceration and/or experiencing poverty and homelessness, persons living with HIV/AIDS, adults living with mental illness, and older adults in long-term care facilities. In turn, community-based staff and organizers from the 6 partnering organizations aided in designing the interview questions by offering suggestions of questions that would be important to address, providing feedback to ensure the wording was appropriate, and partaking in the initial interviews as participants where they introduced concepts that were incorporated into the interview scripts for their members to later elaborate on. The staff collaborators also offered recruitment recommendations for potential community members who may be interested in participating in the study, although they did not engage in any members’ individual interviews. The collaborators were community partners who we have worked with for many years and who were in administration of service positions for their organizations. Potential participants were recruited to partake in individual interviews to gain insight into their experiences accessing oral health care services and information, as well as their dental needs during the temporary service curtailment and the COVID-19 pandemic. All participants provided informed consent verbally, and 18 participants also provided written consent. Inclusion criteria were those older than 19 y of age and able to communicate in English. Recruitment posters with information on the study details were distributed and advertised at each of the 6 participating community-based organizations. We aimed at purposefully selecting participants with a diverse representation of age, gender, and life experience within and between the different organizations. Although the participants were all affiliated with organizations serving specific targeted underserved populations, there was considerable heterogeneity among the participants. Approval for this study was obtained from the UBC Behavioural Research Ethics Board (BREB #H20-02070-A001). Interviews Data Analysis In-depth, semistructured, at-a-distance individual interviews were conducted with community-based staff or organizers from each community organization and with their members over the phone or via Zoom between July and December 2020. Interview guide questions were framed to identify how community organizers and members were addressing oral health issues as they arise, what barriers and facilitators they were experiencing in accessing needed services, how they were coping with potentially reduced access, how they were coping with any oral problems, what resources they were relying on, and what types of services they would find essential and necessary now and in the future. Two authors (VJ, LD) conducted 7 interviews to calibrate interview conduct, and then the first author (VJ) conducted the remainder of the 24 interviews. Each interview lasted between 30 and 60 min and was audio-recorded. Participants were offered a $25 honorarium for their time devoted to the study.
All interview audio-recordings were transcribed verbatim and coded for emerging themes using NVivo 12 software (QSR International) and thematic analysis. Participants’ names and other identifiers were anonymized. Transcripts were reviewed multiple times by the first author (VJ) for familiarization, and then patterns of meaning within the transcripts were identified and interpreted through coding. Coding is an inductive process whereby a code in the form of a word or phrase is assigned to an excerpt of a transcript that describes the essence of what is being said. Two authors (VJ, LD) individually coded 2 of the same interviews, then compared codes for consistency and reached consensus. The first author proceeded to code 10 transcripts, discussed the developing list of codes with the other two authors (MB, HvB), and coded the remaining 19 transcripts. The analysis led to a total of 121 codes, and then similar codes were grouped into 21 categories that describe an aspect of a shared particular occurrence. Examining the similarities and finding relations between categories gave rise to 4 overarching themes and 12 subthemes that were organized sequentially to explain the process in participant experiences. The researchers employed reflexivity throughout data collection, data analysis, and reporting by continually recognizing their socioeconomic positions that enabled them to avoid personal experiences with discrimination and access to dental care issues, as well as being conscious of the potential power imbalances between researchers and members, researchers and staff, and staff and members. To address these potential biases and power imbalances, the researchers built rapport with the participants and performed active listening during interviews, as well as internally reflected on personal assumptions and values throughout data analysis. To improve trustworthiness and achieve rigor in this study, interviews were conducted until data saturation was reached when no new information was being discovered. After the initial data analysis, the study results were presented to each community organization, and all organizations were invited to provide feedback on the findings. Moreover, all participants were provided with the opportunity to review, clarify, and add further insight to their own personal transcripts and the initial data analysis, although only 1 participant provided feedback on the analysis of their own transcript.
A total of 31 interviews with 13 community-based staff or organizers and 18 members were conducted, resulting in 18 h of audio recordings and 250 pages of written transcripts that were analyzed thematically. A summary of the participant demographic characteristics, including gender, staff or member position at the community organization, and the organization’s target population, is described in the . Four main themes with nine subthemes emerged from the analysis: concerns (COVID-19 anxiety, COVID-19 safety protocols), access to services (dental service needs, disruptions of care), coping (accessing community support or medical services, self-management of dental issues, not dealing with dental issues), and needs and suggested solutions (information, resources).
COVID-19 Anxiety COVID-19 Safety Protocols The COVID-19 pandemic had significantly affected people’s daily lives as public health orders were introduced to reduce the spread of the virus. These disruptions to regular life, the uncertainty amid the novelty of the pandemic, and the potential threat of the virus induced feelings of fear and stress as voiced by the participants: The first few months . . . we were kind of like very intimidated by the whole thing. And I was scared. I was washing my hands so often. . . . And I was afraid of going outside. . . . And I saw on the news people were being bagged up in body bags. . . . Six hundred people at a day dying. . . . I was crying inside but I was like just too freaked out to really even cope with that. (Male member, organization 5) The fear of contracting COVID-19 led some members to avoid or hesitate to access various types of services in order to reduce their risk. As 1 staff participant stated, “The amount of women accessing our [community] service was reduced as well and just limited. I think in light of COVID there may be more of a hesitation to access services. Not just dental but all services” (female staff, organization 2). Members with underlying health issues, such as those with respiratory conditions or HIV/AIDS, were particularly more cautious with reducing possible exposure to COVID-19 since they were considered to be at risk of greater disease severity. One male member from organization 6 told us, I was kind of worried about going into the dental office . . . because I have underlying issues that makes me more susceptible to COVID so . . . I was worried about catching COVID. A female staff from organization 1 similarly expressed concern for the health of their members who were medically compromised: [My member] is such high respiratory risk that she’s not going anywhere . . . she normally travels by handy dart but they’re sort of deeming that still too high risk for her so she’s not going to receive any dental care for the next little while. . . . Because the population that we work with . . . [have] many underlying health conditions and also sometimes addictions . . . I feel like if anybody got sick with this virus unfortunately they would be very sick and they wouldn’t be asymptomatic.
The new safety protocols implemented to prevent and control the transmission of COVID-19 in dental offices include increased screening and enhanced personal protective equipment (PPE), although participants had mixed perspectives on the changes. Concerns about the sufficiency of dental offices’ COVID-19 safety protocols were raised as some participants did not know the level of safety or risk of transmission when accessing dental services. As 1 member indicated, “I don’t know what they have in place right so that makes me very anxious. Was someone sitting in the waiting room, is it contaminated, has it been cleaned, how often does it get cleaned? . . . All that not knowing is a big concern for me” (male member, organization 6). In contrast, other members trusted that dental offices would adhere to safety protocols and standards in order to provide care safely: As a patient I have [no concerns], because I know that the Ministry [of Health] and the dentists and the dental review boards will be the ones that are looking and scrutinizing all the care. . . . I know that if a dentist will look at me that that dentist feels comfortable in the environment that he’s in. . . . So it’s kind of like I’m relying on everyone else to do their job. (Male member, organization 6) Approximately half the staff participants similarly felt comfortable resuming dental services during the pandemic either themselves or for their members since they trusted that the dental professionals would follow appropriate safety protocols. This increased confidence in the safety protocols among staff participants may be attributed to their greater understanding and familiarity with COVID-19 regulations given their own organizations’ protocols: I don’t have concerns because I understand what you’re doing . . . and because we had to initiate some of these [regulations], we’ve been living and breathing COVID for the last four months. So we’re quite familiar with what COVID is all about, how it’s transmitted, the steps that we have to take ensuring our safety and we’ve been successful here and we’ve gone for four months without an issue here. (Male staff, organization 5) The new COVID-19 safety protocols also included raised minimum requirements for PPE worn by dental professionals to improve their safety, including a level 3 surgical mask or higher, gown, and face shield or goggles. However, the enhanced PPE may have caused communication difficulties between members and dental professionals, depersonalized interactions, or triggered increased stress among members with negative past experiences in medical environments. As 1 staff participant indicated, If [members] don’t know what you look like and you’re just wearing a mask and that’s what you see it’s quite intimidating. . . . It’s just hard I think for them especially if you’re uncomfortable already in a medical setting. (Female staff, organization 4) Nonetheless, most members and staff were well adapted and not concerned with the enhanced PPE worn by dental professionals, which reassured safety: “I think [enhanced PPE] would probably make you feel less worried about it if I saw that. Because then I know they would be doing steps to keep themselves safe and myself safe” (female member, organization 2). Over time, the fear of and anxiety for the threat of COVID-19 were decreased by the new regulations and enhanced PPE, as reflected by a female staff: “At the beginning [members] were always asking me for masks because their staff [were] wearing them but they’re a measure to keep them safe. . . . And I think a lot of the anxiety and maybe paranoia around it [like] why do you need to wear them and we don’t has lessened” (female staff, organization 5).
Many medical, dental, and community service providers implemented changes to their regular practices to reduce the risk of transmission of COVID-19. The new COVID-19–related changes and restrictions to services led for the most part to reduced access to services and introduced new disruptions to care, such as delays and denial of care. Dental Service Needs Disruptions of Care ost participants agreed that the dental needs did not change during the pandemic. As 1 male member from organization 6 remarked, “The [dental] needs are the same,” but access to dental services has changed: “it’s just harder to access now” (male staff, organization 1). Many members wanted to continue the same routine dental services as prior to the pandemic or complete previously planned treatments: “I had the cleanings halted. . . . I was supposed to go in before the pandemic but then everything shut down after the pandemic” (male member, organization 6). However, staff participants also emphasized that existing dental problems could progress and worsen during this time when only essential dental services were being provided, potentially increasing the needs: I would say [dental needs] actually got amplified because there were many people that had processes in place up until the shutdown and then all the sudden all the offices were just closed. . . . I mean I know the emergency [dental care] was still taking place but there’s lots of people kind of caught in that little area where it’s not quite enough to be considered an emergency. So you know maybe they’ve had a temporary [filling] now on four or five months rather than you know a few weeks and how long is that going to last. (Male staff, organization 6)
Half of the member participants and three-quarters of the staff participants reported having their dental appointments delayed, cancelled, or rescheduled, which the majority attributed to dental offices being closed or limiting service earlier on. As 1 male staff from organization 6 commented, Everything’s been put on hold because dental offices were closed except those few that remained open for emergency services. I had my own appointments canceled around me because of the closures that were ordered and I’ve yet to get back to seeing anyone yet. Participants also discussed the issue of being an essential worker and accessing care: “Frequently you’re not getting [dental services] because you’re an essential services worker and have a higher risk factor of being exposed to COVID” (male staff, organization 6). This experience may indicate dental offices’ reluctance to provide care to those who were at high risk of exposure to COVID-19 in order to minimize the risk of transmission in their offices. Not being a patient of record was cited as another reason for not being able to access needed care, as we were told that “[members] would phone around and other dentists would say ‘sorry you’re not one of our regular patients so . . . we can’t slip you in as an emergency.’ So I have had people that are suffering with the pain” (female staff, organization 4). Although such reasoning left members without a regular dental home with fewer options during the curtailment period, over one-third of the member participants and the majority of staff participants informed us that they were able to access the services as restrictions eased off. As 1 male member from organization 5 said, “I went to the dentist . . . as soon as they started practicing again.”
Members who did struggle to access needed oral health care services described various coping mechanisms to deal with their dental issues. These coping mechanisms included accessing community support, primary care, or urgent care services, self-management of dental issues, and not dealing with dental issues altogether. Accessing Community Support or Medical Services Self-management of Dental Issues Not Dealing with Dental Issues Altogether When experiencing dental pain and unsuccessfully accessing dental services, some members turned to community staff for support, urgent care, and/or primary care services to address their dental problems. However, community staff similarly struggled to support members with immediate dental needs and could only provide them with reduced-cost dental clinics’ contact information. In addition, primary and urgent care offered services limited to analgesic or antibiotic medication as they could not treat the source of the dental pain: We have had a few members successfully receive emergency dental care but even the emergency dental care was like OK I’ll see you in eleven days. This guy is screaming in pain . . . so we ended up even bringing some dental emergencies to the urgent care center . . . for dental stuff because the pain was so bad. . . . We tried to call that member’s primary care physician [who] said like “Well I can’t prescribe you antibiotics over the phone because I haven’t seen you in over a year” . . . and so then you know that’s a phone thrown at a wall and a door slammed in the face. . . . So then that was when my coworker took that member to the urgent care and he was prescribed antibiotics. . . . In both cases that I knew of it took about two weeks of pain, which is horrible. (Female staff, organization 1)
Members experiencing dental pain without access to dental services during the pandemic also described their reliance on analgesic or antibiotic medications, illicit drugs, and oral products to cope with the suffering: [I took] like a tooth solution . . . it cleans your mouth and it numbs your gum like kill the nerves or whatever and heal the pain. . . . [And I took] Advil and Tylenol like painkillers because you know when you have a toothache you feel pain yeah so basically I took that and it helps. (Male member, organization 1) For others with a history of substance use or addiction, the potential to revert to using substances as a coping mechanism for stress or pain during the pandemic was apparent: Even just like stress coping with all these [COVID-19] rules . . . a lot of people that we work with just have coping tools that they lost . . . or they’re not conducive to living a life that they would like to live. Also like I mean drugs are a great coping tool and especially because they’re immediate but then it contributes to the problem so like how do we move towards healthier coping skills in terms of stress . . . [for] people dealing with like addiction particularly that’s really [a] coping tool. (Female staff, organization 1) Unfortunately, 1 staff participant noted an increase in members expressing that they would perform self-extractions to resolve their dental pain during the pandemic: It’s so painful . . . they do crazy things. I’ve had members pulling out their own teeth because they figured that’s what’s going to help. (Female staff, organization 4)
Some members also indicated that they avoided dealing with dental problems, especially if they were no longer being the source of pain: [My dental pain] started again like after the pandemic started. I was like Oh my God. I’m in so much pain and I couldn’t go to the dentist but it went away. . . . I didn’t go to the dentist again after that . . . I’m like whatever it’s going to stop the pain it’s going to stop the pain it’s going to stop and it actually did stop like it wasn’t painful anymore like there’s no more holes in my mouth. . . . I found it so odd too, it just doesn’t hurt anymore. I’m like what’s going on with my body, but it’s good it wasn’t hurting anymore it went away on its own. (Female member, organization 5) While the above coping mechanisms may temporarily help people manage pain, some may also lead to harm, such as the use of illicit substances. In turn, participants identified resources and services that could help them throughout the COVID-19 pandemic and beyond.
Various resources, information, and services were suggested to be necessary and essential by the members and staff. Information Resources Providing members and staff with appropriate and current information about the pandemic, available dental services, and ways to prevent dental problems would help address expectations and dental concerns, as well as ease anxiety. After the temporary suspension of nonessential dental services, participants discussed the need for reconnecting members with services, so that routine dental services could be reestablished and new treatment could be initiated: [Members] really need the help to reconnect with services. Because [dental needs are] not always prioritized. . . . Information about who is already open, who is more available and likely to do that kind of stuff could be helpful. (Male staff, organization 1) Another suggestion was to prepare members for the COVID-19 safety protocols in dental offices. When discussing preparation for members, 1 male member from organization 6 discussed how “being shown at the appointment or prior to the appointment what the new implementations are” would increase their comfort with resuming dental care. In addition, participants recommended informing members about the safety of receiving dental care, as mentioned by a female staff from organization 1: I think just like access, how to access resources during like while keeping people safe. . . . Information around . . . the risk of transmission and how could it be transmitted through a dental clinic . . . because I think there’s a lot of fear in general. . . . It also helps [staff] fall back on so if people have anxiety it’s like “but these are the facts” . . . rather than letting our brains do the fear mongering.
In addition to information, participants requested necessary resources to support underserved populations throughout the pandemic, including reduced-cost or low-barrier dental clinics when nonessential dental services were temporarily suspended: I think having, especially right now with COVID, again a list of places that they can go to if they need further dental care. Free dental care so like referrals to dentists nearby, not too far. (Female staff, organization 4) Participants also discussed how having a contact point of information to answer dental-related questions would be helpful: Being given the tools to be able to refer somebody, to ask the questions they have, to be given the tools on access. . . . I think having being able to say “Well actually there’s a place you can phone that can give you some basic answers. They’re available to you.” I think that would mean a lot . . . and I think it would change people’s opinions about accessing dental as well. Knowing that somebody actually would listen to them and give them an answer. . . . Having again that dental line or having somebody to contact that’s huge. (Female staff, organization 4) Finally, many participants emphasized the importance of dental providers building rapport and supporting positive, cooperative relationships with their members from underserved populations. This would likely improve member comfort, particularly during the pandemic when greater anxiety was experienced: Just the whole “How are you? How’s your day been?” That level of empathy or sympathy, however it needs to be expressed is something that I think is just definitely necessary for all of us during this [COVID-19] time period. When I’m going into a dental appointment and I see that they have their hazmat suit on and I’m ready to get my dental care done, just the typical before we even start, “How’s your day been” or “Hi how are you” that moment where we can remember we’re still human before we even start anything, I think is definitely [important] for me personally. (Male member, organization 6)
As dental services were temporarily curtailed in British Columbia at the beginning of the COVID-19 pandemic, we aimed to explore the experiences of underserved populations and their community organizations when accessing oral health services and information in British Columbia and to identify the coping mechanisms they employed to deal with oral health care problems and what resources they required during that period. As we posed our research question, “H” we found that the pandemic raised concerns and hesitancy among these populations and further reduced access to care. In turn, those with unmet dental needs resorted to coping mechanisms, including substance use, turning to community support, urgent care, or primary care, and persevering with dental pain. Community organizers and members outlined needed resources such as assistance navigating the dental care system, having a contact for dental-related questions, and member preparation for dental service changes while emphasizing the importance of positive relationships with dental providers. As the COVID-19 pandemic brought significant disruptions to daily lives, many participants felt heightened anxiety that led some to avoid accessing services, particularly those with increased susceptibility to the COVID-19 virus. Increased anxiety and psychological distress related to the COVID-19 pandemic among the general public have been well documented ( ). Moreover, also found that having a history of medical problems has also been associated with greater levels of depression and anxiety during the pandemic, possibly due to feeling more vulnerable to contracting SARS-CoV-2. The fear of contracting the virus led participants in this study to avoid or hesitate to access various services, including dental and medical services similar to other studies ( ; ; ; ; ; ). Those who did try to access dental services during this time might have experienced delayed diagnosis, untreated oral diseases, and compromised health ( ). Although there have been very few documented cases of COVID-19 infection during dental procedures ( ; ), the theoretical association between COVID-19 infection and the production of aerosols indicates a risk of occupational transmission ( ; ). Overall, it is imperative to address public concern about the safety of accessing dental services and the importance of the protocols during the pandemic to facilitate patient return to services. Participants, including those with dental pain, might have been denied dental care due to office standards or not being a patient of record, which may have increased oral infections, periodontal problems, and dental caries during the pandemic ( ). found that the pandemic led to temporary decreases in both urgent and nonemergency patient visits and, like our participants, reinforced the need of maintaining accessibility to dental services for pain-driven urgent dental care. Fortunately, the restrictions on nonessential dental services in British Columbia were implemented only for a few months, and most participants were eventually able to access dental services again. Participants with dental problems who were unable to access to services resorted to coping mechanisms, including accessing community support or medical services, not dealing with the issue, and self-management of dental issues, as reported by others even before the COVID-19 pandemic, although these strategies do not resolve the underlying dental issue ( ). The fact that some members resorted to performing their own extractions is not new and has been documented previously when access to dental care is unavailable and oral pain is significant ( ). A number of resources, information, and services were deemed necessary by participants during the pandemic in our study. However, there is limited evidence beyond commentaries and opinion pieces that explore how to better support underserved populations with access to dental services during the pandemic. as well as echoed the importance of improving communication regarding patient safety as they may feel anxious and hesitate to resume dental services. also added how dental providers must follow safety protocols to improve patient confidence and the patient–provider relationship. Messaging to support patient return to regular dental care should include the significance of maintaining oral health and its impact on systemic health ( ). In addition, highlight the need for dental providers to show genuine empathy to patients, especially given the difficulties many faced during the pandemic. Having a dental professional to turn to during times such as this was discussed by many of our participants, who suggested improving access to needed information in a safe manner, likely via telehealth, as recently presented by when introducing a preparedness model of oral health care. Virtual platforms such as teledentistry have been proposed to address access disparities since it has the potential to increase the reach of dental providers to typically underserved low-socioeconomic status, disadvantaged groups and rural populations ( ; ). Teledentistry strategies can include remote screening, caries detection, diagnosis, consultation, triage, treatment planning, and education ( ; ). During periods when dental offices are closed and people mainly stay at home, information and communication can be delivered through teledentistry to reduce the burden on hospital emergency rooms ( ). The need of virtual resources, including teledentistry for the underserved populations, requires further investigation.
Our study has several limitations, including low level of feedback from member participants, small sample size, and limited representation for underserved populations. The study results were presented to the community organizations, and all staff collaborators agreed with the findings and did not provide additional feedback. The 1 participant who provided feedback on the analysis of their personal transcript agreed with the author’s interpretations of their transcript and provided clarification regarding the number of members they witnessed who expressed intent on performing self-extractions before compared to during the pandemic. The low level of feedback on the study results from participants may be due to several factors, including the long delay between the interviews and the offerings to provide feedback, the voluntary aspect of feedback participation as we specifically requested clarification from the 1 participant who did provide feedback, being unable to contact a few member participants, and the possible trauma or difficult experiences of the participants during the pandemic that they would not want to revisit. For greater participation of underserved populations in member checking, it may be ideal to ask participants during their interview if they would like the opportunity to review their interview transcript and request their contact information to involve them in that process. The findings in this study were derived from a small purposive sample composed of 31 staff and members from 6 organizations in British Columbia, Canada, thereby limiting any generalization. The experiences and perspectives of other underserved populations such as Indigenous communities and people living in rural areas were not captured. As the inclusion criteria included those able to communicate in English, other underserved populations such as newcomers and refugees may not have been represented in this study. In addition, the remote delivery method of the interviews may have decreased rapport with the participants and may have led to missing nonverbal information compared to if the interviews were done in person ( ).
Underserved populations who already face barriers to oral health care services experienced increased difficulty in addressing their oral health needs and concerns during the beginning of the COVID-19 pandemic. Strategies aimed at reaching out to this population and those who support them are needed to help mitigate negative coping strategies and increased oral health disparities. Further investigation into how to reach underserved populations through various forms of virtual applications is warranted.
V. Johnson, contributed to the data acquisition, analysis, and interpretation, drafted and critically revised the manuscript; M. Brondani, contributed to conception, design, and interpretation, critically revised the manuscript; H. von Bergmann, S. Grossman, contributed to design, critically revised the manuscript; L. Donnelly, contributed to conception, design, data acquisition, analysis, and interpretation,.
sj-docx-1-jct-10.1177_23800844221083965 – Supplemental material for Dental Service and Resource Needs during COVID-19 among Underserved Populations Click here for additional data file. Supplemental material, sj-docx-1-jct-10.1177_23800844221083965 for Dental Service and Resource Needs during COVID-19 among Underserved Populations by V. Johnson, M. Brondani, H. von Bergmann, S. Grossman and L. Donnelly in JDR Clinical & Translational Research
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Endometrial Receptivity–Lessons from “Omics” | a6186310-ca27-40d4-b9e3-8f47bec64f31 | 11764156 | Biochemistry[mh] | During the menstrual cycle, the endometrium transitions from a non-receptive to a receptive state through complex molecular and cellular changes, including endometrial remodelling, decidualization of stromal cells, and the recruitment of immune cells such as uterine natural killer (uNK) cells, which collectively establish a tolerogenic environment in preparation for successful embryo implantation . This highly coordinated process occurs transiently during a period of the menstrual cycle known as the “window of implantation” (WOI), typically between days 20 and 24 of the 28-day cycle . Endometrial receptivity (ER) refers to the state of the endometrium during the WOI where the luminal epithelium (LE) plays a critical role as the first point of contact between the maternal endometrium and the embryo . Dysregulation of mechanisms controlling ER may lead to implantation failure and infertility . A comprehensive understanding of ER is essential for development of novel diagnostic tests and therapies for infertility . While bulk omics (e.g., genomics) have revealed key mechanisms regulating ER, single-cell omics, particularly in the area of transcriptomics, are starting to provide a more detailed, high-resolution view of molecular changes on the individual cell level that may lay the foundation for the development of personalized treatments . This review will highlight advances, gaps in knowledge, and future directions from various branches of omics on the endometrium, with emphasis on (1) the regulatory mechanism for ER; (2) the dysregulation of ER in infertility; and (3) the targets for clinical translation. Genomic investigation in the context of ER examines the entire genome, including coding and non-coding regions of DNA. Genome sequencing of the endometrium during the WOI have provided an opportunity to discover Receptivity Associated Genes (RAGs), as well as genetic variants (e.g., single nucleotide polymorphisms—SNPs) that may affect ER and fertility outcomes. 2.1. Genes and Endometrial Receptivity2.2. Genetic Alterations Genetic alterations are changes in the DNA sequence or structure that can affect genes and their expression. These alterations can occur at various levels, including single nucleotides (i.e., SNPs), segments of DNA, entire genes, chromosomes, or as epigenetic modifications. SNPs have been identified in many elements of the complex network that regulate ER. In terms of cell adhesion and signalling, a polymorphism in the human Mucin 1 ( MUC1 ) gene may alter its expression in endometrial epithelial cells, potentially reducing embryo implantation . In terms of endometrial hormonal signalling, SNPs in the progesterone receptor ( PGR ) and estrogen receptor ( ESR1 or ESR2 ) genes may lead to abnormal receptor expression, resulting in abnormal hormone receptor signalling, and ultimately leading to inadequate endometrial preparation. For instance, a polymorphism in human PGR (i.e., +331G/A polymorphisms) has been shown to increase the risk of implantation failure in women undergoing in vitro fertilization (IVF) . In contrast, the impact of a polymorphism on an estrogen receptor in regard to ER remains to be elucidated. Whilst the polymorphism in estrogen receptor 1 ( ESR1 ) may be associated with conditions characterized by abnormal ER (i.e., endometriosis), when assessed in the context of IVF outcomes for infertile women, SNPs in ESR1 provided limited predictive value . With regard to cell cycle regulation and apoptosis, SNPs in the human tumour protein 53 (i.e., TP53 Arg72Pro ) may lead to dysregulation of the genes involved in apoptosis and cellular remodelling, leading to lower implantation success in women undergoing IVF . A number of other SNPs in the human genome, including nuclear factor kappa beta ( NF-kβ ), leukemia inhibitory factor ( LIF ), vascular endothelial growth factor ( VEGF ), VEGF receptor 2 ( VEGFR-2 ), tumour necrosis factor-alpha ( TNF-α ), interleukin-1 beta ( IL-1β ), interleukin-6 ( IL-6 ), and the signal transducer and activator of transcription 3 ( STAT3 ), have been linked to recurrent implantation failure (RIF), potentially causing dysregulation of angiogenesis and cytokine production . A comprehensive resource, the Human Gene Expression Endometrial Receptivity database (HGEx-ERdb), has catalogued 19,285 genes for their expression in the human endometrium. Within this database, 179 genes have been consistently identified as RAGs . The regulation and expression of these genes and their impact on ER is discussed in a subsequent section of this review. Whilst alteration to gene expression can stem from changes in DNA sequences (e.g., SNPs), the field of epigenomics investigates various mechanisms regulating gene expression without altering the underlying DNA sequence. Key epigenetic mechanisms include DNA methylation and histone modification . Aberrant DNA methylation and histone modification may disrupt the expression of genes essential for ER . 3.1. DNA Methylation and Demethylation3.2. Histone ModificationStable DNA methylation requires a balance between methylation and demethylation processes. DNA methyltransferases (DNMTs) methylate DNA by adding methyl groups to DNA. In contrast, DNA demethylation is facilitated by ten-eleven translocation (TET) enzymes that remove or modify methyl groups from DNA . Alternatively, passive demethylation may occur if a maintenance DNMT (i.e., DNMT1) is inactive or downregulated . During early embryonic development, nearly all methyl marks in the embryonic genome are removed in a process known as global DNA demethylation that is facilitated by both active and passive demethylation . Following this event, a new pattern of DNA methylation is re-established by DNMT3A and DNMT3B (de novo DNMTs) during embryo implantation . After cell fate specification is completed during embryogenesis, the DNA methylation patterns established by DNMT3A/B are maintained by DNMT1 during subsequent cell divisions . The expression of de novo DNMTs (i.e., DNMT3A/B) rather than a maintenance DNMT (i.e., DNMT1) varied across the menstrual cycle, suggesting that DNA methylation in the human endometrium is dynamic, correlating to the dynamic expression of genes vital to ER . Furthermore, the DNA methylation patterns in the human endometrium may be influenced by the menstrual cycle phases where the late secretory phase and menstrual phase showed the most pronounced changes in terms of methylation profiles . Genome-wide DNA methylation profiling of the human endometrium indicated that, while the overall methylome remained relatively stable during the transition from the pre-receptive to the receptive phase, 5% of the CpG sites showed differential methylation during this transition. Methylation changes affected pathways in extracellular matrix (ECM) organization, immune response, angiogenesis, and cell adhesion, highlighting the nuanced role of epigenetic regulation in preparing the endometrium for implantation . Differential methylation was observed in genes associated with endometrial function and implantation, such as Transforming Growth Factor Beta 3 ( TGFB3 ), Vascular Cell Adhesion Molecule 1 ( VCAM1 ), and C-X-C Motif Chemokine Ligand 13 ( CXCL13 ) . Aberrant DNA methylation patterns during the WOI may lead to impaired ER, contributing to implantation failure and infertility. Whilst we have not identified any investigations exploring the global DNA methylation patterns in women with RIF, altered DNA methylation of ER-related genes, such as the Homeobox A10 ( HOXA10 ) gene, have been reported in the eutopic endometrium of women with endometriosis . Reduced endometrial epithelial and stromal HOXA10 expression during the luteal phase have also been reported in a number of other conditions associated with infertility, such as RIF, recurrent miscarriage, and endometriosis . From human and animal studies, hypermethylation of the promoter and surrounding regions of the Hoxa10/HOXA10 may reduce its expression, thereby implicating abnormal methylation in infertility. Specific levels of DNA methylation have been reported although the range of levels is broad, limiting its utility in disease prediction. For instance, the mean methylation rate of HOXA10 in the eutopic endometrium from women with endometriosis varied between 4 and 70% depending on the gene regions analyzed; F1-3 regions of the HOXA10 promoter, as well as the 5′untranslated region (5′UTR) . Hypermethylation Hoxa10/HOXA10 have also been observed in animal models of endometriosis. In a baboon model of surgically induced endometriosis, complete and partial methylation in the F1 region of the HOXA10 promoter was found in the eutopic endometrium . Similarly, partial methylation of 10.7% in the promoter region of Hoxa10 has been reported in the eutopic endometrium of a mouse endometriosis model. Collectively, data from human and animal models suggest that even a relatively low level of methylation in the promoter region of Hoxa10/HOXA10 is abnormal and sufficient for disrupting normal gene expression . As TET enzymes play a role in maintaining the balance of DNA methylation and demethylation during embryonic development and cellular differentiation, dysregulation of these enzymes (e.g., TET1) has been associated with the hypermethylation of DNA in various diseases . A mid-secretory-phase reduction in TET1 mRNA expression in the eutopic endometrium of infertile women with endometriosis may be a potential mechanism for the hypermethylation of HOXA10 . Interestingly, mid-secretory-phase upregulation of TET3 mRNA expression in the eutopic endometrium of infertile women with endometriosis has also been reported . In a recent study, using a human endometrial stromal cell (ESC) model, Liu et al. demonstrated that elevated TET3 levels in miR-29a-inhibited ESCs increased demethylation of the Collagen type 1 alpha 1 chain (Col1A1) promoter, thereby increasing the Col1A1 expression that ultimately impaired the in vitro decidualization of ESCs and reduced embryo implantation rates in a mouse model . Collectively, these studies highlight the importance of TET in maintaining normal methylation balance in the context of ER. In the context of these epigenomic insights, a recent study aimed to determine whether DNA methylation patterns of genes associated with the WOI in cervical secretions can predict ongoing pregnancy outcomes in patients undergoing in vitro fertilization-embryo transfer (IVF-ET) . Out of 158 genes, 15 differentially methylated probes in 14 genes were identified as important to ongoing pregnancy. These include Serpin Family E Member 1 ( SERPINE1 ), Serpin Family E Member 2 ( SERPINE2 ), and Transgelin-2 ( TAGLN2I ), which showed consistent methylation patterns associated with pregnancy. Validation of the three genes in an independent sample set showed consistent methylation differences between groups (i.e., ongoing pregnancy and no pregnancy). Machine learning models using the methylation data effectively classified these patterns with high predictive power (i.e., AUC ranging from 86 to 91%). A non-invasive methylation analysis of cervical secretions may serve as a diagnostic tool to predict ER and improve IVF success rates. Further validation with larger datasets is required to confirm these findings and refine the prediction models. Histone modification refers to a broad category of chemical changes made to histone proteins that regulate chromatin structure and gene expression. For the purpose of this review, we will focus on histone acetylation and its role in ER. Histone acetylation is a type of histone modification that involves the addition of an acetyl group to lysine residues located on the protruding tails of histones . This type of modification is typically linked to transcriptional activation and is regulated by two opposing enzyme groups: histone acetyltransferases (HATs), which add acetyl groups, and histone deacetylases (HDACs), which remove them . Histone acetylation varies during menstrual cycle. For example, a significant increase in Histone H4 acetylation at lysine 8 (H4K8ac) expression in the human endometrium was observed during the receptive phase, correlating with differentiation and preparation for implantation . These acetylation changes may be under hormonal control, as demonstrated by a recent retrospective cohort study using archived endometrial samples from 40 women (20 with high progesterone levels and 20 with normal progesterone levels on the day of hCG administration). In this study, histone acetylation was assessed via immunohistochemistry, which showed an increase in Histone H3 acetylation at lysine 9 (H3K9ac) expression in the human glandular epithelium in the high-progesterone group . In addition to histone acetylation expression in the endometrium, a recent study explored the regulatory mechanisms behind Histone H3 acetylated at lysine 27 (H3K27ac)-mediated gene activation during decidualization, specifically focusing on its role in regulating the Insulin-Like Growth Factor Binding Protein 1 ( IGFBP-1 ) gene in human ESCs. Tamura et al. first isolated and induced decidualization in human ESCs, then performed Chromatin Immunoprecipitation Sequencing (ChIP-seq) and ChIP-qPCR to assess H3K27ac levels and transcription factor recruitment . Decidualization of human ESCs induced a significant increase in H3K27ac levels at the distal promoter of the decidualization marker IGFBP-1, ultimately leading to upregulation of its expression . Using a similar cell model and also performing ChIP-seq, Katoh et al. further demonstrated how upregulation of key genes (e.g., WNT4 ) in decidualization requires not only an increase in histone acetylation (e.g., H3K27c) at promoter regions of a gene but also removal of histone methylations, such as the trimethylation of lysine 27 on histone H3 (H3k27me3), to allow for transcriptional activation . These findings suggest that the resolution of repressive methylation is just as important as deposition of activating acetylation for gene expression. While we have not identified any direct evidence linking H3K27ac specifically to endometriosis and RIF, the role of H3K27ac in decidualization suggests that aberrations in this histone modification could potentially contribute to implantation failures. Further research is needed to understand the role of H3K27ac in various disease phenotypes associated with infertility. While both HDACs and histone methylation can contribute to gene silencing, HDACs primarily achieve gene repression by deacetylation, which condenses chromatin and directly blocks transcription. Studies have shown that HDAC1, HDAC2, and HDAC3 mRNA were consistently expressed in the human endometrium without significant cyclical variation . However, HDAC2 protein levels exhibited a slight but significant increase during the secretory phase compared to the early proliferative phase. In contrast, HDAC1 and HDAC3 protein levels remained relatively constant throughout the cycle . The observed increase in HDAC2 protein during the secretory phase may be associated with endometrial preparation for potential embryo implantation. Aberrant histone acetylation and HDAC expression have been implicated in implantation failure and endometriosis . HDAC1/2 gene overexpression has been reported in endometriotic lesions compared to normal human endometrial tissue. HDAC1/2 proteins were expressed in both diseased and control tissues, with stronger staining being observed in endometriotic lesions . The findings suggest that dysregulation of HDAC expression in endometriotic cells may lead to hypoacetylation and abnormal gene silencing, contributing to pathogenesis. A subsequent study showed that endometriotic lesions exhibited a global hypoacetylation of histone H3but not H4 compared to controls; specific lysine residues (H3K9 and H4K16) were significantly hypoacetylated in endometriotic lesions . Furthermore, Monteiro et al. observed hypoacetylation at the promoter regions of genes (e.g., HOXA10 ) previously also implicated in ER . Despite the theoretical potential, there are currently no clinical trials or approved treatments involving HAT modulators for infertility . The complexity of epigenetic regulation and the challenges in developing specific and safe HAT modulators have limited progress in this area. HDAC inhibitors (HDACis) such as trichostatin A (TSA) have been reported to enhance decidualization in human endometrial stromal cells by increasing histone acetylation and upregulating decidualization markers . There are no registered clinical trials investigating HDACis specifically for the treatment of female infertility. Preclinical research suggests the potential therapeutic applications of HDACis in endometriosis; however, randomized clinical trials (RCTs) are essential to confirm the safety and efficacy before clinical implementation . Other forms of histone modifications that have been implicated in abnormal ER and are potentially amenable to modulation include histone ubiquitination and lactylation. For instance, inhibition of the monoubiquitination of histone H2A (H2AK119ub1) disrupted decidualization and resulted in pregnancy failure in a mouse model . Similarly, a reduction in lactylation of H3K18 (H3K18la) was associated with pregnancy failure in a sheep model . Collectively, these studies highlight the complex roles histone modifications play in orchestrating ER and the need for future investigations to better understand their implication in human infertility. A transcriptomic investigation of ER examines the complete set of RNA transcripts, including messenger RNA (mRNA) and non-coding RNA (ncRNA) expressed during the WOI. While the mRNA expression ER-related genes have been identified and extensively studied, post-transcriptional regulation of mRNA has become an area of intense research in recent years. ncRNA such as microRNA (miRNA) and long non-coding RNA (lncRNA) have been shown to play a key role in post-transcriptional regulation of the expression of genes involved in a variety of cellular process essential for ER . 4.1. messengerRNA4.2. microRNA4.3. Long Non-Coding RNA4.4. Single-Cell and Spatial Transcriptomics Single-cell and spatial transcriptomics have provided us with new tools to investigate the molecular mechanisms regulating ER. By analyzing gene expression at the individual cell level, researchers have identified molecular maps of the endometrium during the WOI. Two recent studies have utilized single-cell RNA sequencing (scRNA-seq) to compare endometrial samples from women with RIF and those from fertile women . Both studies have shown that scRNA-seq is superior to traditional bulk RNA sequencing because it allows for the analysis of cell-specific expression (e.g., disruptions to estrogen and progesterone signalling in epithelial cells), cell–cell interactions (e.g., WNT5A-SFRP4 signalling between stromal and epithelial cells), gene expression in rare subpopulations of cells (e.g., natural killer cell subsets such as CD49a + CXR4+ cells), and gene expression within a specific cell population over time . Furthermore, a recent study combined scRNA-seq with a spatial transcriptomics approach to characterize the human endometrium . This combined approach allowed the analysis of cell specific gene expression to be mapped to defined spatial regions within the endometrium. Overall, these studies demonstrated novel approaches to explore cell behaviour in complex and heterogenous tissue, like the endometrium, that traditional bulk methods could not achieve. Of the 179 RAGS identified from the HGEx-ERdb, 151 were consistently upregulated during the receptive phase . Since this earlier study, we have found that the number of genes implicated in ER varies across studies, reflecting the complexity of this biological process. Transcriptomic analyses have identified between 107 and 2878 genes that are differentially expressed when comparing pre-receptive and receptive phases of the human endometrium . These genomic insights have paved the way for advanced diagnostic tools and personalized strategies to improve implantation success. One such innovation is Endometrial Receptivity Analysis (ERA), a genomic diagnostic test that evaluates the expression of 238 genes to determine the ER status. By pinpointing the ideal timing for embryo transfer (ET), ERA may enhance the success rates of assisted reproductive technologies (ART), offering hope to individuals facing implantation challenges . Despite the promise of ERA, current evidence is insufficient for supporting its routine clinical application . More recently, a systematic review was performed which included studies that investigated ERA-guided euploid embryo transfer cycles focusing on live birth rate (LBR), ongoing pregnancy rate (OPR), implantation rate (IR), clinical pregnancy rate (CPR), biochemical pregnancy loss rate (BPLR), and miscarriage rate (MR). The review included 11 studies with 7581 patients, of which 1663 underwent ERA. The authors found that ERA did not significantly improve reproductive outcomes, such as LBR, OPR, or CPR, in either the general infertile population or patients with a history of RIF . miRNAs are small, non-coding RNA molecules, typically 20–24 nucleotides long, that regulate gene expression at the post-transcriptional level. They function by binding to complementary sequences in the 3′ untranslated regions (3′-UTRs) of mRNAs, leading to either mRNA degradation or translational repression. A number of miRNA and their respective targets, as well as putative roles in ER, have been identified in recent years. provides some key examples. Earlier studies demonstrated that miR-30b and miR-30d are upregulated in the receptive endometrium whilst miR-494 and miR-923 are downregulated in the receptive endometrium . By comparing miRNA target predictions with previous mRNA microarray data, 12 genes were identified as potentially significant for ER, including Calpastatin ( CAST ), Cystic Fibrosis Transmembrane Conductance Regulator ( CFTR ), Fibroblast Growth Factor Receptor 2 ( FGR2 ), and LIF . A more recent meta-analysis of transcriptomic studies has validated 19 miRNAs (e.g., members from the miR-30, and miR-200) with 11 corresponding upregulated meta-signature genes involved in ER . From animal model and human cell line experiments, miRNAs have been shown to regulate RAGs by different mechanisms (e.g., overexpression) across different cell types (e.g., endometrial stromal cells) involved in ER. A cellular transition process that is critical to ER is mesenchymal–epithelial transition (MET), where stromal cells adopt an epithelial phenotype characterized by improved adhesion and polarity. During decidualization, maternal endometrial stromal cells undergo a MET to support embryo implantation. Using both mouse and human stromal cell models, Jimenez et al. showed that, during decidualization of endometrial stromal cells, the miR-200 family upregulates epithelial markers and downregulates Zinc finger E-box-binding homeobox 1/2 ( ZEB1/2 ), promoting MET. Conversely, inhibition of miR-200 maintains ZEB1/2 expression, preventing MET . Similarly, using both the mouse in vivo model and a human epithelial cell model, upregulation of miR-494-3p was shown to impair ER by targeting LIF and modulating the PI3K/AKT/mTOR pathway. . Furthermore, the regulation of ER is not confined to the endometrium alone. Vilella et al. demonstrated that Hsa-miR-30d was significantly upregulated during the WOI compared to other menstrual phases . miR-30d was secreted by endometrial epithelial cells primarily as an exosome-associated molecule. Mouse embryos internalized both free and exosome-associated miR-30d through the trophectoderm, which led an increased expression of genes involved in cell adhesion (e.g., Integrin subunit beta 3). Embryos treated with hsa-miR-30d exhibited a significant increase in adhesion to the endometrial epithelial cell monolayer compared to control embryos . Aberrant miRNA expression has been linked to a number of conditions associated with endometrium-related infertility, including RIF, endometriosis, and polycystic ovarian syndrome (PCOS) . For instance, downregulation of miR-9 and miR-34 families in the eutopic endometrium of women with endometriosis may delay progesterone-induced shifts to secretory phase endometrium by maintaining the persistent expression of anti-apoptotic (e.g., BCL2) and proliferation genes (e.g., Cyclin E2), thereby impairing ER . Similarly, downregulation of other miRNA families has been implicated in the dysregulation of ER. A recent study analyzing endometrial samples from RIF patients showed that the downregulation of miR-30d-5p corresponded to an elevated level of the suppressor of cytokine signalling 1 (SOCS1—an inhibitor of LIF) expression and reduced the expression of LIF and p-STAT3 (markers critical for ER and implantation) . While no miRNA therapy is currently available, miRNA-based classifiers using miRNA expression profiles from 200 IVF patients have been created to identify WOIs to assist in identifying the optimal time for ET in IVF cycles. The classifier is based on 21 miRNAs that were differentially expressed across three time points after progesterone administration (e.g., 108 ± 5 h). These miRNAs were linked to critical pathways in ER and embryo implantation. The classifier demonstrated high accuracy (93.9% training set, 88.5% testing set) in identifying the optimal WOI for ET . miRNAs are stable and robust biomarkers that may serve as alternatives to traditional mRNA-based receptivity assays (e.g., ERA). Using the miRNA-based classifier for WOI determination may improve IVF outcomes. This approach has the potential to be expanded to study other endometrium-related infertility conditions such as RIF and endometriosis. lncRNAs are transcripts longer than 200 nucleotides that do not code for proteins but regulate gene expression at various levels . Unlike miRNA, lncRNAs do not exclusively regulate gene expression at the post-transcriptional level, and they are capable of also regulating gene expression at the pre-transcriptional, transcriptional, and post-translational stages. One well-known mechanism of action of lcnRNA relevant to ER regulation is its ability to act as a molecular sponge, sequestering miRNAs, thereby preventing the inhibition of target mRNA expression . For example, lncRNA H19 can inhibit the activity of miRNA let-7 by acting as a sponge/decoy or competitive endogenous RNA (ceRNA) ( A–C). This inhibition prevents let-7 from downregulating its target genes, such as integrin β3 ( ITGB3 ), thereby reducing adhesion and the invasive ability of trophoblastic cells . While previous studies have shown that downregulation of H19 lncRNA and ITGB3 in the human endometrium is associated with unexplained pregnancy loss and RIF, the exact role of H19/let-7/ITGB3 axis in unexplained pregnancy and RIF remains to be determined . While lncRNA can inhibit miRNA expression, reciprocal inhibition has also been reported ( D). Overexpression of miR-200c suppressed the expression of lncRNA MALAT1 in endometrioid endometrial cancers, subsequently downregulating the expressions of mesenchymal proteins (e.g., Vimentin) essential for epithelial–mesenchymal transition (EMT), a critical feature of tumour progression and metastasis . While dysregulation of this MALAT1-miR200c axis has been shown in endometrial cancer, its impact on ER and embryo implantation remains to be determined. While the mechanisms by which lncRNA regulates miRNA is gaining clarity, the factors that regulate lncRNA in endometrial epithelial cells remain elusive. Takamura et al. exposed human endometrial epithelial cells (HEECs) to blastocyst-conditioned media (BCM) from embryos that either implanted or failed to implant after IVF to investigate how a lncRNA—Phosphatase and Tensin Homolog Pseudogene 1 (PTENP1) regulates HEECs’ adhesive capacity . The authors found that PTENP1 was upregulated in HEECs exposed to BCM from successfully implanted embryos, by contrast silencing PTENP1 significantly decreased HEEC adhesion to trophoblast spheroids . The study highlighted that PTENP1 may be an important regulator of ER and that embryos may modulate ER through secreted factors that fine-tune cellular and molecular pathways. More recently, Huang et al. explored the molecular mechanisms of RIF by constructing a ceRNA network to identify potential hub genes linked to poor ER. Analysis of the GSE111974 dataset revealed 1500 upregulated mRNAs and three lncRNAs, and 1022 downregulated mRNAs and four lncRNAs in RIF samples compared to controls. The ceRNA network pinpointed five hub genes, such as Gap Junction Alpha-1 ( GJA1 ). While the hub genes themselves are mRNAs, the constructed lncRNA-miRNA-mRNA ceRNA networks indicate that specific lncRNAs interact with these hub genes indirectly through competitive binding with shared miRNAs. These lncRNAs may regulate the expression of the hub genes and influence the pathways involved in RIF . Key hub lncRNA that were differentially expressed in the endometrium of women with RIF compared to control include prostate androgen-regulated transcript 1 (PART1), microRNA-17 Host Gene (MIR17HG), H19, and Long Intergenic Non-Protein Coding RNA 173 (LINC00173) which may play a role in cell adhesion and motility . Lin et al. identified eight other lncRNAs that may be unique to women with RIF, highlighting their involvement in ER . Furthermore, three candidate drugs (miconazole, terfenadine, and STOCK1N-35215) were proposed for targeting the ceRNA networks associated with RIF . Nuclear Paraspeckle Assembly Transcript 1 (NEAT1) is another lncRNA that has been implicated in RIF. NEAT1 binds to CCCTC-binding factor (CTCF) to suppress HOXA10 expression via histone modification, which may impair endometrial epithelial cell proliferation and receptivity . In contrast, downregulation of NEAT1 may enhance ER . Several other lncRNA important in endometrial stromal cell decidualization have been implicated in RIF in recent years, including Long Intergenic Non-Protein Coding RNA 2190 (LINC02190) , HOXA11 Antisense RNA (HOXA11-AS) , and Lung Cancer-Associated Transcript 1 (LUCAT1) . Furthermore, Long Intergenic Non-Protein Coding RNA 1960–201 (LINC01960–201) is another lncRNA that has been implicated in the decidualization of endometrial stromal cells in women with endometriosis-associated infertility during the WOI. Abnormal expression of LINC01960-201 may disrupt the LINC01960-201/ADAMTS7/miR-608 axis, impairing ER and potentially leading to recurrent miscarriage. . Collectively, these findings highlight the complex regulatory roles of lncRNAs in ER and their potential as therapeutic targets. Proteomics analysis involves studying the entire protein complement of a cell, tissue, or organism, including protein expression, structure, interactions, and modifications. The most commonly used method in proteomics is mass spectrometry (MS), often coupled with liquid chromatography (LC-MS/MS) . This combination is considered the gold standard in proteomics due to its versatility, sensitivity, and ability to provide comprehensive insights into protein identification, quantification, and characterization. Proteomic analysis can be performed on endometrial biopsies and endometrial fluid aspirate (EFA). The first study on endometrial fluid protein content was conducted by Beier and Beier-Hellwig in 1998 . In recent years, EFA has become more common due to the distinct advantage it has over endometrial biopsies, as it is associated with less discomfort and risk for patients and better represents the local uterine environment leading up to implantation. However, a significant limitation to using EFA is the lack of standardization in collection methods and timing. Standardization may help to reduce variability in sample quality and biomolecule concentration in order to improve the reliability of proteomic analyses and facilitate the identification of potential biomarkers for fertility-related disorders. By analyzing endometrial tissue, Chen et al. identified 196 proteins differentially expressed from the endometrium during the mid-proliferative (MP) versus mid-secretory (MS) phase . Differentially expressed proteins identified were linked to biological processes essential for ER, such as cell structure, motility, immunity, and developmental processes. Specific pathways, including c-Jun N-terminal kinase (Jnk) signalling and Epidermal Growth Factor (EGF) signalling, were enriched during the MS phase, highlighting their role in preparing the endometrium for implantation . Examples of validated MS-phase endometrial epithelial proteins that may play a role in ER and implantation include Rho GDP-dissociation inhibitor alpha (Rho-GDIα) and Chloride Intracellular Channel Protein 1 (CLIC1) . Similarly, proteins were differentially expressed in EFA during MP compared to the MS phase of the menstrual cycle. More importantly, EFA proteins were differentially expressed between fertile and infertile women, highlighting that EFA proteins are linked to receptivity and fertility. Key endometrial epithelial proteins, such as antithrombin III and alpha-2-macroglobulin, were implicated in implantation . Another study profiled the endometrial secretome using EFA collected during the luteal phase. The authors identified 82 proteins differentially expressed between prereceptive (LH+4) and receptive (LH+9) phases . Proteins upregulated in the receptive phase (LH+9) were primarily involved in host defence and immune responses, while downregulated proteins were linked to stress response and cell structure. Fourteen proteins also showed evidence of altered post-translational modifications, highlighting the complexity of proteomic regulation during the WOI . More recently, Kasvandik et al. compared the proteomic landscape of EFA obtained from fertile women and women with RIF. The authors found that, in women with RIF, 21 proteins showed expression levels similar to the early secretory phase in fertile controls, indicating a displacement of the WOI . Furthermore, a 4-protein panel consisting of Progesterone Reception (PGR), Nicotinamide N-Methyltransferase (NNMT), Solute Carrier Family 26 Member 2 (SLC26A2), and Lipocalin 2 (LCN2) demonstrated high specificity and sensitivity (91.7% and 96.6%) for distinguishing between receptive and non-receptive endometrium, a model that, as such, may help identify the receptive endometrium and optimize the timing for ET in IVF cycles . A number of other approaches have been developed to evaluate the proteome of the receptive endometrium. One recent study identified 82 proteins relevant to the implantation process from extracellular vesicles (EVs) secreted by human endometrial epithelial cells. Key proteins linked to the process of implantation include Annexins (ANXA2, ANXA4, ANXA5), Integrins (ITGA1, ITGA2, ITGAV, ITGB1), and Mucins (MUC1, MUC4, MUC16) . Another approach utilized isobaric tags for relative and absolute quantitation (iTRAQ—a multiplex approach used in quantitative proteomics) to analyze pre-receptive (LH+2) and receptive (LH+7) endometrial tissue . The authors identified 173 differentially expressed proteins with integrated analyses, highlighting five hub proteins, the most significant being Acyl-CoA Synthetase Long Chain Family Member 4 (ACSL4), which was increased in the receptive phase and reduced in patients with RIF . Knockdown of ACSL4 reduced the expression of key ER markers such as HOXA10, LIF, and Cyclooxygenase (COX) and impaired the adhesive capacity of epithelial cells in vitro . Using a variety of the aforementioned methodologies, such as iTRAQ and LC-MS/MS, a number of studies investigating receptive phase endometrial tissue and fluid have consistently identified differential protein expression between fertile women and women with RIF. Notably, Annexin-6 (ANXA6), PGR, and metalloproteinase-2 and 9 were differentially expressed in the endometrium of women with RIF . Proteins such as Mucin 16 (MUC16, also known as CA125), glycogen phosphorylase B (PYGB), and Tubulin Polymerization-Promoting Protein Family Member 3 (TPPP3) were identified as potential biomarkers for assessing ER and predicting IVF outcomes . Collectively, these proteomic insights are crucial for developing diagnostic tools and therapeutic strategies aimed at improving implantation success rates. Lipidomics is the comprehensive study of lipids within biological systems. Lipid mediators secreted by the endometrium include triglycerides and eicosanoids such as prostaglandins, thromboxanes, leukotrienes, endocannabinoids, and sphingolipids . Among these, endocannabinoids, lysophosphatidic acid (LPA), and prostaglandins (PG) have been investigated extensively. Two key endocannabinoids involved in implantation are anandamide (N-arachidonoyl ethanolamine, AEA) and 2-arachidonoylglycerol. Abnormal levels of these lipids are associated with delayed implantation and poor pregnancy outcomes in a rodent model . In another animal study, high uterine fatty acid amide hydrolase (FAAH) expression reduced AEA levels, creating a favourable environment for embryo implantation . Conversely, low FAAH activity results in elevated AEA concentrations, which can hinder implantation . By comparing plasma anandamide (AEA) levels and components of endocannabinoid system (ECS) between viable and non-viable first-trimester pregnancies in asymptomatic women presenting for early pregnancy ultrasound, plasma AEA levels were significantly higher in non-viable pregnancies compared to viable pregnancies. However, non-viable pregnancies also showed increased FAAH expression in trophoblasts and decidua in contrast to the low FAAH activity seen in rodent model, possibly as a compensatory response to elevated AEA . A number of differences may account for the discrepancy between the studies, including localized versus systemic effects, sampling timing (i.e., pre-implantation in animal model), differences in species, and the measuring of FAAH activity versus expression. These differences highlight the complexity of endocannabinoid regulation in early pregnancy and suggest that AEA-FAAH dynamics may operate differently in viable vs. non-viable pregnancies. Future studies should aim to measure AEA levels, FAAH expression, and enzymatic activity at both local (uterine) and systemic levels to reconcile these findings. Lysophosphatidic acid (LPA) is a water-soluble phospholipid and a signalling molecule with diverse functions across organs including regulation of FAAH expression and activity . In earlier mice studies, LPA3 signalling was shown to regulate prostaglandin biosynthesis via cyclooxygenase-2 (COX-2) to ensure proper implantation timing and embryo spacing . Subsequent animal studies also demonstrated that LPA enhanced COX-2 expression and prostaglandin E2 (PGE2) production, as well as increased FAAH expression and activity, thereby reducing endocannabinoid levels (i.e., AEA) which are harmful at high concentrations during implantation . LPA also increased markers of decidualization (e.g., Insulin-Like Growth Factor Binding Protein 1) and vascularization (interleukin-10), highlighting its role in endometrial preparation . LPA3 mRNA and protein levels were significantly higher in the early and late secretory phases compared to the proliferative and mid-secretory phases. LPA3 protein was localized in the cytoplasm of luminal and glandular epithelial cells during the early secretory phase. Elevated LPA3 expression in the early secretory phase may contribute to endometrial preparation by influencing COX-2, MMPs, and collagen production . By comparing the expression of enzymes and signalling molecules involved in prostaglandin synthesis in endometrial biopsies obtained during the WOI from patients with RIF undergoing IVF versus fertile controls, Achache et al. found evidence of impairment in the prostaglandin synthesis pathway (e.g., significant reduction in COX-2) and LPA3 downregulation in the secretory endometrium . In contrast, increased levels of PGE2 and PGF2α were identified in the EFA collected during the WOI . More recent studies have identified lipid species and metabolic pathways associated with successful implantation as well as biomarkers to predict ER. For instance, Matorras et al. profiled the entire lipidome from EFA collected from women undergoing IVF to identify lipids that were important in implantation . Eight lipid species were significantly altered in cycles without implantation, including seven glycerophospholipids (e.g., lysophosphatidylethanolamines—LPE and phosphatidylcholines—PC) and one omega-6 polyunsaturated fatty acid (docosapentaenoic acid). Cycles without implantations showed lower levels of certain lipids, such as LPE (20:5), while others like PC (40:8) were higher. Altered lipid profiles likely reflect changes in metabolic pathways related to inflammation, membrane composition, and nutrient availability in the endometrium. A support vector machine (SVM) algorithm using the eight significant lipid metabolites achieved an AUC of 89.3% . In contrast, another study evaluated lipid ion ratios in EFA as non-invasive biomarkers for assessing ER and distinguishing between receptive (positive pregnancy outcome) and non-receptive (implantation failure) cycles . The authors identified 13 lipid ion ratios were significantly elevated in the non-receptive group and identified phosphatidylethanolamines, diacylglycerols, and sphingolipids as key contributors to non-receptivity. The findings suggest that steroid metabolism and LDL remodelling play a role in ER. Furthermore, Braga et al. demonstrated that lipid ion ratios can be used as an non-invasive prediction model in freeze-all cycles with an AUC of 84% . Collectively, by analyzing lipid signatures, clinicians may better determine the WOI for ET in IVF cycles to improve implantation outcomes. Metabolomics is the comprehensive analysis of metabolites within biological systems. Whilst some evidence suggests that the metabolomics of ECM could predict embryo viability and implantation rates better than traditional morphology-based assessments, a recent systematic analysis found that RCTs using a metabolomics approach did not show a significant impact on key outcomes such as CPR or LBR . Currently, metabolomics is not supported for clinical use in terms of improving fertility outcomes. However, by examining the metabolic profiles from endometrial tissues or serum samples, researchers are beginning to identify specific metabolites and pathways that may be important in ER and implantation. In a recent study, an untargeted metabolomic profiling of endometrial samples was conducted using ultra-high-performance liquid chromatography-tandem mass spectroscopy (UPLC-MS/MS) to identify associations between metabolomes and causes of infertility . Endometrial samples were collected from women diagnosed with infertility, categorized based on etiology such as endometriosis, RIF, unexplained infertility, or male factor infertility. A total of 925 metabolites were identified, with lipids being the most abundant, especially polyunsaturated fatty acids (PUFAs) like linoleate and linolenate . Women with endometriosis or RIF exhibited lower levels of PUFAs compared to those with male factor or unexplained infertility. The study highlighted the potential role of PUFAs in ER and contribution it may have in the pathogenesis of endometriosis and RIF. In contrast, Zheng et al. aimed to discover metabolites involved in endometrial transformation and RIF from serum samples . Serum was collected from women undergoing hormonal replacement therapy (HRT)-frozen embryo transfer (FET) cycles on the day of progesterone administration and on the third day of progesterone administration. Controls include 19 RIF patients and 19 controls. Gas chromatography–mass spectrometry (GC-MS) was utilized for the metabolomic analysis. A total of 105 serum metabolites were identified initially, with significant reductions in 76 during the initial 3 days of endometrial transformation, which was associated with reduced amino acid metabolism and suppression of the tricarboxylic acid (TCA) cycle. The reduced metabolic activity was associated with lower serum levels of amino acids (e.g., glutamic acid, ornithine, proline) during endometrial transformation, particularly in patients with RIF . In terms of potential clinical applications, eight metabolites (e.g., indol-3-propionic acid) were highly predictive of RIF, with a strong discriminative ability between controls and RIF patients (i.e., AUC > 70% for these metabolites) . These findings suggest that serum metabolite profiling could serve as a non-invasive method for assessing ER and predicting implantation outcomes in FET cycles. Furthermore, biomarkers like malic acid and indol-3-propionic acid could guide personalized interventions to optimize implantation success. Whilst specific interventions are not currently available to modulate the endometrial metabolome, dietary interventions (i.e., Mediterranean diet) have been suggested . A diet rich in Omega-3 fatty acids (e.g., fish, flaxseeds, and walnuts) may support a favourable endometrial environment since Omega-3 and Omega-6 (PUFAs) play a role in PG production, which is important in ER. Excessive Omega-6 intakes may disrupt the balance necessary for proper endometrial transformation and implantation by promoting pro-inflammatory responses . In a study by Molina et al., the authors reported that a high adherence to the Mediterranean Diet positively correlated with beneficial metabolites like progestin steroids and ceramides and negatively correlated with potentially harmful metabolites like bile acids and acylcarnitines in women with endometrial-factor infertility . Linolenate levels were notably higher in women with high Mediterranean Diet adherence and no endometrial-factor infertility compared to women with low Mediterranean Diet adherence and endometrial-factor infertility . Adopting a Mediterranean Diet could positively modulate the endometrial metabolomic profile, potentially improving fertility outcomes. The endometrial microbiome is the community of microorganisms residing in the endometrial lining. Over the past decade, research has increasingly focused on the relationship between the endometrial microbiome and ER, particularly exploring how the microbial environment of the endometrium influences fertility outcomes. A healthy endometrial microbiome is typically dominated by Lactobacillus species. Studies have shown that a Lactobacillus-dominant microbiome (LDM) was associated with higher implantation and pregnancy rates for women undergoing IVF . Non-Lactobacillus-dominant microbiome (NLDM) with potentially pathogenic species such as Gardnerella, Streptococcus, Atopobium, Burkholderia, and Prevotella was associated with poorer reproductive outcomes from IVF cycles . Similarly, an increased presence of NLDM was associated with RIF and recurrent miscarriage . Verstraelean et al. identified a Bacteroides and Proteobacteria dominance in endometrial brush samples from 19 non-pregnant women with a history of RIF or recurrent miscarriage . A subsequent analysis of paired EFA and vaginal secretion samples collected during the luteal phase from 28 RIF patients and 18 infertile women linked the presence of Burkholderia with RIF . Collectively, assessing for LDM may serve as a biomarker for predicting implantation success. Furthermore, assessing and potentially modifying a NLDM (e.g., probiotic or targeted antibiotic) may present as a potential therapeutic options to improve reproductive outcomes. Clinical trials have explored the utility of antibiotics and probiotics to address implantation failure by modulating the endometrial microbiome. In one pilot study, patients with NLDM received antibiotics followed by prebiotics and probiotics to restore Lactobacillus dominance. Whilst the intervention successfully restored the LDM in a small number of patients, the reproductive outcomes were not as expected, as untreated patients showed higher pregnancy rates than the treated group, highlighting the need to consider better risk stratification (i.e., by severity of dysbiosis) in order to identify subgroups of patients who would benefit the most from treatment . In a prospective pilot study, 117 infertile women with RIF and 55 infertile women without RIF (control group) were treated with oral enteric-coated lactoferrin to restore a LDM with the aim of improving reproductive outcomes. Lactoferrin supplementation changed NLDM to LDM in 43.2% of women with RIF. Furthermore, an improved LDM was associated with higher CPR and LBR . Whilst the study highlighted the potential of microbiome-targeted therapies, RCTs are needed to confirm these results and optimize lactoferrin dosage and duration. In addition to evaluation of novel therapies, a number of fundamental biological questions remain to be answered, particularly regarding the mechanism by which an altered endometrial microbiome negatively impacts ER and implantation. The prevailing theory suggests that the influence an endometrial microbiome has on ER and implantation is due to its interaction with local immune cells . For instance, a healthy microbiome may promote immune tolerance by enhancing the activity of regulatory T-cells (Tregs) and reducing pro-inflammatory responses . In a recent study, He et al. revealed several ways by which bacterial populations may improve ER and reproductive outcomes . Using an animal model of chronic endometritis (CE) first, treatment with Lactobacillus crispatus significantly reduced CE-induced endometrial inflammation and improved uterine morphology. Implantation rates were restored to near-normal levels in the combined treatment group (i.e., antibiotics and probiotics) . When the combined treatment (antibiotics + L. crispatus vaginal administration during IVF) was given to 100 infertile women with CE, the authors of the study observed higher CPR than with antibiotics only or control groups . Furthermore, a histological analysis showed reduced inflammatory cell infiltration and improved endometrial structural morphology in samples from women who received combined treatment . In addition to influence on the local immune environment, L. crispatus upregulated ER-related proteins such as progesterone, VEGF, and MMP9, highlighting the complexity of the microbiome’s influence on ER. Whilst microbiomics continue to provide new insights into ER, significant challenges in research methodology remain to be addressed: (1) variability in sample collection, analysis methods, and patient selection across studies has hindered the comparability of results; (2) standardization is critical to defining a healthy endometrial microbiome and its clinical implications . The authors proposed a standardized pipeline to (1) focus on consistent sampling during the same menstrual cycle phase; (2) using advanced sequencing methods (e.g., next-generation sequencing, metagenomics) targeting specific 16S rRNA regions (V3-V4); (3) employing controls to prevent contamination and ensure reproducibility . Single-omics studies (e.g., transcriptomics) often provide a limited view of a system because they focus on one layer of regulation. The term “integratomics” refers to the comprehensive integration of various ’omics’ data in order to achieve a holistic understanding of complex biological systems. This integrative approach is increasingly common in contemporary research and is gradually becoming the standard approach to unravelling the complex cellular processes involved in ER. While the concept of a multi-omics approach is no longer novel, its application in specific contexts provides valuable insights and has demonstrated the method’s power to address complex biological questions. As an example, a recent study by Tang et al. integrated transcriptomics, proteomics, and functional assays to investigate the role of Cell Division Cycle 42 (CDC42) deficiency in RIF . Whilst transcriptomic analysis identified the pathways and genes impacted by CDC42 loss, proteomics and functional assays validated these findings at the protein and cellular function levels. Integrated analyses uncovered the interplay between CDC42, Wnt signalling, and decidualization, providing a mechanistic understanding of CDC42’s role in RIF . Another recent study also exemplified the multi-omic approach as it integrated multiple data layers, including morphology, transcriptomics (single-cell and spatial), proteomics, and protein–protein interaction (PPI) analysis to comprehensively investigate the cellular and molecular mechanisms underlying decidualization resistance (DR) . Each omic level validated and extended findings from the other, enhancing reliability and offering deeper mechanistic insights, showing that disrupted responses to progesterone and oestrogens, along with cytoskeletal and oxidative stress imbalances, are the main drivers for DR in the context of severe preeclampsia . These findings have implications beyond preeclampsia, extending to conditions like endometriosis and RIF, where DR has also been implicated. Collectively, the aforementioned examples underscore the utility of an integrative multi-omics approach in elucidating the complex biological underpinnings of ER and conditions that impact ER, paving the way for more accurate diagnostic tools and personalized therapeutic interventions. In summary, various disciplines of the omics have revolutionized our understanding of ER, uncovering key targets for advancing diagnostic and therapeutic strategies in reproductive health. Genomic studies linked SNPs in genes such as PGR and TP53 to poorer IVF outcomes, while SNPs in NF-kβ , LIF , VEGF, VEGFR-2, TNF-α, IL-1β, IL-6 , and STAT3 are implicated in RIF. Epigenomic studies revealed a range of potential targets that may be amenable to modulation, including DNMTs, TETs, HDAC1/2, H3K9, H4K16, H2AK119ub1, and H3K18la. A transcriptomic analysis revealed key miRNAs and lnCRNA implicated in endometriosis and RIF, including miR-9, miR-34, and miR-135a/b miR-30d-5p, PART1, MIR17HG, H19, LINC00173, NEAT1, and LINC01960-201. Proteins such as PGR, MMP2/9, and ANXA6 may be targets for further research due to their association with RIF. Lipid targets, including endocannabinoids, prostaglandins, LPA, and PUFAs offer additional avenues to improve reproductive outcomes. Finally, a Lactobacillus-dominant microbiome emerges as a critical element for the establishment of a receptive environment for implantation. Collectively, these insights provide a robust foundation for developing personalized diagnostic and therapeutic strategies to enhance implantation success and improve reproductive outcomes. Future research should aim to integrate multi-omic datasets to achieve a comprehensive understanding of the complex biological processes underpinning ER. |
Evaluation of Transcutaneous Non-Invasive Blood Gas Analysis for
Monitoring Gas Exchange in Pediatric Cardiac Surgical Patients Post
Extubation | 81ae1eba-22f3-43db-a418-5d99bfc72b41 | 11952150 | Surgery[mh] | Measurement of arterial blood gases is an integral part of monitoring of respiratory
status of the patient. Extubated, postoperative pediatric cardiac surgical patients
may become unstable rapidly if they are not monitored closely for hypercapnia and
hypoxia. Arterial blood gas analysis remains the “gold standard” monitoring.
Transcutaneous partial pressure of carbon dioxide (TcPCO 2 ) monitoring has
been done during neonatal transport and in pediatric patients (four years or older) receiving
mechanical ventilation for respiratory failure . The American Academy of Sleep Medicine recommends
monitoring and reporting of hypoventilation in adults and pediatric population, and
arterial partial pressure of carbon dioxide (PaCO 2 ), TcPCO 2 ,
or end-tidal partial pressure of carbon dioxide (PCO 2 ) can be used for
detecting hypoventilation during a diagnostic study in both adults and
children . However,
limited literature is available in extubated postoperative pediatric cardiac
surgical patients. The purpose of this study was to observe the correlation of
transcutaneous blood gases (transcutaneous partial pressure of oxygen
[TcPO 2 ], TcPCO 2 ) with arterial blood gases in such
patients.
This study was conducted at a tertiary care hospital, in the postoperative pediatric
cardiac surgical intensive care unit, after obtaining informed consent from parents
and was approved by the Institutional Ethical Committee of the Sri Jayadeva
Institute of Cardiovascular Sciences and Research, Bangalore, India. Inclusion Criteria Exclusion Criteria Statistical Analysis Four-month-old to three-year-old pediatric patients who got extubated after
cardiac surgery in postoperative pediatric cardiac surgical unit, had an
arterial catheter in place with stable hemodynamic parameters, normal sinus
rhythm, no residual shunts after cardiac surgery, no signs of cardiac and or
respiratory failure, were normothermic, and had a low inotropic score of
≤ 5 were included in the study.
Patients with unstable hemodynamic parameters, post-repair residual shunts,
palliative procedures related to either single ventricle pathology or cyanosis,
arrhythmias, signs of respiratory failure, low cardiac output, skin edema, and
on high vasopressor support were excluded. Transcutaneous monitoring (TCM) and arterial blood gas monitoring were started
one hour after extubation and continued for four hours by using Draeger
TcPO 2 and TcPCO 2 monitor. Transcutaneous probe
calibration was done according to the manufacturer instructions (TINA TCM4,
Radiometer, Copenhagen, Denmark). Before placement of transcutaneous probe,
calibration was done with gas cylinder which was provided with the instrument.
The probe was attached to the dry skin of the right or left upper chest. The
working temperature of the probe was kept at 43°C, and the monitor site was
changed every two hours to prevent any thermal injury to the patients. The probe
was recalibrated before placing it to a new site. To minimize the inter-rater
variability, probe calibration, placement, site change monitoring, and recording
of data were done by a single observer, and the observer was unaware of arterial
blood gas values which were taken every 30 minutes. Arterial blood gases
(PCO 2 and partial pressure of oxygen [PO 2 ]) were
recorded at an interval of 30 minutes, and transcutaneous gases
(TcPCO 2 and TcPO 2 ) were recorded simultaneously, for a
period of four hours. The transcutaneous gases’ values were displayed on Draeger
Infinity delta XL monitor. A set of eight samples for arterial and
transcutaneous gases were recorded for each patient.
Sample size was calculated based on a previous study , considering correlation coefficient r
= 0.9, alpha error = 0.05 with power = 80%. A total of 30 patients
were included in the study. Pearson’s correlation was done to analyze the
correlation coefficient r between transcutaneous gases and arterial gases. A
linear regression r 2 and a Bland-Altman
analysis were
performed to compare the transcutaneous and arterial blood gas values. The bias,
measured by Bland-Altman, represents the systemic error or variability between
two techniques and is defined as the mean difference between values.
Bland-Altman graphs were plotted for visual observation, and 95% confidence
limit (limit of agreement) was estimated. In addition, folded cumulative
distribution plot (Mountain plot) described by Krouwer and Mont A were also plotted. A Mountain plot measures the difference of the value obtained by the standard
method (arterial blood gases, i.e., arterial partial pressure
of oxygen [PaO 2 ], PaCO 2 ) and the method under
investigation (transcutaneous gases, TcPCO 2 , TcPO 2 ) on the
x-axis and the percentile of differences on the y-axis. The resultant plot is
inevitably a “mountain.” The benefits of the Mountain plot are that it is easier
to find the central 95% of the data and easy to estimate percentile for large
difference between methods. All the statistical analyses were done with MedCalc
software version 12.2.1 (Ostend, Belgium).
A total of 30 patients were included, from whom 240 paired samples between
transcutaneous and arterial blood gases for both carbon dioxide (CO 2 ) and
oxygen were analyzed. There were 17 male and 13 female patients, their age varied
from four months to three years, and their weight varied from 4.4 kg to 17 kg (mean
± standard deviation -10.08 ± 3.15). They underwent various types of
intra-cardiac repair ( ). The TcPCO 2 was higher than PaCO 2 with mean difference of 2.6
± 1.96 mmHg (PaCO 2 -TcPCO 2 ). Pearson’s correlation
coefficient r -value between TcPCO 2 and PaCO 2 was 0.9519, and linear regression analysis showed r 2 -value of 0.9060 ( P <0.001) ( ). Bland-Altman showed a bias of 2.579,
and 95% limit of agreement between PaCO 2 and TcPCO 2 was -6.4
to 1.3 ( ). The mean difference between PaO 2 and TCPO 2 was 20.2 ±
1.96 mmHg (PaO 2 -TcPO 2 ), and PaO 2 was higher ( ). The r -value between
PaO 2 and TcPO 2 was 0.8942, and linear regression analysis
showed r 2 -value of 0.7996
( P <0.001) which indicates a strong correlation between
PaO 2 and TcPO 2 ( ). Bland-Altman analysis of PaO 2 and TcPO 2 showed
a bias of 20.171 and 95% limit of agreement of -0.5 to 40.9 ( ). The Mountain plot, which is generally used as
complimentary to Bland-Altman plot, also showed similar results where the median
PaCO 2 and TcPCO 2 was small (2.57) and showed small tail
( ). The median PaO 2 and
TcPO 2 was large (20.17), with long tail ( ).
After the analysis, we found that TcPCO 2 was accurate and in close
agreement with arterial PaCO 2 in our postoperative pediatric cardiac
surgical population study. Pearson’s correlation coefficient r -value is 0.9519 ( P <0.001), which shows a
strong positive correlation between PaCO 2 and TcPCO 2 , and
Bland-Altman analysis shows a bias of 2.6 and 95% confidence limit of agreement of
-6.4 to 1.3 mmHg. The American Association of Respiratory Care (AARC) clinical
practice guidelines has cited as clinically acceptable agreement between
TcPCO 2 and PaCO 2 of ± 7.5 mmHg or 1 kPa for TCM of
CO 2 and oxygen ,
in 2012. Mountain plot shows median of -2.0, which is very close to “0” and small
tail, i.e., less bias and more precise. In recent studies, Karolina Weinmann et al. did continuous transcutaneous CO 2 monitoring to
avoid hypercapnia in complex catheter ablations under conscious sedation and found
that it is feasible and precise with good correlation ( r =0.60–0.87, P <0.005) to arterial blood gas CO 2 analysis under
conscious sedation and may contribute to additional safety. Wang W et al. found, in pediatric
laparoscopic surgery, that a close correlation ( r 2 =0.70, P <0.01) was established between TcPCO 2 and
PaCO 2 . Compared to end-tidal CO 2 , transcutaneous
CO 2 can estimate PaCO 2 accurately and could be used as an
auxiliary monitoring indicator to optimize anesthesia management for laparoscopic
surgery in children, however, it is not a substitute for end-tidal
CO 2 . Michel Toussaint et al. assessed the quality of peripheral oxygen saturation (or SpO 2 ) and
PCO 2 recordings overnight via TCM in children with neurological
conditions (out of 64 children, 42 used positive pressure respiratory support). They
were able to make satisfactory clinical decisions in 91% of cases and concluded by
saying that the quality of transcutaneous sensor recordings was acceptable, and
clinical findings were deemed as satisfactory in the large majority of cases. Many
studies have not only shown a strong correlation between the TcPCO 2 and
PaCO 2 , but also positively validated accuracy, high degree of
interchangeability, and that sometimes and it may provide a better estimate of
PaCO 2 than end-tidal CO 2 in pediatric
population [ , , , , ] . In 2019, a systemic review and meta-analysis for precision and accuracy of
transcutaneous CO 2 monitoring by Aron Conway et al. has identified that there may be
substantial differences between TcPCO 2 and PaCO 2 depending on
the context in which this technology is used in clinical practice, but in their
meta-analysis, the population limits of agreement between transcutaneous and
arterial CO 2 in pediatric intensive care unit and surgery was -5.1 to 4.4
mmHg, which was an acceptable agreement between TcCO 2 and
PaCO 2 (± 7.5 mm Hg or 1 kPa) . Regarding transcutaneous oximetry (TcPO 2 ), we found Pearson’s correlation
coefficient r =0.894, and linear regression analysis showed r 2 -value of 0.7996 ( P <0.001)
which has a strong positive correlation between TcPO 2 and
PaO 2 , and when comparing with Bland-Altman analysis, it revealed a bias
of 20.17 and wide limit of agreement. On Mountain plot analysis for PaO 2 and TCPO 2 , the median between PaO 2 and TcPO 2 was
20.17, with long tail, indicating less precise and poor interchangeability. Several studies have demonstrated that TCPO 2 is not generally
reliable and have also found to have a
poor correlation, wide limit of agreement between TcPO 2 and
PaO 2 , and suggested that TCPO 2 cannot be surrogate to
PaO 2 . TCM of gases really measures TcPO 2 and TcPCO 2 , not
PaO 2 and PaCO 2 , that could be the possible reason for the clinically
acceptable difference between PaCO 2 and TcPCO 2 be ± 7.5
mmHg, as per AARC clinical guidelines . TcPO 2 is an indirect measurement of PaO 2 and does not reflect
oxygen delivery or oxygen content. Complete assessment of oxygen delivery requires
knowledge of hemoglobin saturation and cardiac output. TcPCO 2 is an
indirect measurement of PaCO 2 , but knowledge of delivery and content is
not necessary to use TCM (TcPCO 2 ) for assessment of ventilation. TCM has traditionally been done by placing a heated sensor on the skin that increases
the capillary blood flow and amount of oxygen diffusing to the sensor. Due to
different diffusion rates, monitoring TcPCO 2 can typically be achieved
using lower temperatures of 38-42°C, which is not feasible for TcPO 2 ,
where temperature has to be kept at 43-44°C to achieve precise results . Epidermal and dermal cells consume oxygen and produce CO 2 , therefore
TcPO 2 is lower than PaCO 2 and TcPCO 2 is higher
than PaCO 2 irrespective of the sensor measuring temperature. This
influence is minimized by applying a temperature-specific constant and a metabolic
factor by the manufacturers . Arterial blood gas analysis is a gold standard technique but provides only momentary
status. It is time consuming, and repeated sampling might lead to blood loss and
anemia especially in neonates and pediatric postoperative cardiac surgical patients.
Liebowitz RS et al. concluded
that there is low but measurable morbidity associated with arterial catheterization
as well. TCM is a continuous, noninvasive method, but transcutaneous probe placement
requires expertise — improper placement, damaged membranes, trapped air bubbles, and
inappropriate calibration techniques may affect its accuracy. Patient problems such
as tissue hypoperfusion, the presence of edema, low cardiac output, and hypothermia
may affect the measurements. Several studies have documented that vasoactive
substances like dopamine, epinephrine, dobutamine, and norepinephrine did not affect
TcPCO 2 /TcPO 2 measurements [ , , ] . Limitations Limitations of the study were: this is a single-center, observational study, only
patients with stable hemodynamic parameters without residual shunt and
arrhythmias were studied, monitoring of the patients for a very limited time,
and the fact that pH, base excess or deficit, serum electrolyte, hematocrit, and
lactate cannot be obtained by this instrument. We need further larger randomized
control studies to assess whether trends of changes in transcutaneous gases
values can be reliable in post cardiac surgery pediatric patients.
CO 2 values obtained from TCM are interchangeable with those obtained from
arterial blood gas analysis in our population study, unlike oxygen measurements
which are not interchangeable. Hence TcPCO 2 values can be used as a
surrogate for arterial PaCO 2 measurements in postoperative pediatric
cardiac surgical patients. However, arterial blood gas analysis should be performed
when transcutaneous gases do not appear consistent with clinical findings.
|
Pediatric ultrasound practice in Italy: an exploratory survey | e92a118b-ff44-43a9-90bf-34279a6b3775 | 11163714 | Pediatrics[mh] | The growing interest toward ultrasound (US) in recent years is basically due to a non-invasive and portable nature of this imaging tool . Being free from ionizing radiation and painless, US is suitable also for neonatal and pediatric age, with a wide range of applications supported by research literature . Pediatric consultants are becoming more and more self-confident in US practice, which is becoming integral to the physical examination and may help physicians for decision-making process, follow up management of acute diseases and therapeutic procedures . Despite a remarkable body of evidence, a routine use of US in pediatrics cannot be recognized yet . Moreover, literature investigating US application almost entirely relies on the emergency and critical medicine experience while deficient data exist for its use in different pediatric settings, such as non-intensive pediatric units or outpatient clinics. Significant gaps still need to be addressed in order to implement US dissemination. To date, the lack of core infrastructural elements is one of the most perceived barrier in US widespread development. Different practice environments may affect the limitation size even if literature is controversial. Some authors report that divisions with larger units invest more resources for technology, have an adequate number of faculty and a greater possibility of collaborating with specialists (i.e., cardiology, radiology) . Conversely, Conlon et al. found that limited access to US machines is independent of the division size . The lack of or inadequate diagnostic imaging equipment is reported as a major barrier for US implementation also in low- and middle-income countries, as underlined in a recent sistematic review . Definitely, the availability of US equipment and machines throughout various clinical settings should be enhanced, also favoring the use of portable and handheld devices with lower cost but good resolution . Nevertheless, the US application is limited by another main factor that is education. Although US has become a powerful tool for treating clinicians, it is dependent on the user’s skills and training. A great push towards the importance of training and standardized educational curriculum has been made through national guidelines publications . Despite this, at present, a standardization of training pathways for both pediatric residents and faculty has not been established and US curricula and credentialing processes deeply vary worldwide . Furthermore, even if a brief educational intervention has been demonstrated to be effective in increasing proficiency on US , the lack of learning time as well as the paucity of skilled trainers are also reported limitations in literature . As the use of US continues to increase, the need for early training also increases: numerous pieces of evidence report how starting upstream training from the medical school period would ease US application postgraduating . The objective of this national survey is to investigate the use of US among pediatricians and pediatric residents and to describe the characteristics of the US machines, the number of scans carried out, the years of experience of the performers and the main US applications. across different pediatric settings. Another main purpose of the survey is to identify training needs and possible barriers to US implementation. The present study is a national, cross-sectional, web-based survey. The questionnaire was emailed to all members of the Italian Society of Pediatrics, including pediatric residents and it was distributed for completion from December, 2021 to March, 2022.In order to optimize the return rate of the survey, reminder e-mails were sent 3 times during the period and the subsequent analysis of the results was performed only for surveys which were fully completed one month after the third reminder. Written consent was not required due to the anonymous and voluntary nature of the questionnaire. Ethical consent was not required due to the study design and local regulations. The estimated time to complete the questionnaire was 6 min. The questionnaire ranged from24 to 27 items, depending on whether pediatricians or residents answered, respectively. The full version of the survey is reported as annex. The questions were formulated as multiple-choice response, with some exceptions (four-point Likert scales and open questions). For questions with “other” category, a free-text response was solicited. Data were extracted from the SurveyMonkey platform and statistics was performed using IBM SPSS for Windows (Version 24.0, IBM Corp ). The survey was completed by 1098 respondents with an overall response rate of 11%. Descriptive characteristics of survey respondents are reported in Table . Most of participants (87.1%, n = 956) declared to have an US machine available within the department. The predominant model resulted to be cart-based (66.9%, n = 516), followed by portable (28.5%, n = 220) and handheld (4.5%, n = 35). Linear and convex were the most available probes (33.8%, n = 551 and 30.1%, n = 491,respectively). Nearly one third of respondents (42.8%, n = 330) assessed that US machines had been provided from 1 to 5 years prior to the survey while only 12.2% ( n = 94) from more than 10 years. Lung and neonatal cerebral US were the most frequently performed by participants (18.7%, n = 289 and 14.1%, n = 218, respectively). Details of US applications are reported in Figs. , and . Regarding US utilization, 707 (84.1%) pediatricians reported any use of US in clinical practice while 51 (44.3%) residents denied it. Among pediatric residents, 71.8% ( n = 46) asserted to use US with less than one year of experience. Moreover, more than half residents(59.4%, n = 38) declared a frequency of scans less than 10 per month. On the other hand, 36.4% of pediatricians ( n = 257) stated to have more than ten-years experience and 46% ( n = 325) referred to perform more then twenty exams per month. Personal position and expertise about US utilization and differences between university/not university and public/private settings are summarized in Tables , and . There were no statistically significant differences in the use of US by dividing the sample by working department and geographical area (north, central or south). More than half respondents (55.7%, n = 430) affirmed to use ultrasounds in the emergency room, for most cases in the suspicion of pneumonia (78%, n = 390), or for patients with respiratory distress (66%, n = 330). 44% ( n = 360) of participants used ultrasounds for trauma cases while 38% ( n = 311) for undifferentiated shock Among family pediatricians, 177 (95.2%) declared to use US routinely, mainly for lung and kidney/urinary tract regions (16.9%, n = 30 and 12.9%, n = 23, respectively). With regard to US training and certification, data are reported in Table . In addition, 59.4% ( n = 38) of residents claimed that US practice was not included in the training program while 94% ( n = 36) of them considered desirable the insertion of a specific course during the residency. When asked to express about the main difficulties in using US, most participants complained a lack of a well-defined training program (57.1%, n = 627), 17.9%( n = 196) unavailability of the US machine, 15.9% ( n = 175) legal responsibility concern, 8.1% ( n = 89) non-collaboration of colleagues. In “other” category (1%, n = 11) participants listed as possible obstacles also insufficient educational time for learning US, lack of trained faculty to rely on and resistance to pediatric US application from other departments such as radiology or surgery (Fig. ). Almost all participants (98%, n = 1076) considered US a useful tool for clinical practice and they deemed US very or quite relevant in guiding clinical decisions (83.5%, n = 644 and 15.7%, n = 121, respectively). 169 (60.1%) of pediatricians working in ED have increased the use of ultrasound during the SarsCoV2 pandemic compared to 26.4% (216) of those working in other departments ( p = 0.001). 153 (54.4%) of pediatricians working in ED have increased their knowledge during the COVID period compared to 111 (13.6% ) of those working in other departments ( p = 0.001). Since the use of US has increased exponentially worldwide in the last few decades, also for the pediatric age , we aimed to provide data on the current Italian state of pediatric US practice. US equipment and applicationsUS practice among different pediatric settingsUS and educationBarriers to POCUS applicationsPOCUS during the COVID-19 pandemicLimitations and strenghts Our study had a number of limitations and strenghts. The first limitation is intrinsic to the nature of the survey which is self-reported designed. Another limitation low response rate, which may reflect a lack of participant involvement and motivation on this topic. Actually, a potential response-bias could be due to the overestimation of US users among respondents, as those more interested in US technique may have been more like to respond. Regarding possible strengths, this study is one of the few survey which investigated the US application not only in pediatric emergency department but also in outpatient clinics. Moreover the survey was not targeting to a specific population sample, including pediatric residents, pediatric hospitalists and family pediatricians. The US spread is strictly related to the availability of machines: the majority of participants declared to have a US machine available for use within the department, with percentages in line with the literature . Particularly, convex and linear probes have been found to be the ones most obtainable for US examinations. US practice seems to be relatively recent tool in pediatrics , considering that machines have been provided mostly 1–5 years prior to the survey. Nevertheless, the vast majority of US machines have been found to be conventionally cart-based, at the expense of newer models such as portable and handheld systems , which have been demonstrated to improve patient outcomes also in environments with limited resources . These results focus on the urgent need to direct more resources for improving US equipment and advanced scanning technologies in pediatric units. Concerning the type of examination, lung resulted to be the most common scanned region. We also found that participants working in the emergency room, mostly use US in suspicion of lung pathologies. Similar results were also highlighted for family pediatricians. A possible explanation for this finding may be the steep learning curve which also allows novices to be able to perform lung US . It should be also underlined that nearly one third of respondents assessed to use US for cardiac and abdominal application: traditionally, the use of US for these two anatomic regions is a prerogative of cardiologists and radiologists, respectively . This finding may be considered as the beginning of a growth path to be pursued in the future for the entire pediatric personnel . Some differences on US practice between pediatric residents and pediatricians have been found in this survey. Among residents, only half declared to use US, mostly with little experience and low scanning frequency. On the contrary, the majority of attending pediatricians routinely perform US, although less than half of them with experience over ten years and a scanning frequency more than twenty per month. Controversial data are reported in literature about disparity in US application between residents and pediatricians : regarding the Italian situation, we can speculate that, despite the growing interest on US in last decades, the method still needs to be spread and implemented in clinical practice, especially during residency. The vast majority of the surveyed family pediatricians report to perform US routinely. As far as we know, the present survey is the first study investigating US application in pediatric family care, so we are not able to compare our data with similar findings in literature. Our results even seem to be in contrast with those described by a recent survey which reports a low percentage of US scans performed among family medicine residents and practicing physicians . We hypothesize that the high percentage of US utilization among family pediatricians may reflect a bias selection. Finally, we did not find statistically significant differences for US practice dividing the sample by geographical area (north, central or south) or working department. We definitely believe that this last result should be verified on a larger study sample. We were not even able to compare our data with those in literature since, as far as we know, there are no studies comparing the US use in the private, public or university setting nor for different geographical areas of the same nation. The section of the survey dedicated to training underlined interesting issues. To date, an informal experience-based training or theoretical-practical courses have been found to be the most frequently adopted while a credentialing process was missing in most cases. Similar findings are reported in literature, pointing out the lack of specific training pathways and non-homogeneous programs . Learning methods also vary based on different realities. In middle and low-income countries, e-learning methods have been reported to be the commonest method, related to the high costs of face-to face training . Nevertheless challenges relating to poor internet connectivity still affects access to study platform and communication with supervisors . The standardization of training plans, starting preferably during residency rather than early under graduation medical period, might allow to create a US curriculum as guarantee of educational pathway and quality assessment . Furthermore medical school/residency directors and their institutions should consider the curriculum as a core requirement for the implementation of US technique . More than half respondents within the pediatric residents subgroup declared that a specific training program has yet to be described; however, almost all of them showed a positive attitude towards US, supporting its endorsement during residency, as previously underlined in literature . Our survey points out possible barriers to POCUS applications. Among these barriers, the lack of well-defined training plan has been identified as a main obstacle to US expansion by respondents: we strongly believe that an educational US curriculum should be mandatory not only for residents but also for attending physicians, as reported in recent literature . Despite the dramatic rise of US use, it is noteworthy that the unavailability of US machine is still considered as a barrier for daily practice in both our study and literature . Actually, some major reasons for complaints from physicians are the expensiveness of US machines and the subsequent difficulty in affording to purchase them by institutions, the scarce use of low-cost ultraportable devices, the US equipment deficiency and the inaccessibility to US machines for bedside use, often due to resistance from other departments . The lack of technological devices for performing US is reported to be a barrier to the implementation of the method even in middle and low-income countries where clinicians often deal with the high costs of equipments, adverse climatic conditions, power instability, and inadequate maintenance service . If the unavailability of US machines is indeed an absolute requirement for US practice, proper education still remains a main issue to be acknowledged . First of all, sufficient time for training should be given since a number of reports indicates a lack of time to learn . Moreover, a well-structured educational program could implement the small number of trainers within various institutions . Last but not least, an ongoing education should be mandatory for all credentialed US physicians in order to improve and consolidate their sonographic skills. Regarding the liability concerns, they may be closely related to the lack of credentialing plan and quality assurance program in case of misinterpretation or misdiagnosis leading to malpractice claims . Again, healthcare professionals should be aware that literature provides non-apprehensive data about litigation directly related to US application in the last decade . Furthermore, to improve ultrasound skill and reduce concerns about the sense of responsibility, with the possibility of using US more in daily clinical practice, it is not only necessary to train ultrasound skills but also training to imaging evaluation starting, for example, from the study of CT findings or anatomical information. From the perspective of survey respondents, US has been highly scored in term of usefulness in order to integrate the patient’s clinical evaluation and guide clinical decision, confirming positive attitude toward US application . The last two questions of the survey investigated the perception of US role during the outbreak of the COVID19 pandemic. Italy has been the first european country to deal with COVID-19, serving a resilient assistance both in emergency departments and outpatient clinics nationwide . Despite the enormous efforts to better understand clinical features of COVID-19 disease , participants assumed that the pandemic does not seem to have implemented neither the use nor the knowledge expansion of US in pediatrics. This unexpected issue may be due to possible challenges in logistics of US examination (e.g. lack of portable ultrasound machines, high risk for contracting the COVID-19 infection) . On the other hand, the further analysis for the subgroup of pediatricians working in ED found that both US use and knowledge were improved during the pandemic maybe due to a focused attention on emergency and COVID patients. is survey underlines a striking interest towards pediatric US for both pediatricians and residents nationwide. Pediatric residents support US training within the residency period in order to improve knowledge and confidence on the method. Nevertheless, the technique still needs to be implemented so that everyone can easily access it. Future US development would benefit from addressing more resources for up-to-date equipment as well as standardized education plan |
Label-Free Proteomics Reveals the Response of Oat ( | 88bb59ec-5df0-40b2-b266-637ce72d3b54 | 11942509 | Biochemistry[mh] | Abiotic stress includes salt stress, temperature stress, and drought stress . Saline soil represents a major concern, with about 95 million hectares of soil globally, or about 20% of agricultural land, affected by salinity . The total area of salinized land in China is about 36.658 million hectares, and the salinized area of cultivated land reaches 9.209 million hectares, with NaCl and Na 2 SO 4 as the main components . It is now understood that salt stress inhibits plant growth and development , eventually leading to yield reduction and limiting the sustainable development of agricultural productivity in salinized areas. As a dual-purpose grain and feed crop, oat demonstrates tolerance to saline–alkali and barren conditions and drought resistance. It is mainly planted in semi-arid agricultural and pastoral areas in northwest China and high-altitude mountainous areas in southwest China. Importantly, oat has emerged as a leading crop for improving saline–alkali soil . In recent years, a large number of studies have analyzed the effects of salt on plants at the physiological and molecular levels. Current evidence suggests that soil salt causes plants to suffer from osmotic stress, ion toxicity, oxidation stress , and other secondary reactions, resulting in various metabolic pathway disorders, including signal transduction, energy metabolism, and hormone synthesis . Roots are the primary organs through which crops absorb water and nutrients, and they are also the first point of contact with salt stress . Their growth and physiological metabolism directly affect the plant’s overall development, yield, and quality. When exposed to salt stress, plant roots adjust their morphology and biomass, trigger physiological and biochemical reactions, and initiate signal transduction pathways to adapt to the environment . As the basis of all life activities, respiratory metabolism provides plants with energy and raw materials for biosynthesis. It involves four key pathways: the Embden–Meyerhof–Parnas (EMP) pathway, the tricarboxylic acid (TCA) cycle pathway, the pentose phosphate pathway (PPP), and the cytochrome pathway (CCP) . During seed germination, the EMP and PPP are dominant, with the PPP playing a leading role when mitochondria are active. Conversely, when mitochondrial ATP synthesis is blocked due to hypoxia, the substrate enters the EMP pathway for catabolism . Increasing the PPP-to-EMP-TCA ratio can improve the active oxygen scavenging ability of papaya , while studies suggest that the EMP-TCA pathway can promote the TCA cycle and produce more ATP for stress resistance . To prevent cell damage under hypoxic conditions, plant cells initiate various stress responses, including major metabolic rearrangements, hormonal regulation, changes in mitochondrial biology, and gene expression reprogramming . These responses involve differences in genes, proteins, transcripts, etc. Through glycolysis, plant cells can be influenced by alcohol dehydrogenase (ADH) and pyruvate decarboxylase (PDC) to simultaneously downregulate genes related to ATP consumption pathways, such as lipid metabolism, secondary metabolism, transport, signaling, and redox regulation . It has been established that salinization makes the soil structure compact, reducing permeability and leading to decreased plant root oxygen concentration and the disruption of EMP metabolism, ultimately affecting plant respiration and metabolism . Studies have shown that salt stress can increase the plant respiration rate, significantly affect the TCA cycle pathway of cucumber seedlings, and inhibit the activities of key enzymes in this pathway [ , , ]. Overexpression of PDC and ADH in Arabidopsis thaliana can improve its tolerance to oxygen deficiency , while ADH and PDC function-deficient mutants of maize, rice, and Arabidopsis thaliana are more sensitive to low-oxygen conditions, highlighting the importance of ethanol fermentation in tolerating low-oxygen stress [ , , , ]. Although studies have investigated the effects of salt stress on respiratory metabolism, differences exist among different crops. For example, sorghum produced a burst of respiration associated with renewed synthesis of biomass from stored photosynthate under salt stress , S. alterniflora adapts to constant salinity through fixed, salinity-dependent structural modifications, such as stomatal density , while salt-tolerant wheat cultivar exhibited higher respiration rates to resist salt stress , therefore necessitating further exploration of changes in respiratory metabolic pathways and key genes. To further investigate the response of oat root respiration metabolism to salt stress, the present study employed a pot experiment to establish three different salt stress concentrations. Two oat genotypes were selected: the salt-tolerant cultivar Bai2 and the salt-sensitive cultivar Bai5. Seedling roots were subjected to various salt stress levels. Root growth, respiratory rate, pathway contribution rates (EMP, TCA, and PPP), and the activity of related enzymes were measured. Through proteomic analysis, the main metabolic pathways and key proteins were investigated to elucidate the mechanism of adaptation of oat to salt stress from the perspective of respiratory metabolism. This research aims to provide a theoretical basis and technical guidance for breeding salt-tolerant oat cultivars and promoting green, high-yield cultivation.
2.1. Growth of Oat Seedlings Under Salt Stress 2.2. Effects of Salt Stress on Potassium, Sodium, Calcium, and Magnesium Ion Contents in Oat Root at Seedling Stage 2.3. Effects of Salt Stress on Oat Root Respiratory Metabolism 2.4. Effects of Salt Stress on Oat Root Proteomics 2.5. GO Functional Annotation and Enrichment Analysis of Oat Root Proteome Under Salt Stress 2.6. KEGG Functional Annotation and Enrichment Analysis of Oat Root Proteome Under Salt Stress 2.7. Screening Differential Proteins 2.8. Fluorescence Quantitative PCR Analysis showed the differences in growth between two oats with different salt tolerance after salt stress. Moderate and severe salt stress had a negative impact on the growth of both oat cultivars, and the more severe the stress, the greater the impact. In addition, the harm caused by salt stress to the growth of salt-tolerant cultivars is relatively small. As the salt stress concentration increased, a gradual decrease in the fresh weight of both oat cultivars was observed. Compared to T1, the fresh weight of T3 and T5 plants declined by 17.8% and 24.4% in the aboveground parts, respectively, and by 45.5% and 54.5% in the underground parts, respectively. Similarly, compared to T2, the fresh weight of T4 and T6 plants showed reductions of 13.2% and 32.1% in the aboveground parts, respectively, and 64.3% and 71.4% in the underground parts, respectively ( ). The dry weight of both aboveground and underground parts followed the same decreasing trend as the fresh weight. Interestingly, compared to T1, T3 exhibited a 2.1% increase in aboveground dry weight, while T5 displayed a 6.4% decrease after T5. Both T3 and T5 exhibited reductions in the root cap ratio compared to T1, with decreases of 55.6% and 33.3%, respectively. Similarly, T4 and T6 displayed reductions of 57.7% and 57.7%, respectively, compared to T2. Notably, under moderate and severe stress conditions, the reductions in dry and fresh weight of both aboveground and underground parts, as well as the root–shoot ratio, were less pronounced in Bai2 compared to Bai5 ( ). The roots were crucial for crops to absorb nutrients, grow and develop, and achieve high yields, while root growth in oat seedlings was inhibited by increasing salt stress concentrations. The total root length, root surface area, root volume, and average root diameter were significantly affected by different salt stress concentrations ( p < 0.05). Compared to T1, T3 and T5 demonstrated reductions of 17.6% and 36.6% in root length, respectively, while T4 and T6 demonstrated reductions of 22.5% and 40.6%, respectively, compared to T2. Similar trends were observed for root surface area, with T3 and T5 showing reductions of 23.8% and 42.8% and T4 and T6 showing reductions of 27.1% and 44.6% compared to their respective controls. Root volume reductions were even more pronounced, with T3 and T5 plants exhibiting reductions of 27.9% and 61.9% and T4 and T6 plants exhibiting reductions of 39.4% and 74.8% compared to their respective controls. The effect on average root diameter was less pronounced, with T3 and T5 showing reductions of 3.2% and 9.7% and T4 and T6 showing reductions of 6.6% and 11.5% compared to their respective controls. Notably, the decrease in root morphological indexes was greater under severe salt stress (T5 and T6) compared to moderate stress (T3 and T4). Additionally, for each stress concentration, Bai2 had higher values for all root morphological indices compared to Bai5 ( ).
Similar to the results of salt stress on oat root morphology mentioned above, in both oat cultivars, the potassium ion content exhibited a decreasing trend with increasing salt stress concentration, as illustrated in A. Compared to T1, T3 and T5 showed reductions of 33.9% and 40.8%, respectively, while the potassium ion content in T4 and T6 was 22.4% and 25.6% lower, respectively, than in T2. A significant difference ( p < 0.05) was observed between the cultivars, with T1 demonstrating a 30.9% higher value than T2. Interestingly, no significant difference between cultivars was detected under moderate and severe salt stress treatments, with T3 being 11.4% higher than T4 and T5 being 4.2% higher than T6. The trend observed for the sodium ion content contrasted that of the potassium ion content, as depicted in B. In both cultivars, the sodium ion content increased with increasing salt stress concentration. T3 and T5 exhibited increases of 386% and 473% compared to T1, respectively, while T4 and T6 saw increases of 377% and 456% compared to T2, respectively. Although the sodium ion content of Bai5 was consistently higher than that of Bai2 across all treatments, the difference was not statistically significant. Additionally, the sodium ion content of T4 increased by 20.8% compared to T3, and that of T6 increased by 19.4% compared to T5. As shown in C, a decline in calcium ion content was observed within the oat roots alongside an increasing salt stress concentration. Compared to T1, T3 and T5 displayed decreases of 36.1% and 21.98%, respectively. The calcium ion content in T4 and T6 was 5.9% and 45.7% lower than in T2, respectively. Notably, the variation in calcium ion content within oat roots differed between cultivars and treatments; the content in T4 was 24.3% higher than in T3, while the content in T6 was 41.3% lower than in T5. As shown in D, the magnesium ion content in oat roots decreased with increasing salt stress concentration. Compared to T1, T3 and T5 exhibited reductions of 4.5% and 6.9%, respectively, while T4 and T6 decreased by 3.9% and 7.5%, respectively, compared to T2. An analysis of magnesium ion content across the two cultivars revealed that Bai2 consistently had higher levels than Bai5. Furthermore, no significant differences were observed among treatments, although T4 displayed a decrease of 1.1% compared to T3, and the magnesium ion content of T6 decreased by 2.3% compared to T5.
A demonstrates the impact of salt stress on the total respiration rate of oat root. As the stress concentration increased, inhibition of the root system’s total respiration rate was observed. No significant differences within the same cultivar were detected under varying salt stress concentrations. However, a significant difference was observed between the CK samples of two cultivars. T2 exhibited a 153% increase compared to T1, indicating the presence of inter-varietal differences. Compared to T1, the total respiratory rates of T3 and T5 were inhibited by 15.6% and 28%, respectively. Furthermore, T4 and T6 displayed inhibitions of 70.4% and 79.0%, respectively, compared to T2. Across all salt stress treatments, Bai2 consistently exhibited higher rates than Bai5. Notably, T3 showed a 12.5% increase compared to T4, while T5 demonstrated a 35.3% increase compared to T6. An increase in salt stress concentration was observed to promote the respiration rate of the oat root glycolysis pathway, as depicted in B. However, the overall respiration rate remained higher in Bai5 than in Bai2. Specifically, the glycolytic pathway respiration rates of T3 and T5 increased by 60% and 2080%, respectively, compared to T1. Similarly, T4 and T6 exhibited increases of 62.5% and 1600%, respectively, compared to T2. While T4 showed a 62.5% increase compared to T3, further analysis revealed that Bai2 contributed 29.6% and Bai5 contributed 54.2% to the total respiratory rate. Similarly, T6 displayed a 24.8% increase over T5, with Bai2 contributing 474% and Bai5 contributing 800% to the total respiratory rate. As the salt stress concentration increased, the respiratory rate of the tricarboxylic acid cycle pathway in oat root was gradually inhibited, as shown in C. Under the same stress concentration, no significant difference was observed between the two oat cultivars. Compared to T1, the respiratory rate was inhibited by 27.8% and 83% in T3 and T5, respectively. Similarly, compared to T2, T4 and T6 exhibited inhibitions of 29.4% and 91.2%, respectively. Interestingly, T3 displayed an 8.3% increase compared to T4, with Bai2 contributing 96.3% and Bai5 contributing 100% to the total respiratory rate. Furthermore, T5 demonstrated a 100% increase compared to T6, with Bai2 and Bai5 contributing 26.1% and 17.6% to the total respiratory rate, respectively. The effect of salt stress on the respiration rate of the pentose phosphate pathway in oat root is shown in D. With increasing salt stress concentration, the pentose phosphate pathway’s respiratory rate was progressively stimulated, with Bai5 exhibiting a consistently higher rate than Bai2 across all treatments. Except for the moderate salt stress treatment, significant differences between the cultivar samples that received the two treatments were observed. Compared to T1, T3 and T5 exhibited increases of 280% and 580%, respectively, while T4 and T6 showed increases of 23.5% and 157%, respectively, compared to T2. T4 demonstrated a 10.5% increase relative to T3, with Bai2 contributing 70.4% to the overall respiratory rate and Bai5 contributing 87.5%. T5 displayed a 58.8% increase compared to T6, with Bai2 contributing 148% and Bai5 contributing 318% to the total respiratory rate. The results presented in E indicate that the PDC activity in oat root initially increased and then decreased with increasing salt stress concentration. Notably, Bai2 consistently exhibited higher activity than Bai5 throughout the entire stress period, with significant differences observed between treatments. Compared to T1, T3 and T5 demonstrated increases of 133% and 26%, respectively, while T4 showed an increase of 116.3% compared to T2. In contrast, T6 exhibited a decrease of 21.6% compared to T2. T3 displayed a 19.8% increase over T4, while T5 presented a 78.5% increase compared to T6. Changes in ADH and PDC activity in the oat roots displayed a consistent trend, as depicted in F. Upon increasing the salt stress concentration, ADH activity was activated, initially rising and then declining, with significant differences observed among treatments. Notably, Bai2 exhibited higher activity compared to Bai5. Specifically, T3 and T5 displayed increases of 384% and 145%, respectively, compared to T1. Similarly, T4 and T6 showed increases of 434% and 157%, respectively, compared to T2. Furthermore, T3 exhibited an 8.3% increase compared to T4, while T5 displayed a 13.7% increase compared to T6. As shown in G, a gradual rise in oat root LDH activity was documented with increasing salt stress concentration. Significant differences were observed between the two cultivars across different treatments, with Bai5 demonstrating higher activity than Bai2. Notably, T3 and T5 displayed increases of 33.4% and 177%, respectively, compared to T1. Similarly, T4 and T6 showed increases of 73.1% and 213%, respectively, compared to T2. Moreover, T4 exhibited a 55.7% increase compared to T3, while T6 displayed a 35.4% increase compared to T5. Notably, the increase in LDH activity was greater in Bai5 compared to Bai2. H demonstrates a decrease in oat root MDH activity with increasing salt stress concentration. Except for the control group, where Bai5 exhibited higher enzyme activity than Bai2, all other treatments resulted in higher enzyme activity in Bai2 compared to Bai5, with significant differences observed among treatments. Specifically, T3 and T5 plants displayed decreases of 17.9% and 53.7%, respectively, compared to T1. Similarly, T4 and T6 plants showed decreases of 52.5% and 79.9%, respectively, compared to T2. Furthermore, T4 displayed a 52.5% decrease compared to T3, while T6 exhibited a 43.5% decrease compared to T5.
A total of 7174 proteins were identified in the oat roots using label-free proteomics, with all treatments achieving a 99% confidence level. In T1, 5441 proteins were identified; in T2, 5366 proteins were identified; in T3, 5425 proteins were identified; in T4, 5388 proteins were identified; in T5, 5416 proteins were identified; and in T6, 5268 proteins were identified. There were 4400 common proteins identified between T1 and T2 ( A), 4506 common proteins between T3 and T1 ( B), 4497 common proteins between T5 and T1 ( C), 4329 common proteins between T4 and T2 ( D), and 4150 common proteins between T6 and T2 ( E). Additionally, based on abundance (FC > 2), 74 proteins were found to be upregulated and 111 proteins downregulated in T1 compared to T2. Between T3 and T1, 236 proteins were upregulated and 328 were downregulated. For T5 versus T1, 215 proteins were upregulated, and 339 were downregulated. Compared to T4, T2 showed 237 upregulated and 189 downregulated proteins. Finally, a comparison of T6 to T2 revealed 247 upregulated and 289 downregulated proteins ( F).
The biological processes impacted by differential proteins between T1 and T2 primarily included the response to stimuli, single-organism processes, metabolic processes, responses to stress, and single-organism metabolic processes. In terms of molecular functions, differences were observed in catalytic activity, electron carrier activity, peroxidase activity, oxidoreductase activity, oxygen binding, transferase activity, isomerase activity, organic cyclic compound binding, heterocyclic compound binding, cofactor binding, and ion binding. The affected cellular components primarily comprised the extracellular space, endoplasmic reticulum membrane, and outer membrane. When comparing the differential protein responses to salt stress of T3 to T1 and T5 to T1, the main changes detected occurred in molecular functions including heme binding, transporter activity, oxidoreductase activity, oxidizing as acceptor, antioxidant activity, and peroxidase activity. Similarly, a comparison of the differential protein response to salt stress of T4 to T2 revealed that it primarily manifested in molecular functions such as catalytic activity, ion binding, metal ion binding, cofactor binding, and heme binding. Biological processes affected included single-organism processes, single-organism metabolic processes, oxidation–reduction processes, organic acid metabolism processes, single-organism biosynthetic processes, and single-organism carbohydrate metabolic processes. Finally, the differential response of T6 compared to T2 proteins to salt stress was primarily reflected in their molecular functions, like heme binding, nucleoside triphosphate activity, oxidoreductase activity, acting on peroxides as acceptors, antioxidant activity, transporter activity, and peroxidase activity. Single-organism processes, single-organism metabolic processes, oxidation–reduction processes, and responses to oxidative stress were the main biological processes impacted ( ).
Differentially expressed proteins in the root tissues of two oat cultivars under different treatments ( ) underwent KEGG pathway analysis, revealing shared metabolic pathways by comparing differential proteins in the groups, including those related to metabolic pathways, biosynthesis of secondary metabolites, and phenolpropanoid biosynthesis. Additionally, following salt stress, starch and sucrose metabolism and glyoxylate and dicarboxylate metabolism were identified as common metabolic pathways in both cultivars. Specifically, under salt stress, Bai2 exhibited enriched glutathione metabolism, amino sugar and nucleotide sugar metabolism, plant–pathogen interaction, peroxisomes, and cysteine and methionine metabolism pathways. Meanwhile, Bai5 displayed enrichment in carbon metabolism, amino acid biosynthesis, glycolysis/gluconeogenesis, alanine, aspartate and glutamate metabolism, RNA degradation, oxidative phosphorylation, citrate cycle (TCA cycle), and pyruvate metabolism pathways after salt stress.
Based on the criteria log2FC > 1 or log2FC < −1, all differentially expressed proteins were screened and classified. This identified a total of 63 differentially expressed proteins within the root of the two oat cultivars ( ). These proteins were categorized into various functional groups including, but not limited to, ion transport, protein synthesis, reactive oxygen species (ROS), carbohydrate and energy metabolism, transcriptional regulation and signal transduction, secondary metabolism, and unknown functions. Notably, protein synthesis and carbohydrate and energy metabolism exhibited a high number of differentially expressed proteins. Furthermore, analysis of the salt-tolerant cultivar Bai2’s root system revealed 111 differentially expressed proteins ( ). These proteins fell into twelve functional categories encompassing ribosome, transcription, signal transduction, secondary metabolism, post-translational modification, protein turnover and molecular chaperone, inorganic ion transport and metabolism, carbohydrate and energy metabolism, cytoskeleton and cell wall components, coenzyme transport and metabolism, amino acid transport and metabolism, ROS, and unknown functions. Notably, post-translational modification, protein turnover, and molecular chaperones, along with carbohydrate and energy metabolism, displayed significant differences in protein quantities. The combined action of proteins within a network environment drives their functions in plants. Differential proteins were identified and visualized using the STRING database and Cytoscape software (3.10.0). The analysis focused on differentially expressed proteins in the salt-tolerant cultivar Bai2 under severe salt stress compared to the control group. Interaction network analysis revealed differentially expressed proteins interacting across five metabolic pathways primarily involving antioxidant enzymes ( A), pyruvate metabolism ( B), glycolysis ( C), the tricarboxylic acid cycle ( D), and energy metabolism ( E). Key proteins identified include the following: (1) catalase (A0A3B6PHD6, I1I9A3, A0A2T7DAI6), associated with functions such as signal transduction, carbohydrate metabolism, energy metabolism, and protein synthesis; (2) acetate dehydrogenase (A0A2S3GZF9), associated with protein synthesis, ribosome function, and carbohydrate metabolism; (3) 3-phosphoglycerate kinase (A0A1J7HFP8), glycerol kinase (A0A3B6B850), and aquaporins (M8A623, A0A3B6DHS5), associated with functions such as carbohydrate metabolism, protein synthesis, quantitative metabolism, amino acid transport, and metabolism; (4) phosphoenolpyruvate carboxylase (A0A1D6QPT3, K3XV32, M1AX28), associated with functions such as signal transduction, carbohydrate metabolism, lipid transport, and metabolism; (5) F-type H + -transport ATPase subunit-α (A0A3B6FZW8) and V-type H + -transATPase subunit-α (A0A0D9XIB2), with functions mainly related to ribosome and protein synthesis.
To examine the expression patterns of related genes, 13 key differential proteins from five pathways of the salt-tolerant oat cultivar Bai2 under severe salt stress were selected for qRT-PCR analysis ( ). The expression trends of these genes were consistent with those of their corresponding proteins ( ).
3.1. Effects of Salt Stress on Physiological Growth Indicators of Oat 3.2. Effects of Salt Stress on Oat Root Respiratory Metabolism 3.3. Effects of Salt Stress on Oat Root Proteomics The inhibition of root growth is the most prominent physiological phenomenon observed in plants under salt stress . The root-to-shoot ratio serves as a crucial indicator for assessing seedling quality . Previous studies have demonstrated that a salt concentration of 0.25 mM NaCl or 1.8 dS/m promotes the growth and development of maize seedlings . Conversely, other studies have reported a gradual decrease in the fresh and dry weight of both the aboveground and underground portions of rice with increasing salt concentration . In this experiment, the dry and fresh weights of both oat cultivars progressively decreased as the salt stress concentration intensified. The root-to-shoot ratio also exhibited a general downward trend. Notably, the root-to-shoot ratio of Bai2 was lower than that of Bai5 following the control treatment. Furthermore, under identical salt stress concentrations, no significant difference in the root-to-shoot ratio was detected between the two oat cultivars. It is highly conceivable that the insufficient supply of essential sugars, proteins, and other nutrients required for root growth exacerbated the inhibitory effect on root development with the increasing salt stress concentration. Consequently, this led to a decrease in biomass and, in turn, a reduction in the root-to-shoot ratio . As Bai2 exhibited greater salt tolerance compared to Bai5, the decrease in its root-to-shoot ratio with increasing salt stress concentration was less pronounced. The root system is the first organ to perceive and respond to salt stress, with metabolic alterations directly impacting the plant’s physiological metabolism. The present study showed that under salt stress, the total root length, surface area, volume, and average diameter of oat cultivars were significantly reduced, aligning with established research findings . Indeed, through its effect on ion toxicity, salt stress can hinder plant growth. Na + , recognized as the primary toxic ion, can be effectively transported to lessen salt damage [ , , ]. Previous studies documented an increase in Na + content within the roots and aboveground parts of plants, while the K + content decreased under salt stress [ , , , , ]. This investigation revealed a similar pattern, with the highest Na + content observed in oat roots under severe stress. Notably, the salt-tolerant cultivar, Bai2, maintained a lower Na + content compared to the salt-sensitive Bai5, potentially indicating superior Na + transport capacity under stress conditions. Conversely, salt stress led to reduced K + content in the oat roots, with the salt-tolerant cultivar samples exhibiting higher K + reserves. This might represent a protective mechanism by mitigating K + loss from the plant body. Maintaining K + /Na + balance under salt stress is another facet of plant salt tolerance . Significant Na+ uptake under stress can trigger the efflux and loss of K + , Ca 2+ , and Mg 2+ . Notably, the establishment of Ca 2+ homeostasis within the cytoplasm is crucial for salt adaptation. This experiment consistently observed a gradual reduction in Ca 2+ content within oat roots under salt stress, mirroring the reported changes in the Ca 2+ content of wheat .
Plants acquire energy for plant growth and development via their respiratory function. Through photosynthesis and respiration, plants convert solar energy into usable forms and store it in their bodies as ATP. This energy fuels their physiological and metabolic activities. Additionally, intermediate products like pyruvate and NADPH, which are generated during respiratory metabolism, are crucial for synthesizing organic substances such as proteins and nucleic acids . Therefore, respiratory metabolism acts as a central energy hub for plants, supplying both energy for life activities and intermediate products for metabolism and synthesis. Higher plants require sufficient oxygen for respiration and other oxidative pathways to maintain normal growth. Saline–alkali land can cause soil compaction, consequently reducing the oxygen supply and placing considerable pressure on plant growth. This leads to an energy crisis, impacting gene expression; cell metabolism, growth, and development; and, ultimately, biomass . Multiple studies have reported changes in the ratio of carbon dioxide (CO 2 ) released to oxygen (O 2 ) consumed (respiration quota) in salt-stressed plants. This suggests that mitochondria might be oxidizing different substrates under saline conditions [ , , ]. In cucumber leaves, salt stress inhibits both the EMP pathway and the TCA cycle, resulting in a decreased respiration rate . Interestingly, some studies have observed an upward trend in the mitochondrial respiration rate of wheat under salt stress in the dark . This study observed that the total root respiration rate of both oat cultivars decreased as the salt stress concentration increased, with a more pronounced decrease in Bai5 compared to Bai2. This difference might contribute to Bai2’s higher salt tolerance. However, the diverse responses observed in different crops under salt stress suggest that stress induces a complex series of coordinated metabolic changes involving numerous pathways, necessitating further investigation. Three respiratory metabolic pathways exist in plants: the glycolytic, tricarboxylic acid cycle, and pentose phosphate pathways. Under normal conditions, the TCA pathway predominantly sustains plants. Studies have demonstrated that temperature stress alters the proportion of respiratory pathways in cotton seedlings, with changes in the PPP closely associated with adverse growth environments . In this study, increasing salt stress concentrations progressively inhibited the TCA pathway while simultaneously enhancing the EMP pathway and PPP. This may result from salt stress-induced insufficiencies in metabolite supply, ATP energy, and reducing NADH, thereby hindering normal cell growth. Inhibition of the TCA pathway obstructs the entry of pyruvate, an oxidative degradation product from the EMP pathway, into the TCA cycle. Consequently, the PPP becomes activated, gradually replacing the TCA pathway as the primary biochemical respiratory pathway . The present study’s findings revealed that salt stress promotes the anaerobic metabolic pathway in oat root. During anaerobic respiration, pyruvate generated by glycolysis is converted into lactic acid under the influence of LDH; it is then further converted into ethanol under the combined action of ADH and pyruvate dehydrogenase. This process ensures the smooth operation of the EMP pathway by oxidizing NADH to NAD+, providing ATP for anaerobic respiration . This aligns with numerous research findings, suggesting that enhanced anaerobic respiration metabolism in oat root and increased PDC, ADH, and LDH activities can improve plant tolerance to salt stress . Under salt stress, the PDC and ADH enzyme activities in the roots of both oat cultivars exhibited an initial increase followed by decrease. However, overall, the salt-tolerant cultivar Bai2 displayed a higher level of enzyme activity compared to the salt-sensitive cultivar Bai5, indicating its primary reliance on pyruvate ethanol fermentation for anaerobic respiration energy supply. Conversely, while LDH enzyme activity in Bai5 continued to increase with increasing salt stress, it remained higher than in Bai2 overall. This suggests that Bai5 primarily relies on pyruvate lactate fermentation for anaerobic respiration energy, and the significant increase in lactate metabolism leads to substantial lactate accumulation in the oat plant, resulting in cytoplasmic acidification and ultimately reduced salt stress tolerance. Due to the design of this experiment and soil conditions, it is unclear whether the effect of salt stress on oat root respiration metabolism is caused by salt toxicity, low oxygen conditions due to salt soil compaction, or both. It is necessary to solve this problem through hydroponic experiments or more detailed experimental designs in the future.
Under salt stress conditions, a total of 7174 proteins were analyzed in the roots of two oat cultivars, resulting in the identification of differentially expressed proteins spanning multiple functional categories. Following an interaction network analysis and the categorization of main functions, it was revealed that the majority of differentially expressed proteins participate in protein synthesis, carbohydrate and energy metabolism, redox processes, and other functions ( ). Plants are known to adapt to stress by maintaining a protein balance, with ribosomes playing a critical role in regulating cell growth and development through protein synthesis . This study identified a substantial number of differentially expressed proteins associated with protein synthesis, leading to the conclusion that it represents a key metabolic process used by oat seedling root systems to cope with salt stress. These differentially expressed proteins primarily included various ribosomal proteins, translation initiation factors, and peptide chain release factors. Notably, most of these proteins were downregulated under salt stress, suggesting a decrease in overall protein synthesis in oat seedling roots, and consequently, in their growth rate. This observation aligns with existing research on kidney beans and suggests an adaptation involving reduced water consumption . Notably, the upregulation of some proteins involved in protein synthesis highlights the complexity of this process . Carbohydrates, which are widely present in plants, are both products of photosynthetic assimilation and substrates of respiration . This study focused on carbohydrate and energy metabolism processes, primarily glycolysis and the tricarboxylic acid cycle. Glycolysis involves breaking down glucose in plants to produce energy. Network interaction correlation and verification resulted in the identification of the main proteins related to glycolysis (A0A1J7HFP8, A0A3B6DHS5, A0A3B6B850, and M8A623), all of which were downregulated under salt stress conditions. Pyruvate dehydrogenase, which is crucial in glycolysis for catalyzing the oxidative decarboxylation of pyruvate to acetyl CoA and NADH, is also a key factor for glycolysis entering the tricarboxylic acid cycle . The upregulation of this enzyme suggests a stronger respiratory function and capacity for energy production in Bai2 under severe salt stress. Indeed, it is widely acknowledged that cellular metabolism primarily relies on the tricarboxylic acid cycle pathway, which is essential for plant life activities and provides significantly more energy compared to glycolysis. Among the three differentially expressed proteins involved in this pathway, only A0A1D6QPT3 was upregulated and the other two were downregulated, indicating inhibited respiration in oat roots under salt stress. When subjected to abiotic stress, elevated reactive oxygen species production in plants hinders their growth . Consequently, ROS clearance becomes a crucial mechanism for plant tolerance to abiotic stress. Antioxidases, such as superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), are a class of differentially expressed proteins involved in ROS clearance. The identified PODs (J3MP40, M7ZH16, A0A077RUR2, A0A0J8BPV6, A0A2T7DXQ7, A0A3B6RDG1, and I1HF19) were all downregulated following salt stress, suggesting their increased sensitivity to this stress. Conversely, CAT (A0A0Q3F350, A0A3B6PHD6, I1I9A3, and A0A2T7DAI6) was upregulated after salt stress, with a more pronounced upregulation in Bai5. Extensive studies in plants like Arabidopsis, rice, wheat, and rapeseed have demonstrated the involvement of proteins like POD, SOD, and CAT in stress responses [ , , ]. The findings of this study support the presence of multiple ROS clearance mechanisms contributing to salt tolerance in oat.
4.1. Overview of Experimental Site 4.2. Test Materials 4.3. Experimental Design 4.4. Determination Indexes and Methods 4.5. Data Processing and Bioinformatics Analysis Microsoft Excel 2010 was used for data calculation and processing and generating charts, and SAS 9.0 software was used for significance analysis ( p < 0.05). We performed one-way ANOVA analysis using the software SPSS (IBM SPSS Statistics Version 19.0) to associate different physiological indexes between the treatments. The identified proteins were annotated using common functional databases, including COG, GO, and KEGG databases. Finally, the differential proteins underwent functional analysis to identify significantly enriched GO terms and KEGG pathways.
This experiment was conducted in the greenhouse of the Oat Industry Research Center of Inner Mongolia Agricultural University. Greenhouse culture conditions included 16 h light at 25–30 °C and 8 h of darkness at 15–18 °C, with natural light and plant supplementary light supplement. Oat plants were grown in plastic barrels with an upper diameter of 24 cm, a lower diameter of 22 cm, and a height of 25 cm. The substrate was taken from the 0–20 cm topsoil layer of a field, with a soil capacity of 6.5 kg per pot. The basic soil nutrients are shown in .
The two tested cultivars, the salt-tolerant cultivar Bai2 and salt-sensitive cultivar Bai5, were selected and bred by the Baicheng Academy of Agricultural Sciences in Jilin Province .
The salt components used in the test were NaCl and Na 2 SO 4 mixed in a 1:1 molar ratio. Three salt stress concentrations were established: control (0 mM), moderate stress (100 mM), and severe stress (150 mM). The two oat cultivars, Bai2 and Bai5, were subjected to these treatments, resulting in a total of six treatment combinations. Each combination was replicated 3 times with 4 pots per replicate for a total of 72 pots. Prior to seeding, 150 kg·hm −2 of diammonium phosphate fertilizer was applied, and 1.5 L of salt stress solution (100 mM or 150 mM) was added to the pot. Control pots received an equal volume of water. When the soil moisture content was suitable, 40 seeds were planted per pot. Every 3 days, 250 mL of hydration was provided. The indexes were determined 3 days after the emergence of oat seedlings.
4.4.1. Determination of Biomass and Root–Shoot Ratio of Oat Seedlings 4.4.2. Determination of Root Morphological Indexes 4.4.3. Determination of Root Ion Content 4.4.4. Determination of Root Respiration Rate 4.4.5. Measurement of Enzyme Activity Related to Respiratory Metabolism 4.4.6. Root Proteome Determination and Methods On the day of sampling, five representative plants were selected from each treatment. Their root substrate was rinsed with tap water, and surface moisture was absorbed with filter paper. The fresh weight of both the aboveground and underground parts was measured. For the aboveground parts, the fresh weight was recorded individually for each plant. After numbering, they were placed in labeled envelopes. To inactivate enzymes, the green parts were subjected to a temperature of 105 °C for 15 min, followed by drying at 80 °C until a constant weight was achieved. The dry weight was measured for each plant, and the average value for each treatment was calculated to obtain the aboveground and underground biomass.
Three representative plants from each treatment and replicate were selected. Their root systems were scanned and stored on a computer using a digital scanner (Epson V700, Beijing, China). WinRhizo PRO 2013 root analysis system software (Regent Instruments, Quebec, QC, Canada) was used for quantitative analysis, yielding data such as the total root length, surface area, volume, and mean diameter.
Dried roots from both Bai2 and Bai5 were digested using a HNO 3 acid solution in a microwave digestion instrument (Multiwave 3000, Anton Paar GmbH, North Ryde, Australia). We determined the sodium, potassium, calcium, and magnesium contents in the digested solution using an inductively coupled plasma–optical emission spectrometer (iCAP 6000 series, Thermo Fisher scientific, Waltham, MA, USA), as per the manufacturer’s instructions. Using the SPSS software (IBM SPSS Statistics Version 19.0), we performed a one-way ANOVA of associations of different element concentrations between the treatments, where p < 0.05 and 0.01 were considered markedly significant and highly significant, respectively.
The root respiration rate was measured as the oxygen consumption rate using an Oxytherm oxygen electrode (Hansatech, King’s Lynn, Norfolk, UK). Fresh root samples (500 mg) from each replicate were used for the measurements. NaF, malonic acid, Na 3 PO 4 , hydroxyzine salicylic acid, and sodium cyanide were employed as inhibitors of the Embden–Meyerhof pathway (EMP), tricarboxylic acid cycle (TCA), and pentose phosphate pathway (PPP), respectively. The difference in oxygen consumption between root treated with and without a specific inhibitor was considered to represent the respiration rate of the corresponding pathway. Basic biochemical pathways and the total respiratory rate (Q) were determined using Oxytherm liquid-phase oxygen electrodes (Chlorolab-2, Hansatech Scientific Instruments, Pentney, UK). Three plants per treatment were sampled, and their roots were immediately washed and stored in a 5 mM MES (pH of 5.5; 1 mM CaSO 4 ) buffer solution for testing. Fresh root samples (0.1 g) were weighed, cut into pieces, and placed in the instrument’s reaction cup. The root respiration rate was expressed as the amount of oxygen consumed per unit fresh weight per unit time. Specific respiratory pathways were determined using the Yu Rangcai method. The following specific inhibitors were employed: 10 mM NaF (enolase, specifically inhibiting the EMP pathway), 50 mM malonic acid (specifically inhibiting the tricarboxylic acid cycle), and 10 mM Na 3 PO 4 (specifically inhibiting the pentose phosphate pathway). Each inhibitor was prepared in 0.05 M phosphate buffer (pH 6.5).
Pyruvate decarboxylase activity determination A fresh oat root sample (0.1 g) was weighed and homogenized thoroughly with 1 mL of Tris buffer on ice. The homogenate was then centrifuged at 16,000× g for 20 min at 4 °C. The supernatant was collected and kept on ice for measurement. The assay was performed according to the instructions provided by Shanghai Qiyi Biotechnology Co., Ltd. (Shanghai, China), with colorimetric measurements at 340 nm. The absorbance values were recorded at 15 and 75 s, and the change in absorbance (ΔA) was calculated as ΔA = A15 − A75. 2. Determination of alcohol dehydrogenase activity Samples were pretreated with PDC. Reagents were added in accordance with the steps provided by Shanghai Qiyi Biotechnology Co., Ltd. After zeroing the instrument with distilled water, colorimetric readings were taken at 340 nm. Absorption values at 15 s and 75 s were recorded, and the difference (ΔA) was calculated as A15 − A75. 3. Determination of lactate dehydrogenase (LDH) activity A fresh oat root sample (0.1 g) was weighed and combined with 1 mL of extract solution. The mixture was fully homogenized under ice bath conditions before it was centrifuged at 4 °C and 8000× g for 10 min. The supernatant was then collected and kept on ice for measurement. Following the instructions provided in the kit from Shanghai Qiyi Biotechnology Co., Ltd., the reagent was added, mixed thoroughly, and incubated at room temperature for 3 min. The absorbance of each tube was determined at 450 nm using distilled water as the blank. Finally, the change in absorbance (ΔA) was calculated as A(determination tube) − A(CK). 4. Determination of malate dehydrogenase (NAD-MDH) activity A 0.1 g sample of fresh oat root was weighed. Then, 1 mL of extract was added, and the mixture was fully ground into a homogenate under ice bath conditions. It was then centrifuged at 4 °C and 8000× g for 10 min. The supernatant was collected and placed on ice for further measurement. Following the kit instructions provided by Shanghai Qiyi Biotechnology Co., Ltd., the reagent was added, and both the initial absorbance value (A1) and the absorbance value (A2) after 1 min of reaction at 340 nm wavelength were immediately recorded. Finally, the change in absorbance (ΔA) was calculated using the formula ΔA = A1 − A2.
Root pretreatment For each treatment, fresh oat root samples were taken. They were washed with tap water, rinsed with deionized water 3–5 times, and dried with filter paper. They were then loaded into 2 mL frozen tubes, quickly frozen with liquid nitrogen, and stored at −80 °C for measurement. Three oat roots were used as a biological replicate for each treatment, with a total of three replicates. 2. Extraction of total protein from oat root Frozen samples intended for measurement were crushed using a liquid nitrogen precooled crusher. The resulting powder was further ground with liquid nitrogen to a fine consistency. Lysis buffer (100 mmol·L −1 NH 4 HCO 3 + 6 mol·L −1 CH 4 N 2 O + 0.2% SDS, pH = 8) was added to the powder in a 1:10 ( w/v ) ratio and mixed thoroughly. Ultrasonication was carried out at an amplitude of 22% with on cycles lasting 0.2 s and 2 s off cycles for a total of 60 s. The mixture was then extracted at room temperature for 30 min. Subsequently, centrifugation was performed at 15,000× g for 1 h at 10 °C. The supernatant was collected, aliquoted, and frozen at −80 °C. 3. Protein quantification Protein concentrations were determined using the Bradford method. The protein concentration of each sample was calculated according to the curve formula (μg·μL −1 ). 4. Filter-aided sample preparation (FASP) Following protein quantification, 200 μg of protein solution was aliquoted into a centrifuge tube. Dithiothreitol (DTT) was added to achieve a final concentration of 25 mM, and the reaction was incubated at 60 °C for 1 h. Iodoacetamide was then added to reach a final concentration of 50 mM, and the mixture was incubated at room temperature for 10 min. The reduced and alkylated protein solution was subsequently transferred to a 10 kDa ultrafiltration tube and centrifuged at 12,000× g for 20 min. The solution at the bottom of the collection tube was discarded. A total of 100 μL of issolution buffer (6 mol·L −1 CH 4 N 2 O + 100 mmol·L −1 TEAB, pH = 8.5) was added, followed by centrifugation at 12,000× g for 20 min. The solution at the bottom was again discarded, and this step was repeated three times. A new collection tube was employed. Moreover, 1 μg·μL −1 trypsin 3 μL and 100 mmol·L −1 TEAB buffer 500 μL were added to the ultrafiltration tube. The reaction was allowed to proceed overnight at 37 °C. The following day, the mixture was centrifuged at 12,000× g for 20 min, and the digested peptide solution at the bottom of the tube was collected. Another 50 μL of dissolution buffer was added to the ultrafiltration tube, followed by centrifugation at 12,000× g for 20 min. The resulting supernatant was combined with the previously collected peptide solution, yielding a total volume of 100 μL of enzymatically hydrolyzed sample. This sample was then freeze-dried for further analysis. 5. Nanoflow reversed-phase chromatography-Q Exactive for protein analysis A solution of 20 μL containing 2% methanol and 0.1% formic acid was prepared for this experiment. The solution was centrifuged at 12,000× g for 10 min, and the supernatant was collected for sample loading. A sample volume of 10 μL was loaded using a loading pump with a flow rate of 350 nL·min −1 for 15 min, while the separation flow rate was 300 nL·min −1 . 6. Mass spectrum data analysis The database uniprot-Pooideae361804_20170619.fasta.fasta (362,934 sequences) was utilized for mass spectrometry analysis, which was conducted using a Thermo Q Exactive mass spectrometer (Thermo Fisher scientific, Waltham, MA, USA). Peptide Spectrum Matches (PSMs) with confidence levels exceeding 95% were deemed trusted PSMs. Proteins containing at least one unique peptide segment (a specific peptide segment) were considered trusted proteins. Only credible peptides and proteins were retained, and FDR verification was carried out to eliminate those with an FDR greater than 1%. For comparisons between pairs of samples, the mean of protein difference multiples in different replication groups was employed as the difference multiple for those samples. Significance was assessed using the t -test, yielding p -values.
study investigated the response mechanisms of different salt-tolerant oat cultivars to salt stress. Label-free technology was employed to quantitatively analyze 18 samples of oat root from both cultivars. We identified candidate differentially expressed proteins and explored key metabolic pathways to provide new references for future research. Interaction network analysis revealed that at a concentration of 300 mM, five differentially expressed proteins interacting with metabolic pathways were detected in Bai2. These pathways primarily involve antioxidant enzymes, pyruvate metabolism, glycolysis, the tricarboxylic acid cycle, and energy metabolism. In the following research, their functions will be validated through genetic modification and other methods, and the research results will be applied to molecular breeding of crops such as oat. By using Oxytherm liquid-phase oxygen electrode, we found that Bai2 primarily utilizes pyruvate ethanol fermentation for its anaerobic respiration energy supply, while Bai5 relies mainly on pyruvate lactate fermentation for the same purpose. This significant discovery revealed for the first time from the perspective of respiratory metabolism that different salt-tolerant oat cultivars adapt to salt stress in different ways to maintain normal growth and developmentThe experimental results provide new insights into plant adaptation to salt stress from the perspective of respiratory metabolism.
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Invasive listeriosis in Finland: surveillance and cluster investigations, 2011–2021 | 6a491fc1-639a-4184-bc90-925102d26215 | 10468812 | Microbiology[mh] | Invasive listeriosis is caused by the foodborne pathogen Listeria monocytogenes. It mostly affects people with a predisposing condition, such as immunocompromised persons, the elderly, pregnant women, and neonates, causing sepsis, infections in the central nervous system, stillbirth, and foetal death . The mortality rate can be as high as 30% . L. monocytogenes is ubiquitous in nature and can withstand variable environmental conditions, such as temperatures from 2 to 45 degrees Celsius, low pH, and high salinity . It has been shown to persist in food processing plants, causing a significant public health burden and possibly prolonged outbreaks over many years . In 2021, listeriosis was the fifth most reported zoonotic disease in the EU and, along with West Nile virus infections, had the highest mortality rate . In that year, Finland had the highest notification rate of listeriosis in the EU, and during 2017–2021, the annual notification rate was two to four times greater than the EU average . ECDC has envisioned whole genome sequencing (WGS) to be the method of choice for typing of microbial pathogens to improve disease surveillance, outbreak investigation, and the evaluation of prevention policies . In Finland, WGS was introduced in 2015 for listeria confirmation and typing, and interviews of listeriosis cases were launched in 2016 at the Finnish Institute for Health and Welfare (THL). In 2015 and before, pulsed field gel electrophoresis (PFGE) was used for the molecular typing of listeria. We collected surveillance data of listeriosis cases notified to the Finnish Infectious Disease Register (FIDR) in 2011–2021, laboratory data of patient samples, and patient interview data. These data were compared to the listeria findings from food and food production plants collected as part of the outbreak investigations to describe how the change in the typing method affected the listeriosis surveillance, and to describe the risk factors for infection.
Listeriosis surveillance Monitoring of L. monocytogenes in foods Microbiological methods Since 1995 in Finland, clinicians and clinical microbiology laboratories report data on culture-confirmed invasive listeriosis cases (gender, age, place of residence, caretaking institution, isolation site of the bacteria, and sampling date) to the FIDR and submit L. monocytogenes isolates (from normally sterile tissue and from foetus, stillborn, newborn, or the mother) to THL . We formed a database of cases notified to the FIDR between 1 January 2011 and 31 December 2021. We linked the laboratory data of patient samples in 2011–2021 and patient interview data in 2016–2021 with the data from the FIDR, and described the cases according to gender, age, and hospital district. The incidence rate ratios (IRRs) with 95% confidence intervals (CI) were calculated for 5-year age groups. Statistical analyses and data description were conducted using the Stata 17.0 software (StataCorp LLC, USA). With Microsoft Excel (version 2202), a linear line estimator for incidence was fitted using a least squares method. Depending on the isolation site, the cases notified in 2011–2021 were classified as septic, meningitis, or materno-foetal. Materno-foetal cases include pregnancy-associated listeriosis or listeriosis in a newborn (infection in both the mother and the newborn was counted as a single case with the mother being included in the data set). For cases notified in 2016–2021, data on underlying conditions, medications, and the consumption of risk-associated food products were collected by local healthcare staff or THL from patients or their family members using an online questionnaire. The questionnaire focused on the consumption of ready-to-eat meat products and cold cuts; cold- or hot-smoked, cured, and raw fish products; unpasteurized milk and dairy products; frozen vegetables; and convenience food consumed within two weeks prior to the onset of symptoms. In addition, the questionnaire asked about travelling in Finland or abroad and eating in a restaurant two weeks prior to the onset of symptoms, as well as knowledge of risk foods, the habit of checking the fridge temperature, and the best-before dates of the food products. Mortality data were obtained from the Finnish Population Registry. To estimate the case fatality rate, deaths within 30 days of sampling were considered listeriosis related.
The local official food control laboratories send L. monocytogenes isolates from food samples to the Finnish Food Authority (before 1 January 2019 Finnish Food Safety Authority Evira) strain collection and, when needed, for further characterization and typing . The samples have been collected as part of official sampling carried out by authorities, or as part of food business operator’s Hazard Analysis and Critical Control Points (HACCP) plan. Additionally, samples taken as part of food- or waterborne outbreak investigations are sent to the Finnish Food Authority. The L. monocytogenes isolates sent by the local official food control laboratories to the Finnish Food Authority were further typed using the same methods as in THL. In 2011–2017, the strains were typed using PFGE, and after 2018, using WGS. The WGS typing was done using the same method and the same schemas as in THL. National surveys to estimate the presence and levels of L. monocytogenes , especially in vacuum-packed smoked or cured fish products and ready-to-eat products, have been performed within the national monitoring of zoonoses . In 2012–2014, sliced heat-treated or cold-smoked ready-to-eat meat products and, in 2015–2016, sliced cheeses were monitored for L. monocytogenes. Samples were taken from retail randomly. All L. monocytogenes isolates detected in these surveys are stored at the national reference laboratory in the Finnish Food Authority.
The detection and isolation of L. monocytogenes from patient samples were performed in clinical microbiology laboratories. The isolates were sent to the reference laboratory (THL) for confirmation and further typing. In 2011–2014, the species confirmation and serogroup determination were performed using PCR and molecular typing using PFGE as previously described . In 2015, WGS was introduced for listeria confirmation and typing, and it was used in parallel with conventional methods for one year. Since 2016, WGS has been the only method for species confirmation and molecular typing for listeria. DNA isolation, library preparation, and sequencing on MiSeq (Illumina Inc.) were performed as earlier described . Ridom SeqSphere+ (Ridom GmbH, Münster, Germany) was used for 5′ and 3′-end quality trimming, assembly using Velvet, and for retrieving serogroup and 7-gene multilocus sequence type (MLST) information. For the determination of serogroups from the sequence data, a Ridom SeqSphere task template based on Doumith et al. and Lee et al. was used. For the determination of 7-gene MLST, a task template based on pubMLST.org was used . For creating a core genome MLST (cgMLST) protocol, the Target Definer function of Ridom SeqSphere+ was used to identify 1503 target loci shared by the L. monocytogenes Finland 1998 strain, which was chosen as a reference (Genbank accession no. NC_01747.1.) , and 45 other complete genomes obtained from Genbank. Default filter settings were used to define the target loci, and the default thresholds (90% of sequence identity and 100% of full length to the reference genome NC_01747.1.) were used for the gene-by-gene analysis. The presence of at least 90% of core genome targets and an average coverage of at least 30-fold were required for each assembled genome to be included in the analysis.
Listeriosis surveillance and case interviews PFGE and WGS results of patient isolates L. monocytogenes in foods Comparison of food and patient isolates There were 16 mutual PFGE AscI profiles in patient samples in 2011–2015 and in food and food production plant samples in 2011–2016 ( ). Of all PFGE AscI typed isolates, 40% (113/282) of patient strains and 41% (182/439) of food and food production plant strains belonged to these 16 mutual profiles. PFGE profiles 21, 70, and 72 were found only in fish, profile 44 only in meat products, and profiles 42 and 68 only in vegetable products. The three most common mutual PFGE AscI profiles, 14, 62 and 96, were found mainly in raw fish or fishery products. The majority (63%, 69/109) of listeria contaminated fish and fishery products were likely to be consumed uncooked (the strain was isolated from cured or cold-smoked salmon). In 2015–2021, 687 strains of L. monocytogenes were sequenced. These originated from patients (490), food items (184), and samples from food production environments (13). The MLST type of seven patient samples and two food samples could not be determined. The strains showed 49 MLST types, 16 of which contained nine or more patient isolates ( ). These 16 most common MLST-types included 408/490 (83%) of patient isolates and 23 clusters defined by cgMLST, each containing five or more patient isolates. Of all 490 patient isolates analysed, 262 (53%) fell into these clusters. In 12 of the clusters, also food or environmental isolates were detected.
In 2011–2021, a total of 722 invasive listeriosis cases (range 42–93 cases/year, annual incidence 0.8–1.7/100000) were notified in Finland ( ). Cases were reported throughout the country, with males and females almost equally represented (48% and 52%, respectively). The median age of cases was 75 years (range 8–101 years), and the incidence rate was 11-fold in those aged >75 years compared to other age groups (IRR 10.6, 95% CI 9.2–12.3). The percentage of septic infections ranged between 74 and 92 per year ( ). Of all cases, 4% (11/722) were materno-foetal. The median age of the mothers was 30 years (range 22–43 years). Serogroups IIa (67%, 487/722) and IVb (22%, 160/722) accounted for most of the cases, while serogroups IIc and IIb caused 6% and 2% of the cases, respectively. The mean annual case fatality rate was 22% (range 13–30%). Interview data were obtained for 68% (304/449) of the cases in 2016–2021. For 38% (115/304) of the interviewed cases, the person who answered the questionnaire was the patient’s spouse or other relative. Twelve cases (4%) did not have any underlying illness, and of them, four were pregnant women. Of the cases with underlying disease(s), 51% (154/301) had heart disease, 29% (87/301) diabetes, 21% (62/301) other cancer than leukaemia, 20% (60/301) lung disease, 15% (44/301) chronic kidney disease, 13% (40/301) rheumatics, 11% (34/301) gastrointestinal disease, 7% (21/301) chronic liver disease, 7% (20/301) immunological illness, 6% (18/301) leukaemia, 4% (12/301) alcohol addiction, and 1% (4/301) had undergone a tissue transplant. In the last three months prior to listeria infection, immunosuppressive medication had been used by 40% (118/296) of the interviewed cases and medication to prevent acidity of the stomach by 50% (148/298). Two weeks prior to start of symptoms, 24% (71/297) of the cases had been in inpatient treatment, 29% (84/294) had eaten in a restaurant, 13% (38/285) had travelled in Finland, and 5% (14/283) had travelled abroad. Ready-to-eat meat cold cuts as well as cured and cold- or hot-smoked fish were the most consumed risk foods reported ( ). Of the cases, 34% (96/286) reported a habit of checking their fridge’s temperature once a week, 9% (25/286) once a month, and 26% (73/286) less frequently, and for 32% (92/286) the information was not available. Furthermore, 63% (179/286) reported throwing out outdated products once a week, 10% (29/286) once a month, and 9% (25/286) less frequently, and for 19% (53/286) the information was not available. Of the cases, 57% (94/166) did not have knowledge of risk foods for listeriosis prior to infection. Since starting the listeriosis case interviews in 2016, eight listeriosis outbreaks could be solved with both epidemiological and microbiological evidence.
In 2015, both PFGE and WGS were used for typing 45 isolates. Eighteen MLST types and 30 PFGE types were identified ( ). Each MLST type contained 1–3 PFGE types. MLST and PFGE were not completely concordant, since two MLST types (ST-155 and ST-18) both contained PFGE types 2 and 5. Three clusters were identified using WGS: ST-155 with five isolates, ST-120 with two isolates, and ST-14 with two isolates. Apart from one isolate, the same clusters were also recognized using PFGE. The ST-155 cluster corresponded to PFGE type Ascl-2, the ST-120 cluster corresponded to PFGE type Ascl-62, and the ST-14 cluster corresponded to PFGE type Ascl-14. One isolate with PFGE type Ascl-5 belonged to the ST-155 cluster in WGS. PCR and WGS were concordant for serogrouping, as all 45 isolates showed same results with both methods. In 2011–2015, 282 strains were typed and a total of 89 different Ascl profiles were detected in PFGE, the most common being 225, 62, and 96 (26, 18, and 18 cases, respectively) ( ). In 2015–2021, 49 different 7-gene MLST types were found from 483 isolates with ST-7, ST-6, and ST-9 being the most common (52, 48, and 41 cases, respectively) ( , Supplementary Table 1 ).
In 2011–2021, the Finnish Food Authority received 4939 L. monocytogenes isolates from foods and food production plants (average 449/year, range 284–572) from 23 local official food control laboratories. Isolated strains represented 3353 food or food production plant samples (average 305/year, range 202–394). In 2011–2021, 3.1% (104/3353) of food samples (3–18 samples per year) contained more than 100 colony-forming units (CFU)/g of L. monocytogenes. In total, 626 (13%) L. monocytogenes isolates from foods or food production plants were genotyped in 2011–2021. During 2011–2017, 105 different PFGE AscI profiles were detected, type 96 being the most common (90/439, 21%). In 2017–2021, when WGS was used as the typing method, 36 different 7-gene MLST types were identified among the strains typed. In 2012–2014, the Finnish Food Authority received results of 793 sliced, heat-treated, or cold-smoked ready-to-eat meat product samples from local official food control laboratories as part of an official survey. Of the products, 94% were manufactured in Finland, and 47% of the food samples contained only domestic meat. Of the analysed food samples, 82% (652/793) were stored in packaging gas and 16% (126/793) were vacuum-packed. In 11% of the samples, the temperature of the product in retail was above the statutory limit of 6.0 degrees Celsius. At the end of shelf-life, L. monocytogenes was detected in 10 (1.3%) samples. In nine of those samples, the count of L. monocytogenes was 10 CFU/g or below. One bacterial count result was missing. In 2015–2016, the Finnish Food Authority received results of 398 sliced cheese samples from local official food control laboratories. The samples were from domestic and foreign, packed, sliced cheeses in retail. L. monocytogenes was not found.
Finland, a slight increase in the incidence of invasive listeriosis has been seen in the past 10 years. A similar trend has been seen in Sweden . At the EU level, the overall trend for listeriosis has been stable during 2017–2021 . In our study, listeriosis was most common in age groups over 75 years old. Additionally, in other EU countries, most cases have been reported among the elderly, which is partly explained by the ageing population and chronic age-related illnesses . In Finland, the proportion of those aged over 64 years is expected to rise from 23% in 2021 to 33% in 2070 . The higher number of people more susceptible to listeriosis may lead to increases in listeriosis cases if appropriate control measures are not taken. In Finland, listeriosis in pregnant women is rare. In 2011–2021, 4% of notified cases in Finland were reported as materno-foetal, all of which were sporadic cases. In England and Wales in 2020, 20% of all reported listeria cases were pregnancy-associated . In the literature, one in seven cases of listeriosis is estimated to occur in pregnant women . Awareness of listeria has been shown to reduce the consumption of high-risk foods; awareness was higher in mothers older than 25 years when compared to younger mothers . In Finland in 2020, the mean age of birth givers was 31 years, with only 1.2% of all birth givers being under 20 years of age . Food recommendations for families with children were published in 2016 by the National Nutrition Council and THL and updated in 2019 . Pregnant women are advised to eat fish products only properly heated and to avoid eating sushi, roe, and foods containing raw fish to avoid the risk of listeriosis. In addition, unheated meat products, raw or unpasteurized milk, cheese made from unpasteurized milk, soft cheeses, frozen vegetables, and processed foods are recommended to be avoided or consumed only properly heated. Unheated meat cold cuts are deemed safe if consumed well before the use-by date. The avoidable risk foods differ somewhat between countries, especially regarding fish products, fruits, and vegetables . In Finland, the recommendations are communicated to mothers at the maternity clinics, which are free of charge for all mothers. According to the Medical Birth Register, they are widely utilized with only 0.2–0.3% of the mothers not attending them during pregnancy . Thus, it can be reasoned that Finnish mothers are probably well aware of the risk of listeriosis. The case fatality rate in Finland was slightly higher (22%) than the EU average (14% in 2021 ). However, in the EU data, the outcome was reported for only 65% of the confirmed cases. In the literature, mortality rates of 20–30% are described . Most of the interviewed cases in our study were immunocompromised due to an underlying illness or medication used. Immunosuppressing treatments and conditions, such as liver and renal diseases, and diabetes mellitus, are known risk factors for invasive listeriosis . According to a population-based Finnish health survey from 2017, 18% of males over 65 years of age had coronary heart disease, 25% had diabetes, and 7% had chronic obstructive pulmonary disease while in women these illnesses were less common (12%, 18%, and 3%, respectively) . Compared to these survey data, the listeriosis cases in our study had more comorbidities than the general population. Despite being immunocompromised, the cases had often consumed high-risk foods, such as ready-to-eat meat products, cured and cold- or hot-smoked fish, and salami or other sliced sausages. Over half of the cases did not have knowledge of high-risk foods for listeriosis prior to infection. In addition, one third of cases checked their fridge’s temperature monthly or less often; one fifth reported throwing out outdated products monthly or less often. This suggests that recommendations about high-risk foods and proper food storage should be highlighted also to other risk groups than pregnant women. THL and the Finnish Food Authority have produced a leaflet on listeriosis for doctors to distribute to patients . However, more proactive communication about listeriosis to risk groups is needed. People at higher risk of listeriosis can reduce the risk by thoroughly cooking food, heating ready-to-eat foods till they are steaming hot, and avoiding outdated products as well as risk foods that are not properly heated. In addition, high-risk foods should not be served for those unable to evaluate the risk of listeriosis themselves such as the elderly suffering from forgetfulness or persons living in institutional care. Since L. monocytogenes is capable of growth in refrigeration temperatures and growth is faster in higher temperatures , it is important to maintain a low temperature in the fridge and consume ready-to-eat products well before the use-by date if not heated. The fridge temperature should be checked regularly, and the proper temperature (<+6 °C; <+3 °C for fish products ) should be highlighted to consumers. Fridge thermometers could be provided for the elderly along with stickers for the fridge door reminding them of the correct temperature. Half of the cases in our study had used medication to treat gastric acidity. Use of proton pump inhibitors (PPI) and other medications that neutralize or inhibit gastric acid are considered to increase the risk of listeriosis, possibly because a low gastric pH is a natural defence against listeria . The same applies also for other enteric pathogens, such as Salmonella and Campylobacter . The potential risks of PPI usage should be better communicated to the public, since their use has increased in recent decades and is more common in the elderly , who are also more likely to have immunocompromising conditions. In over one third of the cases, the patient’s spouse or other relative answered the questionnaire due to the frail condition or forgetfulness of the patient. The incubation period of listeriosis varies by clinical manifestation, but the median is estimated to be 11 days with most cases occurring within four weeks . Old age, forgetfulness, and second-hand information as well as the long incubation period of listeriosis can lead to a recall bias and affect the exposure results gleaned from the interviews. To minimize the recall bias, interviews should be performed as soon as possible after the onset of illness. Using consumer food purchase data helps to solve outbreaks and has been used also in Finland. The serogroup IIa accounted for most of the listeriosis cases followed by IVb. According to the literature, these serotypes account for most human infections . In 2011–2014, serotyping and PFGE were used to genotype the patient samples in Finland. In 2015, WGS was introduced for listeria confirmation and typing in parallel with PFGE. We identified three clusters that were concordant with both methods. However, the methods were not completely comparable, since two PFGE types were included in two MLST types. Since 2016, WGS has been the sole typing method for patient samples in Finland and its usage has enhanced the detection of listeria clusters. Compared to PFGE, cgMLST provides better discrimination and accuracy in typing listeria strains . By using WGS, in 2015–2021 we identified 23 clusters with five or more patient isolates, many of which persisted for years. Since 2018, the Finnish Food Authority started typing food isolates with a similar method to that used for patient isolates, enabling the comparison of listeria strains. We found matching food isolates that gave microbiological evidence to support epidemiological findings in listeriosis outbreak investigations. This indicates that tracing the source of infection is possible if data from patient interviews, production plants, and product distribution are available. Interview data can be used to direct the typing of food isolates stored in the strain collection. Similarly, information on products in which matching listeria isolates have been found can be used to adjust questionnaires for interviews. In the eight listeriosis outbreaks that could be solved with both epidemiological and microbiological evidence since starting the listeriosis case interviews, 133 people fell ill. The causative food items belonged to fish products (cured and cold-smoked salmon, frozen rainbow trout fillet), fresh or frozen vegetables (frozen corn, chopped iceberg lettuce, cabbage salads), and meat products (turkey cold cuts, frankfurter, meat jelly). The outbreaks were long-lasting, with the duration varying from three to seven years. In 2016–2018, a widespread outbreak affecting five European countries was caused by frozen corn . This outbreak was detected, and the frozen corn as the outbreak source was identified, in Finland. The overall occurrence of listeria in food products in Finland is currently unknown. A small proportion of food samples investigated have contained more than the regulatory limit of 100 CFU/g of L. monocytogenes. In 2010, the Finnish Food Authority received 323 food L. monocytogenes isolates of which the majority were genotyped . Since 2014, only strains suspected to be linked to outbreaks have been typed. In the surveys conducted by the Finnish Food Authority, listeria was not found in sliced cheese samples in 2015–2016. In 2012–2014, listeria was rarely detected in sliced, heat-treated, or cold-smoked ready-to-eat meat product samples tested. Aalto-Araneda et al. investigated the occurrence of listeria in ready-to-eat vacuum-packed cured and cold-smoked fish products in 2014–2015 and found 4.2% of the sampled packages to be positive for L. monocytogenes . Since 2016, no national sampling surveys have been conducted. As the indication for typing strains isolated from food is an outbreak investigation, the occurrence of different MLST types and their genetic relationships to one another in contaminated food products and process environments in Finland has yet to be investigated. Typing food isolates is crucial to both outbreak investigations and for controlling the risk of listeriosis in the food industry.
In Finland, the notification rate for listeriosis is higher than the EU average, and the trend is slightly increasing (from 0.8/100000 in 2011 to 1.3/100000 in 2021), probably due to the ageing population and age-related illnesses affecting the immune system. Pregnancy-related listeriosis is rare, due to effective maternity clinic counselling. Recommendations about high-risk foods for listeriosis should be better communicated to other risk groups, as well as to relatives and people taking care of the elderly. Several listeriosis outbreaks have been detected and food sources identified since ongoing patient interviews and WGS were introduced. Patient interviews and testing and typing listeria isolates in foods and comparing them to patient isolates are crucial for solving outbreaks and determining measures to control invasive listeriosis in Finland.
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Histopathological and Immunohistochemical Characteristics of Different Types of Cardiac Amyloidosis | 58fb3785-69f6-4b75-8063-45682f81f0cf | 11476653 | Anatomy[mh] | The heart is one of the predominant organs affected by systemic amyloidosis. Currently, ten different amyloidogenic precursor proteins are known that can affect the heart; nine of them are responsible for systemic amyloidosis, and one is responsible for localized amyloidosis . The most common type of amyloidosis causing amyloid cardiomyopathy is AL amyloidosis, which is considered a variant of lymphoplasmacytic dyscrasia related to the proliferation of abnormal plasma- or B-cell clones in the bone marrow . It is associated with the overproduction of monoclonal-free immunoglobulin light chains that are capable of inducing amyloidogenesis . AL amyloidosis is a systemic rapidly progressive disease that most often occurs in people over the age of 50. It may develop as a nosological unit or co-occur with multiple myeloma in 7–15% of patients . Heart involvement is reported in 50–80% of cases of this amyloidosis type . Transthyretin amyloidosis (ATTR) is the second most common cause of heart involvement in amyloidosis. The precursor protein of ATTR is transthyretin, which is normally produced in the liver and transports thyroxine and retinol . There are two different types of ATTR amyloidosis: wild-type ATTR (ATTRwt) and hereditary (ATTRv) amyloidosis . ATTRwt is characterized by high cardiotropism, and almost 100% of patients show signs of cardiac involvement. In most cases, previously undiagnosed ATTR-wt is uncovered as an incidental finding at the post-mortem examination of the heart in 25–40% of individuals over the age of 80 years, and in 32% of patients older than 75 years who have heart failure with preserved ejection fraction . ATTRv amyloidosis is caused by a mutation in the transthyretin gene and is characterized by heart involvement in 30–100% of cases. So far, over 140 different mutations associated with ATTRv have been described that can cause various phenotypic manifestations affecting individual organs, including the heart and nerves (polyneuropathy with autonomic dysfunction), or give rise to a mixed clinical phenotype . The precursor protein of AA amyloidosis is serum amyloid A (SAA). It is mainly produced in the liver by proinflammatory stimuli such as IL-6, IL-1, and TNFα in response to inflammation and can contribute to tumor development . Cardiac involvement in this type of amyloidosis is considered rare. Also, apolipoprotein A-I, II and IV amyloidosis, fibrinogen alpha chain and gelsolin amyloidosis have been reported . Cardiac involvement occurs in patients with hereditary systemic amyloidosis where the protein β2-microglobulin variant is the causing agent . Isolated atrial amyloidosis (AANF) caused by the accumulation of atrial natriuretic peptide has been detected more frequently over the past several years . The prognosis in patients with amyloidosis is variable and often depends on cardiac involvement. Cardiac amyloidosis is the cause of death in approximately 61% of the patients . As reported in the literature, cardiac amyloidosis is considered a cause of nearly 10% of all non-ischemic cardiomyopathies . The major problem with recognizing cardiac amyloidosis in its early stages is its ability to mimic other disorders . Later clinical manifestations are determined by the involvement of myocardium, endocardium, pericardium, and large and small coronary arteries, and represented by restrictive cardiomyopathy, systolic heart failure, orthostatic hypotension and cardiac conduction system disorders. Frequently, these syndromes co-exist . This study was undertaken to analyze the percentage of the most common types of amyloidosis revealed in two patient groups. The first group consisted of patients with cardiac amyloidosis detected via EMB during life. The second group included patients in whom postmortem histological examination was performed to determine the number of cardiac amyloidosis cases not diagnosed clinically prior to death. 2.1. Endomyocardial Biopsy Findings2.2. Autopsy Findings2.3. The Degrees of Amyloid Deposition To assess the intensity of amyloid deposits in biopsy and autopsy specimens, a grading system was used with three grades of amyloid depositions (Grade 1—0–20% in the field of vision; Grade 2—20–40%; Grade 3—40% and over) based on the clinical significance of the 20% myocardial lesion threshold . The findings demonstrate that the most intense amyloid depositions both in biopsy and autopsy specimens occurred in patients with AL kappa amyloidosis. No cases with Grade 1 amyloid depositions were identified, while Grade 3 depositions were the most common findings (78%). Grade 2 depositions were recorded in 22% of the studied cases. Among the cases of AL lambda amyloidosis, Grade 1 amyloid depositions were found in 19%, Grade 2 in 30% and Grade 3 depositions in 51% of the deceased persons. Most patients with ATTR amyloidosis had amyloid depositions of Grade 2 (42%). Grade 1 was found in 38% and Grade 3 in 20% of cases. In the AA amyloidosis group, the largest proportion of patients had amyloid depositions of Grade 1 (59%), while Grade 2 was identified in 35% and Grade 3 only in 6% of cases ( and ). 2.1.1. Histology and Immunohistochemistry2.1.2. Clinical InformationIn this study, 46 EMB specimens from 28 males (61%) and 18 females (39%) aged 45–91 years (mean 71 years) were evaluated. In all cases, amyloid deposits were identified with hematoxylin and eosin (H&E) stains as homogenous eosinophilic material (homogenous sties of bright pink color). Amyloid deposits also produced a characteristic apple-green birefringence on the black background under polarized light after Congo red (CR) staining . Based on the immunohistochemistry (IHC) typing results, the diagnosis of AL amyloidosis was established in 21 (46%) patients; 6 of them (29%) had the kappa type of light chain amyloidosis (AL kappa) and 15 (71%) had the lambda type of light chain amyloidosis (AL lambda). ATTR was found in 6 women (25%) and 18 men (75%). Based on the EMB results, AA amyloidosis was diagnosed only in one patient. The correlation analysis between patient age and amyloid type in EMB specimens revealed a peak level of AL amyloid type in people aged 70 years or older (48%), while ATTR amyloidosis demonstrated two peaks—during the eighth (54%) and ninth (30%) decades of life . Most EMBs from patients with AL amyloidosis were characterized by a reticular or pericellular pattern of amyloid deposition. In 83% of the AL kappa amyloidosis cases, amyloid deposits were distributed both in the interstitial and intravascular tissues, and in 17% of cases, they were distributed only in the interstitial tissue. Among the AL lambda amyloidosis cases, both interstitial and vascular amyloid deposits were observed in 73% of cases, while an interstitial pattern was found in 27%. In most specimens, ATTR amyloidosis was characterized by the presence of clumpy multifocal amyloid deposits in the myocardial stroma. Interstitial focal amyloid depositions were found in 75% of the examined specimens, and both vascular and interstitial depositions were found in 25%. A single detected case of AA amyloidosis was characterized by moderate amyloid deposits both in the stroma and blood vessel walls . The largest affected areas were found in patients with AL kappa amyloidosis, where a pericellular pattern of amyloid deposition predominated, and cardiomyocytes adjacent to the amyloid deposits were often atrophic. In our study, the clinical diagnosis of amyloidosis was established in patients with the following underlying conditions: monoclonal gammopathy of undetermined significance (MGUS) in nine patients (six AL lambda and three AL kappa) and multiple myeloma in seven patients (four AL lambda and three AL kappa). The medical history of most patients with transthyretin amyloidosis showed that they had restrictive cardiomyopathy (n = 18), severe cardiac arrhythmias (n = 8) and cachexia (n = 3). In six patients who had clinical signs suggesting infective endocarditis, ATTR amyloidosis was verified by pathological morphology examination. The patient with AA amyloidosis had familial Mediterranean fever for seven years. 2.2.1. Histology and Immunohistochemistry2.2.2. Correlation Analysis of Clinical Data and Post-Mortem Findings of the Group of Autopsy Cases Clinical diagnosis of amyloidosis in the group of autopsy cases was established in 16 patients (31%), while in other cases, the disease was identified only at the autopsy (69%). The number of ante- and post-mortem-diagnosed cases of AL lambda amyloidosis was equal (50% each), while clinical diagnosis of AL kappa amyloidosis was established in 67% of patients and found at the autopsy in 33% of patients. ATTR amyloidosis was identified at the post-mortem examination in 90.5% of cases and only in 9.5% prior to the patient’s death. The clinical diagnosis of AA amyloidosis was established in 37% of patients, and at the autopsy, it was identified in 62.5% of cases . In all patients with clinically diagnosed amyloidosis, it was verified in living patients by biopsy findings in the kidneys, duodenum or rectum. In all cases, the diagnosis was confirmed at the autopsy. In patients with AA and AL amyloidosis, the post-mortem examination revealed amyloid deposits not only in the heart muscle, but also in the kidneys, liver, spleen and gastrointestinal system. In ATTR amyloidosis cases, the autopsy results demonstrated the presence of amyloid deposits in the myocardium and pulmonary blood vessels, as well as in the stomach and duodenum walls. Systemic AL amyloidosis as a primary disease was confirmed in five autopsy cases. In six cases it was concluded that the underlying disease was multiple myeloma with the subsequent development of AL kappa (n = 2) or AL lambda (n = 4) amyloidosis. In four cases of AL amyloidosis, COVID-19 was considered a primary diagnosis. As a comorbidity, AA amyloidosis was recognized in patients with the following underlying conditions: rheumatoid arthritis with kidney involvement (n = 5); nephrosclerosis (n = 3); novel coronavirus disease (n = 6); familial Mediterranean fever (n = 1); and Still’s disease (n = 1). ATTR amyloidosis was a comorbidity in patients with such underlying medical conditions as novel coronavirus disease (n = 7), atherosclerotic encephalopathy (n = 4), infectious endocarditis (n = 2), post-infarction cardiosclerosis (n = 5), and cerebrovascular disorders (n = 3). The analysis included autopsy samples of 52 deceased persons—28 females (65%) and 24 males (35%) aged from 50 to 96 years, who had systemic amyloidosis. The mean age was 86 years. Based on the IHC data, ATTR amyloidosis was identified in 21 patients (40%) and AL in 15 patients; AL kappa was seen in 3 (6%) and AL lambda in 12 (23%). A diagnosis of AA amyloidosis was established in 16 cases (31%). AL amyloidosis cases were characterized by a pericellular pattern of amyloid deposition in the myocardial muscle and associated with partial or sometimes complete atrophy of cardiomyocytes . In addition, amyloid masses were detected in subendocardial and subepicardial tissues. In all cases of AL kappa amyloidosis, both interstitial and intravascular amyloid deposits were present. In the specimens with confirmed AL lambda amyloidosis, both interstitial and intravascular amyloid deposits were detected in 92% of cases, and only interstitial in 8%. ATTR deposition was identified as interstitial foci in 71% of cases; in 24% both interstitial and vascular tissues were affected, and in 5% of cases, only intravascular deposits were found. In the group of patients with AA amyloidosis, 69% had an intravascular pattern of amyloid deposition, and in 31% of patients, the amyloid masses were present both in the stroma and walls of blood vessels . Based on these findings, we can conclude that the AL type of amyloidosis is often characterized by a diffuse pericellular and intravascular deposition of amyloid, while the ATTR type usually demonstrates focal interstitial dense amyloid deposits. AA amyloidosis in most cases is represented by the intravascular deposition of amyloid masses ( and ). A correlation analysis of patient age and gender with amyloid type demonstrated that AL amyloidosis was more common in patients during the eighth decade of life (47%), while 48% of cases of ATTR amyloidosis were found in persons during the 9ninth decade of life, and 52% in persons over 90 years of age. Transthyretin amyloidosis was identified more frequently in female patients. AA amyloidosis was found more often in patients in the ninth decade of life (31%), while in the seventh and eighth decades of life, the proportion of patients was the same (25%) . So far, endomyocardial biopsy (EMB) remains a gold standard for diagnosing multiple ischemic cardiac disorders, such as viral myocarditis, cardiosclerosis, and infiltrative or restrictive cardiomyopathy, e.g., cardiac amyloidosis . The indications for EMB include plasma cell dyscrasia in patients with uncertain results of laboratory testing and instrumental methods, as well as when differentiation of AL cardiac amyloidosis from ATTR is required . Though scintigraphy with Technetium-based radiopharmaceuticals (99mTc) is now recognized as the best non-invasive modality for diagnosing ATTR-wt, 20% of patients may have monoclonal gammopathy, which occurs more frequently in AL amyloidosis. In such cases, the differentiation between ATTR-wt and AL amyloidosis is crucial, and EMB with amyloid typing is the only method permitting definitive diagnosis . The grade of amyloid deposition intensity confirmed by endomyocardial biopsy is the key factor for selecting therapeutic management of the disease, especially in patients with AL amyloidosis. As shown, the involvement of more than 20% of the area of an EMB specimen is associated with lower chemotherapy success rates . Thus, increased cardiac involvement in ATTR or AL amyloidosis will be indicative of a worse prognosis . Our study has highlighted the role of EMB as a unique diagnostic procedure for recognizing this important prognostic factor. Despite the recent advances in non-invasive diagnostics of AL and ATTR amyloidosis, a biopsy procedure is still required in some cases. Though the diagnostic sensitivity of the biopsy of subcutaneous fatty tissue is high (84%) for AL amyloidosis, it is lower for ATTR-wt and ATTR-v amyloidosis (15% and 45%, respectively). Thus, in cases of ATTR amyloidosis, EMB remains the most accurate diagnostic tool for identifying cardiac amyloidosis . In our study, the retrospective analysis of 46 EMBs and 52 autopsies of identified cardiac amyloidosis demonstrated that 83% of patients had either AL or ATTR amyloidosis. A high percentage of AL and ATTR amyloidosis (98%) was determined via EMBs. These findings correlate with the data published earlier by other researchers . It is worth noting that the proportions of AL and ATTR amyloidosis in the autopsy group were lower than those in the group of patients with EMBs. Probably, the difference was underpinned by a higher percentage of AA amyloidosis cases. The detection of a single case of AA amyloidosis via EMB can be explained by the fact that cardiac involvement in systemic AA amyloidosis is less common than in AL or ATTR amyloidosis. But even if the heart is affected, such patients do not have cardiovascular manifestations, as this type of amyloidosis is usually associated with kidney problems. Since most patients with AA amyloidosis present with rapidly evolving nephrotic syndrome, they should conventionally undergo renal biopsy. Even in those cases of AA amyloidosis where cardiac involvement is suspected, the diagnosis is usually made after renal or rectal biopsy. Therefore, EMB is not recommended for patients with this type of amyloidosis. Thus, the prevalence of cardiac involvement in AA amyloidosis could be higher than detected in EMB specimens. This assumption has been proven in our study, as the diagnosis of AA amyloidosis in 31% of patients was established only at the autopsy. According to scientific publications, the prevalence of AL amyloidosis is the same in males and females. This trend was also observed in the EMBs examined in our study (men and women were affected equally), but the autopsy reports showed that the ratio of female to male patients was 2:1. Heart failure occurring in patients with AL amyloidosis is associated with extensive amyloid deposits distributed around cardiomyocytes leading to their atrophy, as well as with the direct cytotoxic effect of amyloidogenic immunoglobulin light chains. Amyloidogenic free light chains can impair cardiomyocyte metabolism, causing lysosomal dysfunction, oxidant stress, apoptosis, and the dysregulation of MAP-kinase (MAPK) signaling pathways and autophagy. These data suggest that a direct intracellular cytotoxic effect of amyloidogenic immunoglobulin light chains is also responsible for the rapid progression and poor prognosis of the disease . In our study, ATTR amyloidosis was found more often in the EMBs of male patients (75%), while at the autopsy ATTR amyloidosis was revealed with the same frequency in men and women. Though most of the patients with clinically diagnosed ATTR amyloidosis are men, the percentage of females with this type of amyloidosis is higher in autopsy series . A remarkable finding was the postmortem identification of clinically undiagnosed ATTR amyloidosis in seven patients who died from the novel coronavirus disease. Five of these patients had treatment-resistant rapidly progressive heart failure. A microscopic examination of their heart samples revealed multifocal interstitial amyloid deposits. Thus, the fatal outcome was caused by heart failure and comorbid conditions. Also, the results of our study have demonstrated that ATTR amyloidosis was mostly diagnosed at autopsy (90.5%), suggesting that ATTR may be an underdiagnosed cause of heart failure in elderly patients. ATTR amyloidosis was found three times more often in the EMBs of male patients than in those of female patients, while at the autopsy, ATTR amyloidosis was detected in men and women almost with the same frequency. This retrospective observational study was performed using data collected from Petrovsky National Research Centre of Surgery and Savelieva City Clinical Hospital No. 31 from 2013 to 2023. The study was conductedof Avtsyn Research Institute of Human Morphology of Federal State Budgetary Scientific Institution “Petrovsky National Research Centre of Surgery” No. 6, 21 June 2024. The study included 98 patients with cardiac amyloidosis who were divided into two groups based on the method of examination. The first group consisted of 46 patients with cardiac amyloidosis revealed by endomyocardial biopsies (EMBs), and the second group included 52 amyloidosis patients in whom the morphological diagnosis of cardiac amyloidosis was made at autopsy. Patients from the autopsy group did not undergo EMB prior to death, and the morphological diagnosis of cardiac amyloidosis was established at the autopsy. To collect anamnesis, we used data from medical records, including reports of biopsies from other hospitals where patients were treated. For performing microscopic examinations, all biopsy and autopsy samples were fixed with 10% neutral buffered formalin solution and embedded in paraffin. Subsequently, paraffin sections with a thickness of 3–5 μm were prepared. Then, hematoxylin and eosin (H&E) and Congo red (CR) staining was performed on all tissue sections. CR staining was performed manually according to the standard protocol . To confirm amyloid deposition, Congo red sections were examined under polarized light. All changes were identified via light and polarized light microscopic examination with polarization blocks (Lieca DM2500, Wetzlar, Germany). Immunohistochemical (IHC) analysis was performed with the Leica TM BOND-MAX ® IHC automatic staining system (Leica, Wetzlar, Germany) using monoclonal Anti-Serum Amyloid P/SAP antibody (1:100, Abcam, Cambridge, UK), polyclonal antibody to Prealbumin (1:100, Cloud-Clone Corp., Katy, TX, USA), monoclonal Anti-Kappa light chain antibody (1:200, Abcam, Cambridge, UK), monoclonal Anti-Lambda light chain antibody (1:200, Abcam, Cambridge, UK), and monoclonal anti-AA amyloid (1:100, Clone C3, Cloud-Clone Corp., Katy, TX, USA). The IHC method was used to detect the presence of all amyloid types based on positive staining results with the antibody to amyloid P-component (monoclonal Anti-Serum Amyloid P/SAP antibody). ATTR amyloidosis was diagnosed by positive IHC staining results with the antibodies to TTR (polyclonal antibody to Prealbumin); AL kappa and AL lambda types were determined via positive IHC staining results with the antibodies to κ- light chain (monoclonal anti-Kappa light chain antibody) and λ-light chain (monoclonal anti-Lambda light chain antibody), respectively, and AA amyloidosis was determined via positive IHC staining results with the antibodies to AA amyloid (monoclonal anti-AA amyloid). To assess the intensity of amyloid deposits, a semi-quantitative grading system was used with three grades of amyloid depositions, as follows: Grade 1—amyloid deposition made up less than 20% in the field of vision; Grade 2—20–40%; Grade 3—40% and over. We used these grading criteria to assess the extent of cardiac involvement. This factor could play an important role in clinical practice, as the effectiveness of the administered therapy may decrease in cases in which amyloid deposition comprises more than 20% in the field of vision . Our study has demonstrated that the clinical diagnosis of cardiac amyloidosis is challenging. The fact that patients with ATTR commonly remain undiagnosed prior to death raises the issue of expanding indications for EMB. Another important conclusion is associated with the intensity of amyloid deposition. As shown, the most intense amyloid deposits both in the examined EMBs and in the autopsy reports were revealed in patients with AL kappa amyloidosis, suggesting that the disease has a more aggressive and quickly progressing clinical course, leading to a poorer prognosis for patients with this type of AL amyloidosis. |
Facial pain, health-related quality of life and trismus-related symptoms up to 5 years post-radiotherapy for head and neck cancer | b7c0319e-ecc0-4f1a-a116-1494d5a6408f | 10651533 | Internal Medicine[mh] | Head and neck cancer (HNC) is the fifth fastest increasing cancer type in Sweden and accounts for over 900,000 new cases annually worldwide . Approximately half of the patients with HNC present with late tumor stages, i.e., T3 or T4 . Treatment strategies for HNC include surgery, radiotherapy (RT) and chemotherapy. In early-stage HNC, unimodal therapy is common, whilst multimodal approaches are usually indicated in more advanced stages . Pain is a common symptom among HNC patients, either due to the cancer itself or as a result of acute or late adverse effects of treatment – . Cancer-related pain after treatment has been associated with negative effects on quality of life . Furthermore, trismus is another burdensome complication that may occur in up to 40% of patients with HNC after oncological treatment – . Trismus, defined as a maximal interincisal opening (MIO) of ≤ 35 mm , has been associated with pain, decreased health-related quality of life (HRQL), as well as eating difficulties and maintaining oral hygiene , . Radiation fibrosis of the masticatory muscles is thought to be the primary cause of postradiation trismus , . Few studies have to date described radiation-induced trismus and its association to pain longitudinally. The longest thus far is by this research group, for up to 3 years after completion of radiotherapy in a cohort half the size of the current study , . The study found facial pain to be reported by over half of the HNC cohort before oncological treatment and at 3 years post radiotherapy. Patients reporting facial pain at the 3-year follow-up had a lower MIO than the patients that did not suffer from facial pain. Other studies have reported on oral pain specifically, with cross-sectional or retrospective study designs , and hence, studies investigating long-term prevalence of pain in patients with HNC after treatment are scarce – . As the prognosis of HNC is improving with modern oncological treatments and a large subgroup of HNC are expected to live many years after their cancer treatment, research focusing on mapping pain and improving HRQL after HNC treatment is warranted. Hence, the aim of this prospective observational study is to investigate the prevalence of facial pain, as well as its effect on HRQL and trismus-specific symptoms in patients with HNC for 5 years post-RT. A secondary aim includes investigating the correlation between facial pain and the degree of maximal interincisal opening.
Study participants and design Oncologic treatment Data collection and measurement tools Statistical analyses Ethical considerations This prospective study was conducted in accordance with the Declaration of Helsinki and was approved by the Regional Ethic Review Board in Gothenburg, Sweden. All participants gave their written informed consent before study inclusion.
Participants were recruited from the weekly multidisciplinary tumor board meeting at Sahlgrenska University Hospital in Gothenburg, Sweden between 2007 and 2012. Patients with newly diagnosed HNC that fulfilled the inclusion criteria were consecutively invited for study participation. Inclusion criteria included age ≥ 18 years, planned for primary radiotherapy with curative intent and tumor site in oral cavity, oropharynx, nasopharynx or cervical carcinoma of unknown primary (CCUP). Since the cohort was going to be used for multiple studies regarding postradiation trismus, additional exclusion criteria were applied, i.e., the presence of trismus prior to treatment and a tumor site where one would not normally expect trismus as a complication (larynx, esophagus, hypopharynx and skin malignancy). Patients were also excluded if they had recurrent disease, surgical treatment only, insufficient Swedish language skills and performance status or mental capacity too poor to partake in examinations or respond to questionnaires. Patients were followed prior to radiotherapy (baseline), 3 months, 12 months, and 60 months post-radiotherapy completion.
Traditional external radiotherapy with curative intent was given according to regional guidelines. Between the years 2007 and 2009, accelerated hyperfractionated radiotherapy was administered to patients (about half of the patients), consisting of 1.7 Gray (Gy) doses given twice daily, 5 days/week (total radiation dose of 64.6 Gy). During the time period 2010–2012 however, the other half of patients received accelerated fractioning 1–2 times daily in 2–2.4 Gy fractions, 5 days/week (total dose was 64–68 Gy). Sixty-two percent ( n = 121), mainly those with advanced tumor stages, also received induction (cisplatin and 5-fluorouracil) or concomitant (weekly cisplatin) chemotherapy according to regional guidelines at the time. For oropharyngeal and nasopharyngeal cancers, non-surgical oncological treatment regimens were used. Advanced stage (III–IV) oral tumors were typically also treated with surgical excision, including neck dissections. In cases where the tumor was assessed to be unresectable, chemoradiotherapy was administered instead. Other tumor sites where surgical removal was performed including neck dissections were salivary gland cancers and sinonasal cancers. In selected CCUP cases, neck dissection was performed prior to non-surgical oncological treatment.
Adult Comorbidity Evaluation 27 (ACE-27) Maximum interincisal opening (MIO) The European Organization for Research and Treatment of Cancer (EORTC) Quality-of-Life Questionnaire Core-30 (QLQ-C30) and Head and Neck 35 (HN35) Gothenburg Trismus Questionnaire (GTQ) The GTQ is a validated trismus-specific questionnaire, developed to be used as a complement to the objective measure MIO and was registered at all study time points. It is composed of 21 items in total, where 13 items are incorporated into the domains jaw-related problems (7 items), eating limitations (4 items), muscular tension (2 items) and the remaining single 8 items assess facial pain, pain associated with trismus, trismus affecting work, leisure or social life. In the GTQ, eight items in total are directly related to facial pain. Score ranges from 0 to 100, where a high score indicates a high symptom burden . The primary variable “Intensity of facial pain during the last month” was reported on a 7-point Likert scale where 1 point equals no facial pain and 7 points equates to unbearable pain. Furthermore, 2–3 points are considered mild pain, 4 points moderate pain and 5–6 points severe pain. The GTQ has been previously described in detail . Patients were subgrouped into two groups: those reporting “No pain” (1 point) and those reporting some degree of “Pain” (2–7 points) on the GTQ variable “Intensity of facial pain during the last month”.
Adult Comorbidity Evaluation 27 (ACE-27) was recorded at baseline. The ACE-27 is a validated comorbidity instrument widely used in head and neck oncology. It includes the assessment of 27 elements from twelve organ systems. Based on the amount and severity of comorbidities, it provides an overall comorbidity score of 0–3, where 3 indicates maximum severity .
MIO was measured in millimeters with a ruler and registered at each time point. A maximal interincisal distance of ≤ 35 mm as proposed by Dijkstra et al. was used for definition of trismus.
The EORTC QLQ-C30 was developed for assessing the quality of life of cancer patients. It is comprised of multi-item scales as well as single items, including five functional scales, three symptom scales, a scale regarding global health and quality of life and six single items . An additional Head and Neck disease specific module, the EORTC QLQ-HN35 was also utilized. The EORTC QLQ-HN35 consists of seven multi-item scales assessing pain, swallowing, senses, speech, social eating, social contact and sexuality in addition to 11 single items , . For the EORTC questionnaires, a 4-point Likert scale was used, and scores are then transformed to a scale from 0 to 100, where high points correspond to high function on the functioning scales and Global HRQL, whilst high points correspond to a higher symptom burden on the symptom scales and items , .
The GTQ variable “Intensity of facial pain during the last month” was dichotomized with the purpose of distinguishing those that had some degree of facial pain from completely pain-free patients. Standard procedures were used to calculate descriptive statistics with 95% confidence interval. The SAS version 9.4 for PC was used for analyses. For comparisons between groups, Fisher’s Exact test was used for dichotomous variables, Mann–Whitney U -test for continuous variables, Mantel–Haenszel Chi Square test for ordered categorical variables, and Chi Square test for non-ordered categorical variables. Wilcoxon signed rank test was used for continuous variables within groups over time. Spearman’s correlation test was used to analyze the correlation between the degree of pain and MIO. Cohens’ convention was used for interpretation of correlations. A correlation coefficient of 0.10 represents a weak or small association, 0.30 is considered a moderate correlation and 0.50 or larger represents a strong or large correlation . All tests are two-tailed and conducted at a 5% significance level.
tient demographics and treatment characteristics Prevalence of facial pain over time Patient-reported outcome measures over time MIO group comparison and pain correlation MIO was significantly lower in the pain group at all four time points (Fig. ) compared to the group reporting no pain. The distribution of pain intensity on a scale of 1–7 points in the pain group is presented in Table . A majority of patients reported 2–3 points of 7 (i.e. mild pain) on the GTQ pain question at each time point. At baseline, 43 out of the 97 subjects (44%) in the pain group reported moderate to severe pain the last month. The corresponding figures for the 3-month, 12-month, and 60-month follow-up were 54/129 (42%), 33/90 (37%), and 15/52 (29%), respectively. The subjects reporting higher scores tended to have a lower MIO. However, the correlation between these variables was weak during the first year post-RT ( r = 0.29) and moderate at the 5-year follow-up ( r = 0.48).
Demographic data and treatment characteristics of the patient cohort stratified by reporting “No pain” versus “Pain” during the last month at each time point are described in Table . At baseline, the median age for the whole cohort ( n = 194) was 61 years. The subjects were predominantly male (74%), currently non-smokers (77%), and the most common primary site of tumor was oropharyngeal (63%). In total, 70% of subjects presented with advanced stage disease and 61% received combination treatment with RT and chemotherapy. When comparing the groups at baseline, twice as many women were seen in the “Pain”-group than in the “No pain”-group (32% versus 16%, respectively). Furthermore, at baseline there were more smokers in the” Pain”-group (32% versus 14%). This difference was statistically significant ( p = < 0.01) and similar results regarding smoking was seen at the 12-month follow-up time point ( p = < 0.001). Regarding differences in BMI, a statistically significant difference ( p = < 0.01) was seen between the “No Pain” and “Pain” groups only at the 5-year follow-up where the “Pain”-group had higher BMI scores. A tumor site of oral cavity was also more common in the “Pain”-group than in “No Pain”-group at baseline (27% versus 6%).
Half of the cohort ( n = 97) reported facial pain during the last month at baseline (Table ). This number increased to 70% at 3 months post-RT and decreased to 54% at 12 months post-RT. At 60 months post-RT, 41% of the subjects reported some degree of pain. When comparing no reported pain versus some degree of reported pain at each follow-up time point, there was a statistically significant difference between the groups, only at 3 months post-RT ( p ≤ 0.001).
Tables , , and present the results from the EORTC QLQ-C30, HN35 and GTQ at all four study time points for both groups. The European Organization for Research and Treatment of Cancer (EORTC) Quality-of-Life Gothenburg Trismus Questionnaire Furthermore, the pain group reported significantly worse scores on all GTQ items except for Limited mouth opening before cancer at baseline and 3 months post-RT, and on all items at 12 months and 60 months post-RT completion (Table ).
For the EORTC QLQ-C30 questionnaire, the pain group reported significantly worse scores on all the functioning scales, Global QoL , the majority of single items at all time-points, as well as all symptom scales except nausea/vomiting at the 12-month follow-up ( p = 0.11) (Table ). The pain group also reported significantly worse scores compared to those without pain regarding most EORTC QLQ-HN35 scales and single items at 12- and 60 months post-RT and on most scales at 3 months post-RT (Table ).
This longitudinal observational cohort study describes pain and HRQL in 194 patients with HNC at multiple time points after completion of radiotherapy with curative intent and is to our knowledge the longest prospective study to date examining the association between facial pain and restricted mouth opening in this setting. Study results show that a large proportion of treated HNC patients still suffer from facial pain long-term. Approximately 50% of the study population reported some degree of facial pain at baseline before starting oncologic treatment, which then increased to 70% at 3 months post treatment completion. This decreased to 54% at 12 months post-RT completion and finally dropped to 41% at 5 years post-RT. These results are similar to previous findings from other studies. In a cross-sectional study by Ren et al. , a HNC cohort of 505 survivors with a mean survivorship of 4.6 years and a history of cancer treatment of radiotherapy (81.5%), surgery (62.9%) and chemotherapy (58.1%) filled out surveys asking if they had had pain in the last week. If yes, they were asked to grade the pain on a numerical pain scale from 0 (no pain) to 10 (as severe as it could be). They were given a body map to point out where in the body they experienced the pain. 45% of HNC survivors with a median follow-up of 3 years post cancer treatment reported pain. The locations of pain were mainly reported being in the “neck and throat”, followed by “head and oral cavity” and lastly “shoulder”. In another study by Cramer et al. from 2018, patients were asked to self-report grade of pain, from none to severe followed by a secondary assessment of pain as measured by the University of Washington quality of life questionnaire. The received cancer treatment of the cohort was mostly multi-modal including surgery, RT and chemotherapy alone or in combination, where 83% had received RT and 60% surgery. 45.1% of 175 HNC patients with a median of 6.6 years after diagnosis reported pain. These patients were however included from a survivorship clinic which may theoretically result in an overrepresentation of reported pain in that specific group. Furthermore, type of cancer pain is not specified in the study. A systematic review by Epstein et al. reported that 50% present with orofacial pain at initiation of treatment (chemotherapy and radiation for HNC) and the number increases to 81% during therapy, dropping to 70% at the end of therapy with 36% of patients still presenting with orofacial pain at 6 months post-therapy. Pain was often assessed using validated quality of life questionnaires such as EORT QLQ-C30, EORTC QLQ-HN35, University of Washington Quality of Life questionnaire (UW-QOL), as well as visual analog scale and study specific questionnaires like Oral Mucositis Daily Questionnaire among others. According to the results from the EORTC QLQ-C30 and HN35 in our study, the pain group also reported worse HRQL on most scales and items in both questionnaires. Hence, a clear association was seen between reports of presence of facial pain and negatively affected HRQL. The majority of other post treatment studies regarding HNC, pain and quality of life include pain in assessment of more broadly evaluated quality of life as also noted in the mentioned systematic review by Epstein et al. . Furthermore, prospective follow-up studies for years on the subject of pain after oncological treatment are difficult to find. This further complicates comparison of results. In the above mentioned the cross-sectional Cramer et al. study on patients with HNC following treatment, 46% of the patients reporting pain reported low overall quality of life versus 12% of those who reported to be pain-free . Pain was assessed in two formats, first by asking about presence and degree of pain, then pain was secondarily assessed in the UW-QOL survey. Furthermore, patients who scored higher (worse) regarding pain, also appear to have a decreased MIO. Albeit this correlation was weak during the first year post-RT, a moderate correlation was shown towards the end of the study period. A possible explanation for this change in degree of correlation strength may be that acute treatment toxicities which are not directly linked to mouth opening, including oral mucositis, cause pain and discomfort during the first time-period post-RT . Factors supporting this theory may also be found in the patient-reported HRQL, where scores were worse for symptoms such as Fatigue (EORTC QLQ-C30), Senses and Social eating (EORTC QLQ-HN35) at 12 months post-RT compared to 5 years post-RT in the group reporting pain. A similar pattern was observed for the EORTC QLQ-HN35 single item Dry mouth , again potentially hinting at consequences of acute treatment toxicity. Albeit caution should be advised when drawing conclusions regarding the causative effects of a multifactorial phenomenon such as the perception of pain, our results may point to the possibility of trismus causing pain or vice versa. In an interventional, matched control study by Andréll et al. , HNC patients with post-radiation trismus underwent a jaw exercise program ( n = 50) and were compared to a matched control group who did not undergo the structured jaw exercise program ( n = 50). Fifty-nine percent of the subjects reported facial pain before oncologic treatment and more than half (51%) of the HNC survivors with trismus reported facial pain at three years post treatment completion. The patients with HNC in the Andréll study all had post-RT trismus. The higher prevalence of pain in that study population indicates that there is an important relationship between a lower MIO among HNC survivors and the risk of reporting pain. Another important finding in the Andréll study that underlines the relationship between facial pain and trismus was that the patients undergoing the structured jaw exercise program had 9 times higher chance of being pain-free at 3 years following treatment. Nonetheless, there are numerous other potential causes of head and neck pain after oncologic treatment for HNC, including osteradionecrosis, lymphedema, burning mouth syndrome and neurological damage . The strength of this study lies in its prospective design with a long follow-up period of 60 months. Furthermore, the objective measurement of MIO, the use of validated, well used patient reported outcome measures and the size of the cohort also contribute to its strength. The study may be limited by the fact that the patients who dropped out of the study had somewhat worse overall health at baseline. Recurrent HNC is an important cause of local pain and the patients in the study who developed a recurrent disease were excluded. Hence this potential cause of reported pain was minimized, possibly further potentiating the risk of survival bias. Furthermore, there might be a slight risk of recall bias considering the recall time of the primary variable in this study being 1 month. In the more recent version of GTQ, the recall time for pain has been changed to 1 week. There is also limited information regarding the use of specific pain medication or if other methods have been used for alleviating pain, as well as the exact location and cause of the facial pain. Finally, most of the patients had received chemoradiotherapy, and no direct comparison of pain outcomes was made between those who received chemoradiotherapy and those who received radiotherapy alone. Therefore, the effect of chemotherapy versus radiotherapy alone could not be distinguished in this study. Clinical implications It is clear from the study that pain is a prevalent symptom experienced by irradiated HNC patients both short-term as well as long-term. It also has a large impact on HRQL. Hence, it is of utmost importance to follow these patients over time in order to identify and alleviate pain. Trismus may very well be a contributing factor and mouth opening ability should also be measured and structured jaw exercises initiated when trismus occurs in an attempt to increase mouth opening and reduce facial pain in the long-term within the HNC population.
The study findings demonstrate that facial pain was common among HNC patients before oncological treatment as well as up to 5 years post-RT. Reductions in MIO were associated with significantly more facial pain, particularly in the long term and patients who reported facial pain also reported worse HRQL compared to those reporting no pain. The degree of facial pain needs to be evaluated continuously by clinicians to ensure that other possible methods or medications for alleviations are offered to increase the HNC patients’ chance of optimal well-being.
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Molecular signatures from omics data: From chaos to consensus | 7db31965-44c8-44be-b517-1dfa4f31a7b2 | 3418428 | Pathology[mh] | In recent years, new high-throughput measurement technologies for biomolecules such as DNA, RNA, and proteins have enabled unprecedented views of biological systems at the molecular level. The fields of research associated with obtaining and understanding such measurements – for instance, genomics, transcriptomics, and proteomics – are sometimes referred to in aggregate as omics . Given molecular measurements taken from a biological system, a natural goal is to develop a statistical model that uses these measurements to predict a clinical outcome of interest, such as disease status, survival time, or response to therapy. In this paper, we will discuss the process of using omics data to discover a molecular signature . Here, we define a molecular signature as a set of biomolecular features (e.g. DNA sequence, DNA copy number, RNA, protein, and metabolite expression) together with a predefined computational procedure that applies those features to predict a phenotype of clinical interest on a previously unseen patient sample. A signature can be based on a single data type or on multiple data types . The overall process of identifying molecular signatures from various omics data types for a number of clinical applications is summarized in . Many possible clinical phenotypes might be predicted by a molecular signature; a few examples include prediction of disease risk and progression , response to therapeutic drugs and their physiological toxicity , and time to disease recurrence or death . A successful case of the clinical utility of omics-derived molecular signatures is MammaPrint , a diagnostic test approved by the Food and Drug Administration for clinical use. MammaPrint is a 70-gene expression signature used to predict breast cancer prognosis and to determine the appropriate therapeutic regimen for lymph node negative breast cancer patients with either ER positive or negative. The list of 70 genes was selected based on correlation with clinical outcome (distant metastasis vs. no metastasis), and underwent successful validations on independent patient cohorts . Despite a few notable exceptions such as MammaPrint, the successful discovery of molecular signatures has largely been hampered by limited reproducibility and variable performance on independent test sets , as well as difficulty in identifying signatures that outperform standard clinical measurements like the cardiovascular disease risk C-reactive protein (CRP) . These difficulties can be attributed in large part to the low S/N inherent to omics datasets, the prevalence of batch effects in omics data, and molecular heterogeneity between samples and within populations . These issues are exacerbated by the fact that the datasets used to develop molecular signatures tend to have small sample sizes relative to the number of molecular measurements . Moreover, improper study design, inconsistent experimental techniques, and flawed data analysis can lead to further challenges in the process of molecular signature discovery. Though there has been marked progress in the field of molecular signature discovery in recent years, there remains a clear need for further improvements in the discovery process in order for omics-based technologies to begin to achieve their full clinical potential.
Roughly speaking, the process of molecular signature discovery on the basis of omics data consists of four major stages: (i) Defining the scientific and clinical context for the molecular signature; (ii) Procuring the data; (iii) Performing feature selection and model building; and (iv) Evaluating the molecular signature on independent datasets. In the sections that follow, we will discuss each of these stages in turn. 2.1 Stage 1: Defining the scientific and clinical context 2.2 Stage 2: Data procurement 2.3 Stage 3: Feature selection and model building 2.4 Stage 4: Evaluation on independent datasets We first consider the problem of selecting a suitable omics data type for a molecular signature. A signature intended to distinguish between cancer and normal tissue could be based upon a number of omics data types; for instance, one might base the signature upon gene expression measurements, if it is believed that this type of cancer shows altered expression of some genes relative to normal tissue, or upon DNA sequence data, if samples from this cancer are characterized by particular mutations or copy number changes. However, given a clinical phenotype of interest, certain types of omics data might not form the basis for a sensible molecular signature. For instance, it would not be reasonable to attempt to create a molecular signature to screen for adult onset (type II) diabetes on the basis of DNA sequence data alone because an individual's DNA sequence remains essentially static throughout his or her lifetime, but risk of developing the disease may change. We now consider the clinical context of the molecular signature. A gene expression-based signature that can distinguish between cancer and normal tissues would be of little practical use if a physician can easily make the same distinction using standard (and less expensive) clinical approaches. Similarly, a signature that can distinguish between two subtypes of cancer is useful only if those two subtypes differ in some clinically relevant way, such as in survival time or response to therapy, since otherwise the information about cancer subtype provided by the molecular signature may not serve a practical purpose. As an example, gastrointestinal stromal tumors (GISTs) and leiomyosarcomas (LMSs) are remarkably similar morphologically and were originally classified as being the same cancer. However, it was found that they respond very differently to distinct therapies, and thus a signature that can distinguish between these two diseases based on gene expression in tissue samples can be useful . An example outside of cancer involves the use of metabolomic information from human serum to noninvasively diagnose and monitor Alzheimer's disease (AD) progression .
The development of a molecular signature requires the availability of adequate omics data for which the clinical phenotype of interest is available. In general, there are two ways in which such data can be procured: new data can be collected experimentally for the specific purpose of molecular signature discovery, or else existing data (collected previously for other purposes, and generally publicly available) can be used. There are pros and cons of either approach. Collecting new data has a major advantage, in that all aspects of the experiment can be carefully controlled. On the other hand, data collection is expensive, and given the large sample sizes necessary for successful molecular signature discovery, using existing datasets may be a more feasible approach. There are a number of public data repositories from which omics data and associated clinical phenotypes can be obtained. For instance, a useful source of gene expression data is NCBI Gene Expression Omnibus (GEO), a repository of over 26000 studies that continues to grow at a rapid pace. Other public data repositories include ArrayExpress and Sequence Read Archive . Regardless of how the data are procured, it is crucial that the samples correspond to the scientific and clinical context of interest, as described in the previous section. In order for a dataset to be suitable for molecular signature discovery, the samples must be collected under appropriate experimental and analytical conditions. As an example, any biological factors (such as gender, age, or ethnicity) that may be associated with the clinical phenotype of interest or with the omics measurements should be taken into consideration in the process of data procurement. In addition, to reduce the prevalence of batch effects , factors such as sample collection and processing procedures, laboratory personnel, study run-dates, reagent sources, measurement instruments, and data processing methods should be carefully controlled . Deviations in these protocols can have a surprisingly large effect on the omics measurements obtained, often larger than the effect of the clinical phenotype of interest . Ideally, there should be no association between the clinical phenotype of interest and these factors. For instance, in the case of a molecular signature that classifies tissue samples into tumor versus normal, there should be no difference between the tumor and normal samples in terms of the laboratory personnel who performed the sample preparation, or the sample run-dates. If experimental and analytical procedures are not carefully controlled, they can result in confounding with the clinical phenotype of interest, leading to the development of a classifier that performs very well on the data used in its development, but that will perform poorly on independent test samples. To the extent that analytical and experimental factors do vary among the samples, these factors should be explicitly included in the model used to develop the classifier. Normalization procedures have been proposed that are intended to reduce the effect of measured and unmeasured external factors on omics data ; however, good experimental design remains the best strategy . Exploratory data analysis techniques, such as hierarchical clustering ( ) and principal components analysis ( ) can be useful tools to assess the extent to which covariates that are not of primary interest may have affected the data. When existing data is used for omics-based molecular signature discovery, it is particularly important that sufficient information about the experiment is available to ensure that good experimental design was followed (this will be discussed further in Section 4). For instance, if the run date for each sample is not given, then one cannot be certain that the clinical phenotype of interest is not highly confounded with run date. Unfortunately, many omics studies have sample sizes substantially smaller than would be required for the successful identification of molecular signatures. A molecular signature that is developed on the basis of a small number of samples is more likely to be sensitive to technical and biological sources of noise and variation, and less likely to capture the aspects of the data that are truly associated with the phenotype of interest. This exacerbates the risk of over-fitting, wherein the signature performs well on the samples used for signature development but fails to correctly predict the clinical phenotype of interest in previously unseen samples. In contrast, global molecular characteristics of a particular phenotype may become more apparent as sample size increases. Therefore, having a large sample size, while by no means a cure-all, will greatly improve the odds that a given attempt at molecular signature discovery will prove fruitful. Integrating across multiple datasets of the same phenotypes from different labs can also help to amplify the primary biological signal of interest relative to noise. Of course, whether a given sample size is “large” or “small” depends the type of omics data being used for signature discovery, the clinical phenotype of interest, and many other factors.
Once a scientific and clinical context has been established and one or more datasets have been identified, we can develop a molecular signature through (i) feature selection; and (ii) model building. These two tasks can be performed together or separately. We first consider the task of feature selection. A typical omics experiment simultaneously measures thousands or even millions of biological features (e.g. single nucleotide polymorphisms, RNA transcripts, protein levels) on each patient sample. However, just because thousands of molecular measurements are obtained does not mean that thousands of molecular measurements should be used in the molecular signature. Since financial cost, technical practicality, and measurement robustness are important criteria to select signatures, then if all else is equal, a signature that could be ultimately measured via PCR or Western blot is favored over a signature that requires a technique involving many more protocol steps, such as in omics measurements. In order to reduce the number of features used in molecular signature development, feature selection is performed. Feature selection can be performed in a supervised manner (e.g. the 20% of features that are most associated with the clinical phenotype of interest are selected), or in an unsupervised manner (e.g. the 20% of features with the highest variance are selected). Once a set of features has been selected, only those features are used in the model building process, which is described next. We now consider the task of model building – i.e. the process of developing a specific computational procedure that can be applied to the omics measurements from a future patient sample in order to predict the unknown clinical phenotype of interest for that sample. There are many possible approaches to building such a model, and in particular, the type of model used will depend on the clinical phenotype of interest. For instance, if we wish to develop a molecular signature to predict time to cancer recurrence, then a Cox proportional hazards model might be appropriate. On the other hand, to develop a molecular signature that can distinguish between cancer and normal tissue, one could use a classification approach, such as logistic regression, support vector machines, neural networks, or linear discriminant analysis. Some approaches for model-building involve first performing an unsupervised technique, such as clustering or principal components analysis, followed by a supervised procedure, such as logistic regression. Once we have developed a model, how can we determine whether it is any good? Despite certain drawbacks , the most popular approach for evaluating model performance in this context is cross-validation . (Cross-validation is also often used for tuning parameter selection, though that application is outside of the scope of this paper.) Cross-validation involves repeatedly splitting the samples in the dataset into training and test sets, performing all aspects of feature selection and model building on the training set, and evaluating the model's performance on the test set. Cross-validation can also be used to select from among a small number of possible models: the model with the smallest cross-validation error rate should be chosen. Cross-validation is a simple and intuitive approach to estimating the error rate associated with a model, but it must be performed with care. Most importantly, within each cross-validation fold, no information about the test set can be used in building the model on the training set. For instance, suppose that one performs feature selection by selecting the 10% of features whose t -statistics between cases and controls are largest. One then performs logistic regression, using only these features, to develop a classifier to distinguish between cases and controls. How should the cross-validation error rate be calculated? Consider the following two approaches: Approach 1 (incorrect): identify the 10% of features that differ most between cases and controls, and use only those features henceforth. Perform cross-validation by repeatedly splitting the samples into training and test sets, fitting a logistic regression model on the training set (using just the 10% of features previously identified), and then evaluating the model's performance on the test set. Approach 2 (correct): perform cross-validation by repeatedly splitting the samples into a training set and a test set. Within each training set, identify the 10% of features that differ most between cases and controls, and use those features to fit a logistic regression model. Then, evaluate the performance of this model on the test set. The difference may seem subtle, but it is in fact crucial. Approach 1 will yield a woeful underestimate of the true error rate, because the 10% of features that differ most between cases and controls were identified using all of the samples, including those in the test set, rather than simply the training samples. In effect, if Approach 1 for cross-validation is taken, then perfect error rates can potentially be obtained even on datasets in which the “case” and “control” labels were assigned randomly! On the other hand, in Approach 2, feature selection is performed using the training set within each cross-validation fold, and so the resulting cross-validation error rate is valid. Unfortunately, the difference between Approaches 1 and 2 is often overlooked, and the literature is rife with papers in which extraordinarily low, but grossly inaccurate, cross-validation error rates are reported because some variant of Approach 1 has been performed. The key principle is that in computing cross-validation error rates, within each cross-validation fold only training observations can be used in any aspect of feature selection or model development. Deviations from this principle, even if seemingly innocuous, may result in dramatic underestimates of error. At the end of the feature selection and model building process, the molecular signature must be locked down – i.e. the precise computational procedure used to convert a new omics sample into a prediction of the clinical phenotype must be completely specified. Only then can the molecular signature be fairly evaluated on independent datasets, as described next.
Once a promising molecular signature has been identified, its performance needs to be evaluated on completely independent patient samples. Unlike cross-validation, wherein the test set is drawn from the same population as that of the training set, an independent sample is one that is completely separate from the set of samples used for feature selection and model building. In particular, this means that the test set is not simply a random split from a large dataset (even if sequestered and not used in any training sets). If a molecular signature performs well on a truly independent set of samples, then this provides evidence that it will likely generalize to future patient samples. However, the amount of evidence for a molecular signature's performance based on independent data depends critically upon specific characteristics of the independent dataset. Lower level of evidence. Good performance on an independent dataset collected at the same institution using carefully controlled protocols . This provides evidence that the molecular signature works well in this particular setting, with these protocols, with the patient profile at this institution, etc. However, it may not hold up elsewhere. At the very least, its ability to work in other settings has not been demonstrated. Higher level of evidence. Good performance on multiple independent datasets collected at multiple institutions . Success in this setting is the best evidence that a molecular signature will perform well on future patient samples. This indicates that the signature is robust to the kinds of things that might change between locations: namely, aspects of the biology of the populations that tend to go to particular hospital, sample preparation and measurement techniques used, and so forth. Evaluation of a molecular signature on fully independent patient samples is the gold standard for assessing its performance. Unfortunately, it often is the case that molecular signatures that seem promising in the feature selection and model building stage (i.e. that have very low cross-validation error rates) exhibit poor performance on independent data.
A key principle of science is that other researchers must be able to reproduce the results. In order for a molecular signature to be reproduced, three essential pieces of information are required: (i) the experimental and analytical protocols; (ii) the raw data; and (iii) the source code used to develop the signature. We discuss each of these points in turn. In order for a molecular signature to be fully understood by other researchers, detailed information on the experimental protocol, including the patient selection criteria and experimental and analytic procedures, must be made available. Without this information, one cannot determine the scientific or clinical contexts in which the molecular signature is intended, appropriate, or useful. Second, in order for a molecular signature to be reproduced, the omics data used in its development, as well as the associated metadata and clinical data, must be made available. If the data are not released, then it simply is not possible for other research groups to determine whether the molecular signature is valid. Finally, even if the data are made available, other research groups will not be able re-derive the molecular signature based on the same data used for its discovery, and confirm that the signature does truly work well on independent data, unless all data processing techniques and all analytical and computational methods are made available. Unfortunately, in practice this information often is not provided in sufficient detail. For instance, there is a tendency for authors to publish a list of the features (e.g. genes) involved in the signature, without the detailed mathematical formulas required to understand precisely how the omics measurements are used in order to predict the clinical phenotype of interest. This is a major obstacle to progress in the field, as other research groups cannot reproduce or validate – much less build upon – research that is not sufficiently reported. In order to address this problem, the source code used to develop the molecular signature should be released. Ideally, this code should encompass all aspects of signature development, from processing and normalization of the raw omics data, to feature selection to model building to evaluation on an independent dataset.
Thus far, we have described the development of a molecular signature on the basis of a single dataset, followed by evaluation of the signature on one or more independent datasets. However, in principle, multiple datasets can be used for molecular signature discovery. In fact, this can often lead to more accurate and more broadly applicable molecular signatures. When a molecular signature is developed on the basis of a single dataset and then tested on an independent dataset, its performance tends to degrade severely in the independent dataset relative to its cross-validation error rate in the dataset used for development. This drop in performance can stem from heterogeneity between studies due to underlying variance in the biology of the patients studied, as well as from technical variations in measurement, normalization, and analysis. That is, a signature developed using a single dataset may overfit certain aspects of the dataset that are not of primary scientific interest, leading to poor performance on independent data. This problem can be partially overcome by developing the signature on the basis of multiple datasets, collected at different institutions and at different time points . (However, the primary clinical phenotype of interest, such as tumor versus normal, must be balanced between the datasets in order to avoid confounding between the datasets and the clinical phenotype.)
Given the complexity of biological systems in general and pathological processes in particular, there is an upper limit to how well a molecular signature developed on the basis of a single data type (e.g. genome-wide expression on DNA microarrays) can predict disease phenotypes and clinical outcomes. Integrating multiple types of omics data may allow for the development of increasingly accurate and robust molecular signatures. For example, gene expression data can be combined with copy number variation data or DNA sequence data. Successful multi-scale integration of different types of biological information is one of the current challenges in systems biology . In , we provide brief summaries of a few recently published studies in which multiple data types were used for molecular signature discovery. A number of methods to combine diverse types of omics data across different measurement platforms and laboratories have been proposed [ , , ], in order to more accurately select clinically relevant features or to develop better molecular signatures. For example, English and Butte evaluated data from 49 obesity-related studies that used different experiment types, including DNA microarrays, genome-wide association, proteomics, and RNAi knockdowns . The investigators found that the biomolecules reported to be associated with obesity in individual studies had little overlap with previously known obesity-related genes. The investigators then determined a gene to be obesity-related if five or more studies reported the gene to be obesity-related. Using this approach of feature selection, they were able to identify a higher proportion of known obesity related genes than from any of the 49 individual studies, and also discovered new genes for which there was compelling support of association with obesity . This demonstrated that even straightforward integration of multiple omics data types can substantially improve the feature selection process. In a study by Lu et al. , the investigators integrated data types in order to perform more effective feature selection: they identified 475 genes that were differentially expressed between lung adenocarcinoma and normal tissue, and that were also located in copy number varying regions. This gene set was used to create a predictive model for patient survival, which was then shown to be accurate on three independent patient cohorts. Advances in integrating diverse omics data types may lead to a reduction in spurious signal caused by technical limitations of individual platforms, and an increased ability to identify molecular signatures associated with the underlying mechanistic roles in disease pathogenesis.
The use of network-based approaches is a promising avenue for molecular signature discovery. These networks represent a complex web of interactions among diverse components in a cell, and can be used to develop more reproducible and accurate molecular signatures by exploiting the underlying biology of the system. Network-based approaches extend beyond simple integration of different omics data types, and can involve evaluating complex interactions that can vary due to disease or other perturbations. Most statistical methods for feature selection and model building do not take a network-based approach: they implicitly assume that the features are independent, or that they are only weakly dependent, though this has begun to change in recent years . However, in most biological contexts, the assumption of independent features is certainly violated. For instance, genes regulated by the same set of transcription factors, or genes encoding enzymes for the same metabolic pathway, will tend to show correlated expression. Therefore, rather than treating each feature in an omics dataset individually, it may be preferable to map from the high-dimensional molecular space to a much smaller number of (possibly curated) functional biological networks. Mapping features into functional sets reduces dimensionality, increases the statistical power to detect small but coordinated disease perturbations, and improves the interpretability of the resulting molecular signatures. In order to identify features that are associated with a clinical phenotype of interest, features can be mapped onto a priori defined and manually curated modules or “pathways”. Gene Set Enrichment Analysis (GSEA) is a very widely used approach to investigate pathway-level changes in gene expression data, and more recent proposals have also been made. One recently developed approach to identifying pathway-based molecular signatures for phenotype classification is the Differential Rank Conservation (DIRAC) method . Unlike GSEA or other enrichment methods that usually return p -values for gene set enrichment, DIRAC builds a network-based molecular signature that identifies robust differences in pathway activity between two disease states. However, one major caveat to such pathway-based approaches is that a priori defined pathways do not fully represent the complexity of the underlying biology, and may not be accurate within the particular physiological context. To overcome this limitation, molecular features can be mapped into more comprehensive interaction networks, such as protein-protein or protein-DNA interaction networks, which can be much more comprehensive and unbiased, as well as disease and context specific. Specifically, biological networks can be used as a structured framework to integrate omics data for the purpose of molecular signature development. For example, Chuang et al. integrated microarray gene expression data with protein–protein interaction networks to identify network-based prognostic biomarkers for breast cancer metastasis, and generated novel hypotheses regarding cancer progression. The average sub-network activity, defined in this study as a function of expression levels of genes that compose the sub-network, was used to predict clinical outcome of breast cancer specimens. The network-based markers displayed better predictive accuracy on an independent dataset than markers selected without network information. In another study, Nibbe et al. used proteins that were differentially expressed between normal and cancer colon tissue from proteomics experiments as seeds to identify sub-networks enriched in these differentially expressed proteins from the human protein interaction network. Then, the mRNA expression profiles of the components of these sub-networks were used as input features to a support vector machine in order to classify colorectal cancer and normal samples. The prevalence of these networks being perturbed in colon cancer was demonstrated by these features alone being sufficient to achieve 90% classification accuracy in independent validations. In the particular case of prion disease, a set of neurodegenerative disorders caused by the misfolding of prion proteins in the brain, Hwang et al. analyzed the dynamic network perturbations during the onset and progression of disease. In this study, infectious prion proteins were delivered into the brains of living mice, and were harbored within the tissue for different time-spans of disease progression. At the end of each time-point, gene expression measurements were taken from harvested diseased brain tissue, and subsequently mapped onto physical protein interaction networks for comparative analysis. Intriguingly, this study showed reproducible perturbations that occurred in core networks that could be monitored prior to the manifestation of disease symptoms. In the work summarized above, thousands of feature measurements for static biological states were used to characterize molecular networks. However, a more complete understanding of molecular networks requires perturbing the biological system under study in order to understand how the network components, as well as the clinical phenotype of interest, are affected by those perturbations. For example, stimulating one or more signaling pathways using in vitro cytokine assays can lead to different immunologic and metabolic responses in different diagnostic phenotypes , such as different disease progression levels. In a study by Hale et al. , the investigators used a cocktail of cytokines and mitogens to stimulate whole blood cells from patients with different stages of systemic lupus erythematosus, an autoimmune disease. They then used flow cytometry to measure multiple signaling responses at the single-cell level, generating a highly multiplexed view of intracellular signaling network activity during disease progression. They found that robust changes in signaling protein interactions in response to stimuli were good indicators of disease stage. Therefore, evaluating cell response after an activating stimulus may serve as a compelling approach for incorporating perturbations into patient classification going forward.
Given that two molecular signatures seem to perform well on independent datasets, how can we decide which is better? If all else is equal, we should prefer the molecular signature for which there is a plausible biological mechanism, as such a signature is much more likely to hold up in future patient samples as opposed to having overfit the data used in its development. Ideally, if sufficient numbers of samples were available, then a molecular signature's performance on one or many independent datasets would be the preferred way of assessing its suitability, regardless of whether or not a mechanism for its performance is known. But in reality, sample sizes are limited, and thus a molecular signature for which there is a plausible biological mechanism tends to be more convincing than one for which no such mechanism is known. Such biologically motivated signatures can also hold great promise to be developed as companion diagnostics for therapies, which may be motivated by the underlying mechanism. Thus, while lack of a known biological mechanism underlying a molecular signature certainly does not preclude its use provided that it works well in practice on independent samples, mechanistic information can increase our confidence that the signature will hold up to further scrutiny.
Another major challenge in omics-based molecular signature discovery is the prevalence of overly optimistic accuracies in reported results. This problem is not unique to omics research but is problematic in many data-driven research settings . Such bias can occur for a number of reasons: (i) research groups tend to report only the best results among many attempted approaches; and (ii) only positive results are published. Consequently, across the literature there is an overly optimistic view of how well molecular signatures perform. This pervasive bias is not necessarily the result of faulty science in any particular lab, but rather is a consequence of the way in which science is conducted and reported. This is responsible, in part, for the fact that many reported molecular signatures have not held up in follow-up studies.
In this paper, we have discussed some of the key considerations and challenges facing the discovery of omics-based molecular signatures of clinical phenotypes, such as good experimental design, careful data procurement, avoidance of over-fitting, validation on independent datasets, and integration of multiple datasets and data types. For guidance to the reader, Box 1 summarizes the key steps in molecular signature discovery that were discussed throughout this paper. We hope that this methodological checklist will aid investigators interested in identifying omics-based molecular signatures. Since the emergence of the field of omics-based molecular signature discovery, researchers have developed an improved understanding of how to discover (and how not to discover!) such signatures. The field is still young, and as time passes, best practices in this area will continue to evolve. Currently, the number of validated and useful molecular signatures is disappointingly (but not surprisingly) small relative to the number of signatures that have been reported in the literature. However, we remain optimistic that as experimental and analytical practices improve, as sample sizes increase, and as techniques for data type integration continue to develop, omics-based molecular signatures will indeed transform the practice of medicine. Box 1. Steps for the development of molecular signatures on the basis of omics data Step 1. Establishing the scientific and clinical contex Step 2. Collecting omics data for molecular signature discovery Step 3. Developing molecular signatures through feature selection and model building Step 4. Evaluating performance on independent datasets Step 5. Disclosing information on all aspect of study to enhance reproducibility Step 6. Reporting all performance results to mitigate bias in public literature Encourage the objective assessment of molecular signatures by reporting both positive and negative outcomes (i.e. correct and incorrect predictions, respectively) Make data publicly available after publication
Clearly define clinical phenotypes of interest Ensure that, if discovered, a molecular signature has the potential to be useful in the clinic Only use types of omics data that are suitable for addressing the task of interest Determine acceptable sensitivity and specificity
When collecting new experimental data, ensure that: sufficient sample size can be obtained all aspects of the experimental and analytical procedures are carefully controlled to avoid batch effects no confounding occurs between datasets of different phenotypes from factors unrelated to phenotype of interest > When using existing data, ensure that: sufficient sample size can be obtained sufficient patient information is available for omics samples proper normalization is implemented to make samples comparable across different datasets Consider integrating multiple datasets and data types: approach with caution can lead to molecular signatures that are more accurate and robust
Perform feature selection in either a supervised or an unsupervised manner Choose models that are well-suited for the context of the study and nature of phenotypes of interest Consider mapping features onto biological pathways or more comprehensive interaction networks Consider choosing models that show clear insight into plausible biological mechanisms Ensure that all cross-validation steps are performed correctly Approach favorable cross-validation results with caution
Test promising molecular signatures on independent datasets Independent test sets are not created equal. The strength of evidence from an independent test is based on the characteristics of the independent dataset used (i.e. evaluating on data from multiple, different sites is a more stringent test than evaluating on data from only the same institution)
Encourage the evaluation of the molecular signature by independent research groups Disclose: information on the clinical context in which molecular signature is intended, patient selection criteria, clinical data (i.e. patient information), raw data, meta-data (if applicable), data processing and normalization methods, feature selection and model building methods, experimental protocols, records on study run-dates, lab technicians, reagent sources, etc., analytical methods, and source code
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Preoperative heart rate variability as a predictor of postoperative pneumonia and lung function recovery in surgical lung cancer patients: a prospective observed study | b81599dc-eae2-4920-8397-934e84de00d6 | 11883988 | Surgical Procedures, Operative[mh] | Lung cancer stands as the foremost cause of mortality associated with cancer on a global scale, which has become a malignant tumor with high incidence rate and mortality in China. According to the 2022 global cancer epidemiology statistics, China reported approximately 871,000 new cases of lung cancer and 767,000 lung cancer-related deaths. These figures represent 18.1% and 23.9% of all malignant tumor cases and deaths globally, respectively . Approximately 85% of newly diagnosed lung cancer cases fall within the histological category of non-small cell lung cancer (NSCLC). For stages I to IIIA, surgical resection is considered the preferred treatment option, offering the greatest potential for improved survival outcomes . However, it is important to note that patients with impaired cardiorespiratory function or compromised physical status are at an increased risk of adverse postoperative outcomes, including postoperative pulmonary complications (PPCs) and diminished lung function . Pneumonia is one of the most common postoperative complications following lung cancer surgery, with an incidence ranging from 4 to 20%, and reaching up to 24% in high-risk patients. This complication is associated with an increased risk of morbidity and mortality . A population-based cohort study has demonstrated that patients who developed pneumonia after surgery had a 1.3-fold higher risk of 1-year mortality . Moreover, lung cancer surgery often leads to compromised lung function, resulting in postoperative symptoms such as dyspnea, coughing, and chest tightness. Impaired respiratory function negatively impacts patients’ quality of life . Thus, it is necessary to explore modifiable risk factors or predictors of pneumonia or lung function recovery conditions in which interventions may be carried out to enhance clinical practice. The autonomic nervous system plays a vital role in the maintenance of homeostasis. Various pathophysiological conditions can disrupt the equilibrium within this system. Heart rate variability (HRV) refers to the fluctuations in interbeat intervals that are influenced by respiration and other physiological factors . HRV analysis has emerged as a valuable non-invasive technique for assessing cardiac autonomic modulation across various medical conditions . Previous studies have demonstrated that HRV can serve as an index for the activity of neurophysiological pathways responsible for adaptively regulating inflammatory processes in humans, and HRV monitoring of autonomic inflammatory processes may offer a continuous and supplementary approach to risk management for the early detection of infections or sepsis in both adults and neonates . Recent research has highlighted the direct impact of SARS-CoV-2 infection on HRV test outcomes, indicating that both the severity and prognosis of COVID-19 may be reflected in certain HRV-related parameters . Furthermore, several studies have established a correlation between HRV and pulmonary function, indicating that pulmonary function is positively associated with autonomic control . However, there remains a lack of investigations into the influence of HRV on postoperative lung function recovery and the incidence of postoperative pneumonia (POP) in patients undergoing surgical treatment for lung cancer. The purpose of this study was to assess the value of HRV as a surrogate marker for vagus nerve activity in predicting the incidence of POP in surgical lung cancer patients. In addition, we examined whether HRV was discriminative for lung function recovery after surgery.
Ethical review Population POP criterion Perioperative lung function analysis HRV analysis Statistical analysis Our study strictly adhered to the ethical principles outlined in the Helsinki Declaration (2024). All aspects of the research were conducted in accordance with these guidelines to ensure the protection of human subjects and the integrity of the scientific process. The study received approval from the hospital’s clinical trials and biomedical ethics committee (No. 2023 − 1541) as well as from the Chinese Clinical Trial Registry (ChiCTR2400085997, registered in June 2024). Informed consent was obtained and duly signed by all participating patients.
Consecutive patients who underwent lung cancer surgery were prospectively evaluated in our department. The inclusion criteria for this study were as follows: (1) age between 18 and 85 years; (2) a confirmed diagnosis of primary NSCLC with subsequent anatomic lung resection, including lobectomy or segmental resection. Patients meeting any of the following exclusion criteria were not included in the study: (1) diagnosis of non-NSCLC; (2) undergoing non-anatomic lung resections, such as wedge resections; (3) receiving neoadjuvant therapy.
Pneumonia is defined according to the latest criteria established by the Centers for Disease Control: the presence of new or progressive and persistent infiltration, consolidation, or cavitation observed on chest radiographs. Additionally, at least one of the following criteria must be met: (1) Fever (> 38 °C) without an alternative explanation; (2) Leukopenia (< 4,000 WBC/mm³) or leukocytosis (> 12,000 WBC/mm³); (3) For patients over 70 years of age, a change in mental status accompanied by purulent sputum or alterations in sputum characteristics, along with increased respiratory secretions requiring suctioning; (4) The onset or exacerbation of symptoms (e.g., dyspnea, tachypnea) or clinical signs (e.g., rales, bronchial breath sounds).
Lung function test was performed in the day before surgery and postoperative day 30 (POD 30). The predicted postoperative Forced Expiratory Volume in 1 s%/ Forced Vital Capacity%/ Diffusing Capacity of the Lungs for Carbon Monoxide% (ppo FEV1%/FVC%/DLCO%) was calculated using the preoperative FEV1%/FVC%/DLCO% (pre FEV1%/FVC%/DLCO%), the number of functional lung segments resected (y), and the total number of functional segments available at the time of resection (z). The formula for ppoFEV1 is as follows: ppoFEV1%/FVC%/DLCO%=pre FEV1%/FVC%/DLCO% × [1 − (y/z)]. In cases where patients do not require a redo operation, the total number of functional lung segments across both lungs is 19: comprising 10 segments in the right lung (3 upper, 2 middle, and 5 lower lobes) and 9 segments in the left lung (5 upper and 4 lower lobes). Based on POD 30-FEV1% (FVC% or DLCO%) /ppoFEV1% (FVC% or DLCO%) ≥ 1 or not, the patients were divided into well-recovery in FEV1% (FVC% or DLCO%) (≥ 1) group and poor-recovery in FEV1% (FVC% or DLCO%) (< 1) group.
The HRV test was conducted on the day prior to surgery and on POD 30. The methodology employed for HRV analysis adhered to the standards established by the Task Force. All patients had ECGs acquired using a MedEx iMAC300pro 12-lead ECG analysis system, with electrodes in the same positions as conventional 12-lead-ECGs; the sampling frequency was 1000 Hz, and the sampling time was 5 min; each patient received the same samples in the early morning and late evening moments. One day before signal acquisition, patients are not allowed to consume beverages or food that may affect autonomic function, such as coffee, tea, or cola; 15 min before signal acquisition, patients need to lie still in the hospital bed, and the room temperature is 20 ± 2℃. The patient needs to remain still, keep breathing naturally during this time and avoid talking. The original ECG signal is processed through a hardware band-pass filter with a range of 0.05–150 Hz, followed by a 50 Hz notch filter to eliminate power line interference. All ectopic beats were excluded from analysis, and any missing data points were interpolated using values derived from adjacent valid data. A total of 512 stationary R-R intervals (RRI) were utilized for HRV analysis.Time-domain HRV metrics included mean RRI (mRRI), standard deviation of RRI (SDRR), coefficient of variation of RRI (CVRR), and root mean squared successive difference of RRI (RMSSD), the formula of RMSSD is sqrt(1/N * ∑((RR i - RR i+1 )^2)). The power spectrum of RRI was computed using fast Fourier transformation with Mathcad 15 (Mathsoft Inc.). In accordance with previous research, frequency-domain components were categorized into low-frequency (LF: 0.20 to 0.75 Hz) and high-frequency (HF: 0.75 to 3.0 Hz) bands. Comprehensive HRV analysis was performed utilizing Kubios HRV premium/animal software version 3.2. The software automatically analyzes and outputs important correlates such as High Frequency Power (HFP), Low Frequency Power (LFP) and Low/High Frequency Ratio (LHR).
For non-normally distributed variables, continuous data are presented as the median with interquartile range (IQR), while normally distributed continuous data are reported as the mean with standard deviation (SD). Categorical variables are described using percentages. Appropriate statistical tests include the chi-square test for categorical variables, Fisher’s exact test for small sample sizes, and Student’s t-test for normally distributed continuous variables. Multivariate logistic regression analysis is employed to identify potential risk factors for POP or lung function recovery indicators, incorporating variables that exhibit a p-value of less than 0.2 in univariate analysis. To assess the relationships between independent variables and outcomes, Pearson correlation analysis is used for normally distributed data, and Spearman correlation analysis is applied for non-normally distributed data. The threshold for statistical significance is set at P < 0.05. All statistical analyses were conducted using R statistical software version 3.2.2.
Clinical characteristics of the patients between POP group and non-POP group Risk factors of the occurrence of the POP Receiver operating characteristic curve (ROC) analysis for predicting the occurrence of the POP Correlation between HRV variables and lung function in POD 30 Multivariable analysis for risk factors of postoperative lung function recovery indicators ROC analysis for predicting postoperative lung function recovery Independent factors of postoperative lung function recovery indicators were selected in the ROC analysis. For poor-recovery in FEV1%, among the single indices, the AUC of preHFP and preRMSSD was 0.701 ( P < 0.001) and 0.672preHFP and preRMSSD showed the highest diagnostic accuracy (AUC: 0.708, P < 0.001) but with no significance compared to preHFP ( P = 0.790) or preRMSSD ( P = 0.150) alone. For poor-recovery in FVC% (AUC: 0.734, P < 0.001) or DLCO% (AUC: 0.770, P < 0.001), the combination presented the highest diagnostic accuracy and differ significantly compared to the single indices alone (Table ; Fig. ).
A total of 257 subjects met the inclusion criteria and were ultimately included in the study (Fig. ). 33 patients presented POP, accounting for 12.8% (33/257) of the patients. Higher body mass index (BMI) (24.6 [23.1, 27.4] vs. 22.9 [21.1, 25.0], unit: kg/m 2 , P = 0.001), longer operation time (130.0 [120.0, 145.0] vs. 120.0 [115.0, 130.0], unit: min, P = 0.020), older age (64 [61, 70] vs. 61 [55, 66], unit: year, P = 0.005) and higher proportion of COPD (30.3% vs. 8.0%, P = 0.001) were found in the POP group. Regarding the HRV variables, the POP group had lower preRMSSD (15.0 [11.5, 18.0] vs.18.0 [16.0, 23.0], unit: ms, P < 0.001) and preHFP (138.2 [122.0, 192.8] vs. 238.2 [138.1, 344.1], unit: ms 2 , P < 0.001). Moreover, the POP group had higher LHR (1.6 [1.1, 2.7] vs. 1.0 [0.6, 2.1], P = 0.003). The details were presented in Table .
A logistic regression analysis was conducted to identify potential risk factors for the occurrence of POP, incorporating variables with a p-value < 0.20 from the univariate analysis. The multivariate analysis included age, BMI, smoking status, COPD, operation time, LHR, preRMSSD, and preHFP as candidate risk factors. The results of the logistic regression revealed that BMI (OR: 1.204, 95% CI: 1.035-1.400, P = 0.016), COPD (OR: 6.202, 95% CI: 1.820–21.140, P = 0.004), preRMSSD (OR: 0.812, 95% CI: 0.720–0.912, P = 0.001), and preHFP (OR: 0.990, 95% CI: 0.983–0.996, P = 0.002) were independent risk factors for POP (Table ).
ROC analysis was conducted for the variables to predicting the occurrence of the POP. Among the single indices, the area under the ROC curve (AUC) of BMI, preHFP and preRMSSD was 0.681 ( P < 0.001), 0.729 ( P < 0.001) and 0.757 ( P < 0.001) respectively. For combined indicators, after cross-verification, the combination of BMI, preHFP and preRMSSD showed the highest diagnostic accuracy (AUC: 0.867, 95%CI: 0.819–0.906, sensitivity: 84.4%, specificity: 80.6, Youden index: 0.650, P < 0.001) (Fig. ).
We used lung function indicators/predicted postoperative indicators, for instance, POD 30-FEV1%/ppoFEV1%, to assess the recovery of the lung function after surgery in short-term. Pearson correlation analysis figured out that RMSSD and HFP in POD 30 was positively corelated with the recovery of the lung function (Fig. ). Moreover, POD 30-FEV1%/ppoFEV1% was positively corelated with preRMSSD ( R = 0.15, 95%CI: 0.02–0.27, P = 0.018) and preHFP ( R = 0.35, 95%CI: 0.23–0.45, P < 0.001). Similar trends can be found in POD 30-FVC%/ppoFVC% (preRMSSD: R = 0.22, 95%CI: 0.10–0.34, P = 0.003; preHFP: R = 0.31, 95%CI: 0.19–0.42, P < 0.001) and POD 30-DLCO%/ppoDLCO% (preRMSSD: R = 0.27, 95%CI: 0.14–0.38, P < 0.001; preHFP: R = 0.39, 95%CI: 0.28–0.49, P < 0.001) (Table ).
Based on POD 30-FEV1%/ppoFEV1%≥1 or not, the patients were divided into well-recovery in FEV1% (≥ 1) group and poor-recovery in FEV1% (< 1) group. A logistic regression analysis was conducted to identify potential risk factors for postoperative lung function recovery, incorporating variables with a p-value < 0.20 from the univariate analysis. HRV indicators including preRMSSD (OR: 0.937, 95%CI: 0.892–0.985, P = 0.010) and preHFP (OR: 0.995, 95%CI: 0.992–0.998, P = 0.001) were independent factors. Similar results can be found in POD 30-FVC%/ppoFVC% (preHFP: OR: 0.996, 95%CI: 0.994–0.999, P = 0.007) and POD 30-DLCO%/ppoDLCO% (preRMSSD: OR: 0.945, 95%CI: 0.897–0.995, P = 0.032; preHFP: OR: 0.996, 95%CI: 0.993–0.999, P = 0.016) (Table ).
In this study, we prospectively selected 257 patients from a regional tertiary center with the aim of investigating the factors influencing POP and assessing the recovery status of lung function following surgery. Specifically, our objective was to identify HRV indicators associated with POP and postoperative lung function recovery. The results indicated that both pre-RMSSD and pre-HFP were significantly lower in the pneumonia group. Logistic regression analysis revealed that pre-RMSSD and pre-HFP served as independent predictors for POP, suggesting that RMSSD and HFP are effective HRV indicators for forecasting the occurrence of POP. Furthermorepostoperative 30 days, also serving as independent factors in this context. These findings provide compelling evidence supporting the utility of HRV indicators in predicting both POP and postoperative lung function recovery among surgical lung cancer patients. Surgery remains the primary treatment modality for lung cancer, particularly in patients diagnosed at early stages of the disease or those with resectable tumors . Postoperative complications are well-established as significant prognostic factors, exerting immediate adverse effects on recovery and survival following major lung resections . Patients undergoing lung resection face an elevated risk of postoperative pulmonary and infectious complications, which can result in high morbidity rates. Among these complications, POP is regarded as the most severe infection that may develop after lung resection . Previous studies have suggested that such complications could serve as indicators of increased long-term mortality in patients who undergo surgical intervention for lung cancer . In our presented study, BMI ( P = 0.018) and COPD ( P = 0.003) were identified as independent risk factors for the occurrence of POP, consistent with findings from prior research . Numerous studies have suggested that clinical laboratory data and HRV measures can be utilized to predict the prognosis of various diseases . The interaction between the vagus nerve and the immune system plays a critical role in the pathogenesis of lung diseases. Recent research has indicated that the vagus nerve influences acute lung injury in patients with acute respiratory distress syndrome (ARDS) via an anti-inflammatory cholinergic pathway . Furthermore, stimulation of the vagus nerve has been employed to prevent various conditions associated with acute or chronic activation of the immune system, enhance immune function responses, improve antioxidant capacity, reduce oxidative stress, and increase energy generation efficiency, thus contributing to a lower incidence rate of inflammatory diseases . Recent studies have highlighted the prognostic significance of HRV as a surrogate marker for vagal nerve activity in COVID-19 cases, suggesting that the vagus nerve may play a moderating and protective role by reducing inflammation and potentially improving survival rates in COVID-19 patients . Moreover, HRV indices could predict prolonged postsurgical stay in the intensive care unit (ICU) and mortality, and reported the potential use of pre-HRV in predicting perioperative complications . One of the innovative findings of our study was that preRMSSD and preHFP served as independent predictors for POP, indicating a correlation between HRV and the occurrence of POP. This may be attributed to the fact that a patient’s preoperative vagus nerve activity demonstrates a greater capacity for reduction compared to that associated with pulmonary infection. HRV has its unique advantages as non-invasive method, and the HRV detection can be detected easily. Furthermore, autonomic nerve activity changes are more rapid and sensitive than that inflammatory markers in blood, which were previously used as risk factor of POP, and had positive role in model establishment . In further studies, combining HRV with traditional inflammatory markers may help to improve the accuracy of the model and make clinical applications possible. Of note, HRV is influenced by the patient’s psychological state, and the sympathetic-vagal balance of patients in a state of heightened stress is disrupted, exacerbating postoperative pain and other discomforts, and consequently affecting the recovery of postoperative lung function . Some medications may also influence HRV such as beta-blockers, etc . Therefore, the effects of these factors on HRV need to be further validated.Accordingly, several studies have demonstrated sequential changes in pulmonary function following lung resection. These studies report that lung function experiences a sharp decline until one-month post-surgery, partially recovers by three months, and stabilizes six months after the procedure . Lung cancer survivors frequently experience post-treatment symptoms such as pain, dyspnea, and fatigue, which adversely impact their quality of life (QOL) . Furthermore, multiple studies have indicated that postoperative respiratory symptoms are more prevalent among patients with lower pulmonary function . The relationship between HRV and pulmonary function remains unclear. While the correlation between HRV and pulmonary function has been explored in respiratory diseases such as COPD and asthma , MS Bianchi and colleagues reported that pulmonary function is influenced by autonomic control of cardiovascular function, independent of major confounding factors in healthy adults . In our study, we found that preRMSSD and/or preHFP were positively correlated with postoperative lung function recovery within 30 days post-surgery. These indicators also served as independent predictors, supporting the utility of HRV metrics for forecasting postoperative lung function recovery in patients undergoing surgical treatment for lung cancer. It implies that clinical strategies aimed at enhancing vagal nerve activity could be implemented to promote the recovery of lung function. Such strategies may include pulmonary rehabilitation interventions centered around exercise training, as well as neuromodulation techniques such as electrical stimulation. However, it is essential that these approaches should be supported by more in-depth mechanistic research. There were several strengths associated with this study. To the best of our knowledge, this is one of the first studies to empirically establish a link between a neuro-immuno-modulatory variable—specifically HRV—and outcomes in lung cancer patients undergoing surgery. Given that lung cancer represents a global health crisis, these findings may have far-reaching implications. The measurement of HRV is straightforward and non-invasive, making it feasible for integration into routine clinical practice. This study encompassed all consecutive patients admitted to a single center, resulting in an adequate sample size for analysis. Additionally, the database included a wide range of potential confounders, such as variations among different surgical teams, which allowed us to adjust for suspected confounding variables. Of note, the study has several limitations. First, patients who may not meet absolute contraindication but undergo surgery are not fully investigated, these patients may have abnormal cardiac rhythm, which makes it hard to record precise HRV; Second, it is important to note that some risk factors may not have been identified; consequently, patients without known risk factors were also included in the analysis, potentially introducing bias into the results. Meanwhile, confounding factors during HRV measurement, such as medications that affect autonomic function and the patient’s emotional state on the day, can interfere with the results, although these patients were.in small proportion. For this subgroup of patients, the parameters we measured clearly need to be adjusted to fit. Furthermore, due to limitations in data availability, we were unable to incorporate all variables that could influence POP or postoperative recovery of lung function. In conclusion, this study demonstrates that higher HRV is associated with a lower incidence of POP and improved lung function recovery following lung cancer surgery. Consequently, HRV measurements may serve as valuable tools for the early identification of surgical candidates with lung cancer. Given that impaired vagus nerve activity appears to correlate with hyper-inflammation, future research should investigate the inflammatory levels in relation to vagus nerve activity among these patients. These findings carry significant clinical implications. This is the first study to report a relationship between HRV and both the occurrence of POP and postoperative lung function recovery. Interventions could include pharmacological treatments or non-invasive methods such as transcutaneous vagal stimulation; where feasible, deep breathing biofeedback may also be employed for patients. Therefore, exploring the effects of non-invasive vagus nerve stimulation may enhance postoperative lung function recovery while mitigating POP risk, warranting further investigation in upcoming intervention studies.
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Application of the flipped classroom model based on Bloom’s Taxonomy of Educational Objectives in endodontics education for undergraduate dental students | e56931e5-45f8-49fa-8da0-f871a26bd93f | 11786706 | Dentistry[mh] | In the 1950s, Ralph W. Tyler stated that educational objectives are the foundation and core of the curriculum, and that the curriculum should be based on the establishment of educational objectives . Bloom’s Taxonomy of Educational Objectives, proposed by educator B.S. Bloom’s team in 1956, divides educational objectives into three main areas: cognitive, affective and skill-oriented . At the beginning of the 20th century, described the objectives of the cognitive domain and established the six dimensions of the cognitive process: reading, comprehension, application, analysis, evaluation and creativity. Traditional curriculum divides teaching objectives into three categories: mastery, familiarity and comprehension, but these objectives may not reflect those of the humanities. According to Bloom’s Taxonomy of Educational Objectives, the classification of the educational objectives of a course can clarify teaching goals and ideas, rationalize teaching content, make humanistic content more natural to students, improve student empathy, and establish high quality medical ethics, while strengthening the effect of theoretical teaching and effectively combining theory-based and humanistic education. Lectures represents a traditional form of teaching. When theoretical content of a curriculum is extensive and classroom time is limited, it can be a challenge for teachers to efficiently, clearly, and engagingly convey theoretical knowledge. The flipped classroom model can effectively extend learning time. Using this model, students independently watch an explanatory video based on the curriculum in advance, allowing for classroom time to be used more efficiently for teacher-student and student-student interaction, answering questions and solving problems, and for collaboration and exploration, ultimately leading to improved teaching and learning outcomes . Traditionally, theoretical instruction for students majoring in stomatology is organized around a lecture-based curriculum. Students taught under this model reported that the learning objectives were broad and vague, the theoretical knowledge was difficult to understand and the teaching style lacked interest and engagement. Therefore, an exploration of innovative teaching methods is crucial for the optimization of undergraduate theoretical teaching. conducted a study to determine the effect of an interactive learning method using prepared questions based on Bloom’s Taxonomy on nursing students’ learning of the need for movement, and the authors recommended that studies on interactive learning be repeated in different subjects within nursing education. Another study found that Generative Pre-trained Transformer 4 (GPT-4) demonstrated a strong performance, in regards to Bloom’s Taxonomy, when queried with psychosomatic medicine multiple-choice exam questions . An investigation into teaching reform, led to the proposal of an innovative flipped classroom model based on Bloom’s Taxonomy of Educational Objectives to be applied in the theoretical teaching of endodontics. The present study aimed to compare the flipped classroom model with the curriculum-based lecture method by utilizing questionnaires to compare and analyze students’ learning levels in terms of theoretical knowledge and learning outcomes under the two teaching models. An additional goal was to develop a standardized flipped classroom model that is applicable for undergraduate stomatology curriculum, aiming to implement it in undergraduate education across the country. Study participants and designTeaching methodologyQuestionnaire surveyA normalized teaching process for the flipped classroom model based on Bloom’s Taxonomy of Educational ObjectivesStatistical analysisStudy participants were divided into two groups, each consisting of sixteen third-year students enrolled in the 5-year undergraduate stomatology program at Guanghua School of Stomatology, Sun Yat-sen University. The study was approved by the Ethics Committee of the Hospital of Stomatology, Sun Yat-sen University (No. KQEC-2024-72-01). This approval qualifies for a waiver of review and does not require the signing of an informed consent form. The educational material used for the study was the nineteenth chapter of the national textbook of higher education, “Endodontics,” which covers root canal therapy (5th edition, People’s Health Publishing House). The study duration was 1 year. Sixteen students received theoretical lessons employing a curriculum-based lecture method (Model 1). Under this model students reviewed the curriculum and textbook material prior to class, while the instructor delivered theoretical concepts in the classroom through lectures, including the use of visual presentation aides. The instructor summarized key points at the end of the class and engaged students with questions and answers during and after the lecture. The presentations and audio were recorded in real-time and uploaded to an online platform so that students could review and reflect on the curriculum after class. A second group of sixteen students received theoretical lessons using a flipped classroom model (Model 2) based on Bloom’s Taxonomy of Educational Objectives. The lecturer for both groups was the same professor from the endodontics program who possessed 5 years of dentistry teaching experience. The Endodontics Teaching and Research Department at Guanghua School of Stomatology, Sun Yat-sen University, categorized the teaching objectives of the pilot curriculum into three main areas: knowledge, competence and emotion, with the knowledge area focusing on learning and elaboration of theoretical knowledge, the competence area focusing on clinical application of knowledge, and the emotion area focusing on humanistic goals . The content of Chapter 19 of “Endodontics” was divided into five theoretical modules, for which the Department of Endodontics Education and Research produced five corresponding micro-videos: (1) Overview of the development of root canal therapy and case selection, (2) Anatomical morphology of the medullary cavity, (3) Root canal preparation and sterilization, (4) Root canal obturation and (5) Strategies for the prevention and treatment of complications in root canal therapy. Students reviewed the curriculum based on Bloom’s Taxonomy of Learning Objectives and watched the micro-video before class. During this time, students had the opportunity to independently work on the learning objectives in the knowledge domain, as well as reflect on the learning objectives in the skills and affective domains, ensuring alignment with classroom instruction. In the classroom, the instructor imparted the basic concepts and enhanced the content of the knowledge areas. This was followed by group discussions where the teacher posed questions about the curriculum content. Students responded in groups, and the teacher reviewed the responses for omissions, provided additional information and summarized key points. This process helped clarify the teaching and learning objectives. The teacher concluded by summarizing the discussion using presentations and board notes. The overall focus in the classroom was to provide instruction and facilitate interactive discussions about the objectives related to skill and affective areas. Classroom lessons were also recorded in real-time and uploaded to an online platform for students to review after class. At the end of the lesson, students had the opportunity to review and reflect on the content in relation to the curriculum based on Bloom’s Taxonomy of Educational Objectives. After completing the pilot curriculum, both groups of students completed a questionnaire involving examining, comparing and analyzing the degree of mastery of the theoretical content of this chapter, understanding of the learning objectives, satisfaction with the teaching methods, learning interest and duration and additional metrics . Feedback on the degree of satisfaction and acceptance of the two teaching modes was collected using a five-point Likert scale as follows: very dissatisfied/disagree (1); dissatisfied/disagree (2); unsure (3); satisfied/agree (4); very satisfied/agree (5). Feedback on students’ knowledge acquisition was measured using a 10-point scale, with 10 representing a perfect score and 6 indicating a sufficient score. To compare learning outcomes, the study calculated students’ final exam grades for the chapter exam questions and converted the grades of the two groups to a 10-point scale to standardize analysis. The flipped classroom model based on Bloom’s Taxonomy of Educational Objectives for the theoretical teaching of “Endodontics” Chapter 19, Root Canal Therapy, in the 5-year undergraduate dental program was developed by the Department of Endodontics, Guanghua School of Stomatology, Sun Yat-sen University. After repeated discussions and modifications, a standardized teaching process was developed . This process is summarized as follows: 1. Instead of the traditional lecture-based curriculum, the teaching objectives were divided into three main parts: knowledge, skills, and emotion. These were provided to students for previewing 1 week before class. 2. Teaching content was optimized by dividing the theoretical knowledge of the chapters into five modules. A corresponding micro-video was created for each module and uploaded to an online platform 1 week before the course, and the teaching secretary ensured that all students completed the pre-course study. 3. Students were divided into four groups to discuss and learn from the questions in modules 1, 2, 4, and 5. Module 3 was an extension not discussed in class. 4. In the classroom, the instructor first taught basic concepts and other theoretical content through lecture. Fragmented content, basic definitions, etc ., did not need to be presented in the form of micro-videos. During the teaching modules, questions were posed to students to encourage critical thinking and response. The teacher then provided corrections and summarized any mistakes made by the students. 5. The students asked questions, which were answerd by the teacher before the end of the lesson, and then repeated and summarized through presentations. 6. Questionnaires examining the effectiveness of the lessons were collected 1 week after the course. 7. Teaching was optimized based on student feedback and expert evaluations. SPSS 26.0 software was used to conduct statistical analysis on the questionnaire results from the two groups of students. Comparison of the learning outcomes of the pilot chapter with the learning outcomes of modules 1–4, the results of the theoretical examination, understanding of learning objectives and key points, comprehension of humanities objectives, grasp of medical ethics and morals, interest in learning endodontics, satisfaction with the teaching method, pre-study methods, curriculum, teaching hours and a comparison of study hours prior to class were conducted using chi-square variances and t-tests for independent samples between two groups of data. Comparison of module 5 learning outcomes and post-course learning hours was performed with t-tests for independent samples to assess heterogeneity of variance between the two groups of data. Analyses were evaluated at a significance level of α = 0.05. Comparison of the effectiveness of the two teaching models A total of sixteen students from the lecture method cohort (Model 1) attended the course, and all 16 questionnaires were subsequently completed, representing a response rate of 100%. The students’ personalized comments and suggestions on teaching included: “Less class time, more content, lectures are too fast,” “I hope animations and anatomical modeling are integrated,” “The course content is difficult,” “I hope that more clinical aspects are included,” “I suggest adding more lessons,” “The theory is abstract, animations could enhance understanding” and “I recommend incorporating pictures or 3D models for better comprehension”. A total of sixteen students from the flipped classroom cohort (Model 2) participated in the course, and all 16 questionnaires were subsequently completed, resulting in a response rate of 100%. Students’ personalized comments and suggestions about the class included: “I hope the curriculum is more detailed,” “Increase the number of class hours, otherwise the content density is too high,” “Pictures can be added” and “I hope the introduction of new concepts can be more concrete and vivid”. Statistical analysis of the student questionnaire revealed that students in the flipped classroom cohort showed a 10.9% increase in theoretical knowledge learning compared to students in the lecture-based cohort (7.9 ± 0.7 vs . 7.1 ± 0.8; ). This difference was statistically significant ( p < 0.006). The scores of the five theory modules in the flipped classroom cohort were higher by 9.8%, 13.0%, 13.2%, 10.0% and 10.1%, respectively, with an average increase of 11.2%, representing a statistically significant difference ( p < 0.01). In the final theory exam, students’ exam scores in the flipped classroom group increased from 7.0 ± 0.8 to 8.2 ± 0.7, with an average improvement of 17.1% ( p < 0.001). At the same time, students’ understanding and appreciation of humanistic goals and medical ethics increased significantly, by 11.4% ( p = 0.038) and 13.8% ( p = 0.014), respectively. Students’ satisfaction with the mode of instruction and the duration of teaching also increased significantly, by 11.1% ( p = 0.009) and 14.3% ( p < 0.020), respectively. Students’ interest in learning increased significantly, by 17.1% ( p = 0.004). Pre-class study time was approximately 10 min longer for the flipped classroom group compared to the lecture group ( p = 0.003). However, post-class study time in the flipped classroom group was reduced by approximately 30 min compared to the lecture group ( p = 0.001), a statistically significant difference. An analysis was conducted of the post-class study time after pre-class study as the same time (minutes) in mode 1 (35.5 ± 9.16) and mode 2 (36.14 ± 9.23). The results showed that the flipped classroom model (54.6 ± 11.7) was able to reduce post-class study time compared to the lecture method (85.5 ± 18.9), a statistically significant difference ( p < 0.01) (Data not shown). In the flipped classroom group, students’ understanding and appreciation of the teaching goals increased by 8.3% compared to the lecture group ( p = 0.083), and students’ satisfaction with the preparation method and curriculum increased by 8.1% ( p = 0.081) and 11.1% ( p = 0.072), respectively, compared to the lecture group. There was no statistical difference among the three groups mentioned above. Theoretical teaching is characterized by abundant textual knowledge, often presented in a manner that some students may consider abstract and unengaging. The instructor may incorporate pictures and animations to aid student learning and understanding, but due to classroom time constraints, it can be difficult to strike a satisfactory balance between providing sufficiently detailed explanation and remaining on the academic schedule. Flipped classroom instruction using micro-videos, combined with the characteristics of case-based learning, can effectively extend the classroom before and after the scheduled class time . Micro-videos are brief and concise, employing targeted division of theoretical knowledge, with each video addressing one topic with strong focus, facilitating learning and understanding of the teaching objectives. Micro-videos present theoretical knowledge in a vivid and fresh manner in order to stimulate and attract interest in learning . Micro-video length is generally 10 min or less, tailored to students’ attention span, as well as their physical and mental development characteristics. The availability of micro-videos online enables students to self pace, aiding independent study and review. The core concept of the flipped classroom, as a novel education reform model, is to remove course content from the classroom and allow students to explore and learn independently outside the classroom . In the present study, the flipped classroom model divides and records the learning content into micro-videos for independent pre-class viewing. Maximum student initiative is encouraged while valuable classroom time focuses on addressing questions and points of confusion. Students engage more actively in project-based learning, collaboratively researching and solving problems, which can promote the internalization of knowledge . Improved learning efficiency in endodontics undergraduate teaching have yielded positive results in promoting self-directed learning among medical students, consistent with findings from other related professional practices . Since 2017, the university’s endodontincs program has strove to establish a close connection between knowledge-and-skills-based teaching and values-based education into the curriculum. How to efficiently integrate the content of humanities education into undergraduate teaching, how to organicallly combine theoretical knowledge, clinical practice, and humanities education and how to promote innovations in teaching represent challenges in undergraduate education when considering the target curriculum content and requirements established by professors. Along with the professional curriculum of endodontics, additional focus should be placed on strengthening education on medical ethics and professional demeanor. Emphasis should be placed on education regarding medical benevolence, ensuring that students comprehend the importance of prioritizing the maintenance of oral and maxillofacial health, function and aesthetics, while continuously honing their medical skills. Moreover, it is crucial to prioritize respecting patients during clinical diagnosis and treatment to enhance these students’ ability to empathize. Continuous improvement of doctor-patient communication skills is crucial in building professional trust and reliablity. Bloom’s Taxonomy of Educational Objectives divides teaching objectives into three major categories and six dimensions. By incorporating the teaching of theoretical knowledge and the construction of humanistic education in the curriculum, it effectively provides guidance for teachers to clarify teaching objectives, teaching ideas and the design of academic content. Sanghee Yeo analyzed the response to integrated courses of a medical school that introduced an outcome-based curriculum, and found that most of the verbs used to describe course outcome belonged to the two lower levels of Bloom’s taxonomy: knowledge and comprehension . Pedro Tadao Hamamoto Filho et al. investigated the psychometric properties of items according to their classification in Bloom’s taxonomy and judges’ estimates, employing an adaptation of the Angoff method, and they found that items with high-level taxonomy performed better in discrimination indices, and additionally that a panel of experts may develop coherent reasoning regarding the difficulty of items . These studies confirmed that Bloom’s taxonomy can be applied effectively to humanities curriculum. Aiming to address the pain points and challenges present in current theoretical teaching, the Department of Endodontics at Guanghua School of Stomatology, Sun Yat-sen University, has implemented teaching reforms. This includes the introduction of a flipped classroom teaching model based on Bloom’s Taxonomy of Educational Objectives to help standardize the teaching process and methodology. Using this model, syllabus objectives are categorized into targeted micro-videos that are easily digestible for students. This approach enables students to engage in independent learning before class. Using this system, students can understand the knowledge, skill and emotional objectives of the curriculum prior to class. In the classroom, students are guided to discuss and study the key concepts, helping them to generalize and summarize, with mistakes corrected promptly by the teacher. The purpose of this model is to encourage students to be more receptive to the theoretical content and to stimulate their interest in learning through discussion and interaction. In the classroom, learning objectives are conveyed through clinical cases, doctor-patient communication and other practical scenarios. After class, students can reinforce the teaching objectives by reviewing the syllabus within the context of Bloom’s Taxonomy of Educational Objectives. The current study employed a questionnaire to assess the efficacy of the two teaching models. The students’ self-assessment of theoretical knowledge improved, which was reflected in improved exam scores. Additionally, students’ self-reported understanding of humanistic objectives, medical ethics and morals significantly increased. Moreover, students’ satisfaction with the teaching methods and class duration also improved. Analysis of the questionnaire responses found that the flipped classroom model improved students’ understanding of the teaching objectives, although the improvement was not statistically significant. This result was mirrored in the satisfaction survey of the pre-study process and the syllabus. The methodology of this study presented certain limitations. Due to the academic setting, it was not feasible to randomly divide students of the same grade into two groups, and there existed a memory bias. However, analysis of the questionnaire and the results of the final examination can still objectively and realistically reflect the efficacy of the teaching models. Teaching reform aims to enhance teaching effectiveness without increasing the burden on students and teachers. As technology advances, increasingly sophisticated tools are being integrated into traditional teaching methods, including virtual simulations , micro-videos , apps and artificial intelligence tools such as ChatGPT . The present study found that, under the flipped classroom model, the total study duration, both pre- and post-class, did not differ significantly, but the post-class duration was significantly shorter compared to the pre-class duration. A potential explanation for this is that students spent sufficient time before class to understand, organize and reflect on the teaching objectives. Due to students’ understanding and mastery of the course objectives, as well as adequate communication with the teacher and fellow classmates in the classroom, less time was required for post-class review and study. For teachers, it is essential to invest more time and effort in fostering teaching innovation and implementing teaching reform. This could include creating short and concise micro-videos before classes, as well as revamping lesson plans and syllabi. Teachers can update their teaching concepts and innovate their teaching methods in line with teaching reform. The department can compile a series of micro-videos through this reform, which serve as a valuable teaching resource for online and offline instruction, catering to undergraduate students, graduate students, and international students. Syllabus-based lectures represent a classic teaching method with certain strengths and weaknesses. Students find pre-course preparation relatively easy, while teachers can effectively control the classroom flow, rhythm and time. Additionally, course materials and syllabi are often repeated during multiple iterations of the same course, which reduces preparation time. However, this teaching method often leads to average performance in terms of student participation, teacher-student interaction, classroom engagement and teaching effectiveness. In comparison, the teaching approach of the flipped classroom model based on Bloom’s Taxonomy of Educational Objectives offers several advantages. These include clear teaching goals, improved integration of humanities teaching, innovative and engaging teaching methods, enhanced student interaction and participation, dynamic and engaging classroom environments and positive teaching outcomes. However, students are required to engage in more pre-class thinking and discussion, as well as increase motivation and active participation in classroom discussions and interactions. Additionally, this model requires teachers to invest more time and effort in class preparation and face challenges in controlling the pace and timing of classroom discussions and interactions. Though the flipped classroom model requires teachers to adapt their skillset, this new teaching approach could improve their effectiveness and help them better manage their classroom teaching time. It is necessary to acknowledge this work’s limitations. The small sample size of the study limits the impact and scope of the results. Therefore, subsequent studies should increase the sample size to verify the observed findings. Future studies could also incorporate teaching aides, such as AV (audio/video) and tooth simulation molds to enrich the classroom teaching content. The current study has resulted in the development of a standardized flipped classroom teaching model based on Bloom’s Taxonomy of Educational Objectives. This model, combined with humanistic teaching objectives, can increase the efficacy of teaching, and is worth popularizing and applying in the theoretical teaching of undergraduate students majoring in stomatology. 10.7717/peerj.18843/supp-1 Supplemental Information 1 Raw data and analysis results. 10.7717/peerj.18843/supp-2 Supplemental Information 2 Questionnaire (English). 10.7717/peerj.18843/supp-3 Supplemental Information 3 Questionnaire (Chinese). |
The treatment of continuous double-level lumbar degenerated disease with PE-TLIF: A case report | d679b2d0-2cce-48bc-aee0-ffb3adf4ab2f | 11749712 | Surgical Procedures, Operative[mh] | The percutaneous endoscopic transforaminal lumbar interbody fusion (PE-TLIF) technique was first reported by Osman in 2012. With the development of surgical technique and instrumentation, PE-TLIF was reported to address the variety of spinal disorders by using endoscopic and expandable cages. It allowed directly reaching the disc for achieving simultaneous decompression and fusion without excision of lamina, articular processes, and ligamentum flavum. Several literatureminimally invasive transforaminal lumbar interbody fusion (MIS-TLIF). However, considering the high incidence of surgical complications and long operative time, patients reported in previous papers were single segment involved. In our study, we performed PE-TLIF on 2 patients who had continuous double-level lumbar degenerated disease.
2.1. Case 1 2.2. Case 2 A 57-year-old woman was admitted to hospital with severe low back pain and bilateral leg pain for 6 months. The symptoms have not improved after being bedridden and taking nonsteroidal anti-inflammatory drugs (NSAIDs) for 3 months. Magnetic resonance imaging revealed that L4 spondylolisthesis combined with L4-S1 intervertebral disc herniation (Fig. ). Subsequently, we performed PE-TLIF on the patient. Operative time was 220 minutes, the blood loss was 1371-year-old man was admitted to hospital with severe low back pain and right leg pain for 3. Magnetic resonance imaging revealed that L3-L5 intervertebral disc herniation (Fig. ). Subsequently, we performed PE-TLIF on the patient. The operative time was 190 minutes, the blood loss was 100 mL. Six weeks later, the patient obtained a satisfactory clinical efficacy, What’s more, no surgical related complications was observed.
The incidence of lumbar degenerative diseases is increasing, causing a huge economic burden. Lumbar interbody fusion is the main surgical produce for the treatment of lumbar degenerative diseases and it has been proved to have effective clinical efficacy. However, conventional open surgery may result in greater surgical trauma, as well as increased bleeding and infection risk. Sometimes, it can even cause postoperative back pain. Against this drawbacks, minimally invasive surgical procedures are receiving increasing attention in the field of spine surgery. Foley and colleagues first described the MIS-TLIF technique and it has since become an increasingly popular surgery method for lumbar fusion. MIS-TLIF had the advantages of less blood loss and soft tissue trauma through smaller incision, increase the speed of recovery, and reduce postoperative pain compared with the conventional open technique. However, MIS-TLIF also need to expose paraspinal muscles and excise lamina. PE-TLIF was first described by Osman in 2012. Jacquot et al reported the incidence of surgical complications is as high as 36% and do not recommend it before technical improvements, although it has many advantages. With the development of surgical technique and instrumentation, PE-TLIF has gained much attention. It can be operated under direct visualization under endoscopy, effectively avoiding damage to nerve roots and dural sac. Several literature had reported PE-TLIF had advantages of less surgical trauma, less postoperative low-back pain, and faster recovery compared with MIS-TLIF. However, The locations of the working tube, expandable cage and pedicle screws need to be determined with X-ray during the operation, thus, patients and surgeons are exposed to higher doses of radiation. The learning curve in this technique appeared steep, creating a huge challenge, even for skilled surgeons. The operative time for PE-TLIF is relatively longer and operative time is a risk factor for postoperative complications of lumbar surgery. Di Capua et al investigated 7761 cases and discovered that operative time ≥ 4 hours is related to postoperative complications.
We firstly reported 2 patients whit continuous double-level lumbar degenerated disease who treated with PE-TLIF. Both of them achieved a satisfactory short-term clinical efficacy and the average operative time is 205 minutes, it is completely acceptable for both patients and surgeons. Thus, we suggested PE-TLIF can effectively relieve the pain and improve the lumbar function of patients with continuous double-level lumbar degenerated disease. It is undeniable that PE-TLIF technique requires a steep learning curve that requiring a thorough understanding of foraminal anatomy and rich endoscopic experience. Due to the limited number of cases reported in this article and the short follow-up time, further research is needed for more cases and longer follow-up time. Our cases can provide important clues for Surgeons in selecting appropriate treatment in patients with double-level lumbar degenerated disease. PE-TLIF is a feasible and effective surgical technique.
Conceptualization: Xiaoming Ma. Data curation: Yufeng Chen, Weiwei Jiang. Formal analysis: Yufeng Chen, Weiwei Jiang. Writing – original draft: Yufeng Chen, Xiaoming Ma. Writing – review & editing: Yufeng Chen, Xiaoming Ma.
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Age-specific assessment of initial hemoglobin levels and shock index for predicting life-saving interventions in pediatric blunt liver and spleen injuries | 30fd0874-dd23-4b7e-aaac-385b8c57e7ee | 11903640 | Digestive System[mh] | Abdominal injuries represent about 25% of major trauma incidents in pediatric patients. The majority of these abdominal traumas are of a blunt nature, predominantly affecting the liver and spleen , . Delayed intervention may lead to fatal consequences. Therefore, it is critical to identify simple, reliable, and objective predictors that can be used for the early identification of patients who will most benefit from timely and effective management , . An earlier study suggested that a lower initial hematocrit was associated with intra-abdominal injuries following pediatric blunt trauma . A recent large study also emphasized that initial low hemoglobin level was linked to failure of non-operative management, as well as the need for blood transfusions, in pediatric patients with blunt liver and spleen injuries (BLSI) . Importantly, normal hemoglobin values vary across different age groups, necessitating the evaluation of hemoglobin based on specific age categories . The shock index, defined as the heart rate to systolic blood pressure ratio, is commonly employed as a predictor of mortality in trauma patients. In pediatric trauma cases, the shock index pediatric age-adjusted (SIPA) has proven to be superior to shock index unadjusted for age in identifying severely injured children who require early transfusion, emergency surgery, or are at risk for death , . Of note, SIPA has been demonstrated to be useful in the management of children with BLSI . Considering age-related variations in shock index is crucial for a more accurate assessment in pediatric trauma care, especially in predicting need for urgent interventions. Given these findings, we hypothesized that the combination of hemoglobin levels and shock index in age-specific groups would be more useful in predicting the need for life-saving interventions (LSI) in pediatric BLSI. Accordingly, we seek to analyze the utility of initial hemoglobin levels in conjunction with the shock index based on age-specific categories, as predictors for LSI, using a cohort study of pediatric BLSI cases in Japan.
Study design Study participants Grouping and definitions Outcomes Statistical analysis This was a pre-planned sub-analysis of the SHIPPs (Splenic and Hepatic Injury in Pediatric Patients) study, which was a multicenter retrospective cohort study. The SHIPPs study involved 83 institutions and included 1,462 pediatric patients (≤ 16 years of age) with BLSI from 2008 to 2019 in Japan . The SHIPPs study included patients admitted to an emergency care setting with at least an Abbreviated Injury Scale (AIS) grade ≥ I BLSI, as detected by any imaging method or operative findings. This study conforms to the principles outlined in the Declaration of Helsinki. Okayama University Hospital Ethics Committee approved the study on December 23, 2022. The approval was for the study titled ‘Age-specific assessment of initial hemoglobin levels and shock index for predicting life-saving interventions in pediatric blunt liver and spleen injuries’, under Study ID: K2302-006. Furthermore, the committee waived the requirement for written informed consent. The study followed the Standards for Reporting of Diagnostic Accuracy (STARD) reporting guidelines for diagnostic accuracy studies .
Among the study candidates, we excluded those who had duplicated records due to inter-hospital transfers, as well as cases with an AIS of 6 in any body region or treatment limitations due to severe traumatic brain injury (TBI), defined as a head AIS ≥ 5 we further excluded patients who were younger than 1 year old because the SIPA was not defined for this age group . Additionally, we excluded those with missing data on systolic blood pressure, heart rate at arrival, or initial hemoglobin levels.
Patients were divided into three age-groups based on previous studies regarding SIPA: 1 to 6 years old, 7–12 years old, and 13 to 16 years old , , – . Abnormal SIPA was defined as greater than 1.2 for the 1 to 6-year-old age group, greater than 1.0 for the 7 to 12-year-old age group, and greater than 0.9 for the 13 to 16-year-old age group, based on these studies. In cases where patients were transferred from another hospital, their vital signs and laboratory data from the first-visit facility were used if available.
Our primary outcome was the necessity for LSI. LSI was defined as the requirement for any component of blood transfusion or the need for transcatheter arterial embolization or laparotomy due to bleeding from BLSI , , . Blood transfusion components included packed red blood cells, fresh frozen plasma, platelets, cryoprecipitate, or fibrinogen administered within 24 h of hospital arrival, regardless of BLSI severity. We examined two secondary outcomes: 1) hemostatic interventions for bleeding due to BLSI including transcatheter arterial embolization or laparotomy, and 2) blood transfusions within 24 h of hospital arrival.
Continuous variables are shown as median and interquartile range values, unless otherwise described. Categorical variables are presented as frequencies and percentages. Comparisons between two groups were madeWe evaluated the predictability of the need for LSI by analyzing initial hemoglobin levels and shock index values for all ages and across three age groups. The predictive accuracy in each of these categories was determined using the area under the receiver operating characteristic curve (AUROC). The cut-off value for prediction was established using the Youden index. Next, we performed a binary logistic regression analysis with the need for LSI as the dependent variable and initial hemoglobin levels and shock index as covariates. This analysis yielded predicted probabilities, from which we derived the AUROC to assess the combined predictability of initial hemoglobin levels and the shock index for determining the need for LSI. To interpret the AUROC values, we adopted the following standard categorical ratings: 0.5 to 0.6 as failed, 0.6 to 0.7 as poor, 0.7 to 0.8 as fair, 0.8 to 0.9 as good, and 0.9 to 1.0 as excellent , . When evaluating the performance of two or more diagnostic tests, a higher AUROC value generally indicates better diagnostic accuracy . To conduct statistical comparisons between the AUROCs of different models within the same cohort, we employed the Z statistic for pairwise comparisons, thereby determining any significant differences in their diagnostic accuracy . As sensitivity analyses, we excluded patients transferred to or from other hospitals. Additionally, we conducted another sensitivity analysis excluding those who had severe TBI, defined as a head Abbreviated Injury Scale score of 4 or 5, which can potentially affect the shock index . All tests were two-tailed, and a P value of < 0.05 was considered statistically significant. We performed all statistical analysis using IBM SPSS Statistics 26 (IBM SPSS, Chicago, IL), Prism 10.0.3 (GraphPad, San Diego, CA), and Microsoft Excel for Mac Version 16.79.2.
Among 1,462 patients registered in the SHIPPs study, 1,370 were included in the final analysis as shown in Fig. . Of these, 365 were aged 1 to 6 years, 619 were aged 7 to 12 years, and 386 were aged 13 to 16 years. Patient characteristics Outcomes Primary outcome Secondary outcomes Sensitivity analysis The baseline characteristics of each age category patient cohort are presented in Table and Table S1. Initial hemoglobin levels were significantly lower in the LSI group compared to the no LSI group: 10.7 vs. 12.1 g/dL in the 1 to 6-year age group; 11.8 vs. 12.6 g/dL in the 7 to 12-year age group; and 12.9 vs. 13.6 g/dL in the 13 to 16-year age group, with P < 0.001 for each comparison, respectively. Meanwhile, the shock index was significantly higher in the LSI group: 1.27 vs. 1.10 in the 1 to 6-year age group; 0.96 vs. 0.85 in the 7 to 12-year age group; and 0.83 vs. 0.69 in the 13 to 16-year age group, with P < 0.001 for each comparison, respectively. There were no patients who died from hemorrhagic shock due to BLSI without receiving any LSI.
Table presents the outcomes for specific age groups. LSI were required in 92 out of 365 patients (25.2%) aged 1 to 6 years, 139 out of 619 patients (22.5%) aged 7 to 12 years, and 154 out of 386 patients (39.9%) aged 13 to 16 years, respectively. In terms of hemostatic procedures, transcatheter arterial embolization was more commonly performed than laparotomy across all age groups. A majority of patients in each group received blood transfusions within 24 h of hospital arrival.
Table shows the predictability for all ages and for specific age groups of hemoglobin levels, shock index values, and their combination in predicting the need for LSI. The ROC curves for hemoglobin, shock index, and their combination for the entire cohort and age-specific groups are illustrated in Fig. . Both initial hemoglobin levels and shock index demonstrated improved predictive performance for LSI in age-specific groups compared to the entire cohort encompassing all ages. Cut-off values for hemoglobin, determined by the Youden index, were 10.8 g/dL for the 1 to 6-year age group, 11.9 g/dL for the 7 to 12-year age group, and 12.1 g/dL for the 13 to 16-year age group. Similarly, cut-off values for the shock index were 1.20 for the 1 to 6-year age group, 1.03 for the 7 to 12-year age group, and 0.93 for the 13 to 16-year age group. When combining hemoglobin levels and the shock index, the predictability for the entire cohort was categorized as poor, with an AUROC of 0.665 (95% CI, 0.630 to 0.700). In contrast, predictability was categorized as fair within each specific age group. In the 1 to 6-year age group, the shock index in conjunction with the initial hemoglobin levels demonstrated statistically superior predictive performance compared to the shock index alone (AUROC of 0.770 vs. 0.671, P = 0.025; Z = 2.245).
As for the secondary outcomes, hemostatic interventions including transcatheter arterial embolization or laparotomy due to BLSI was required in 61/365 (16.7%) for the 1 to 6-year age group, 110/619 (17.8%) for the 7 to 12-year age group, and 110/386 (28.5%) for the 13 to 16-year age group. While any blood transfusions within 24 h of hospital arrival was required in 75/365 (20.5%) for the 1 to 6-year age group, 84/619 (13.6%) for the 7 to 12-year age group, and 99/386 (25.6%) for the 13 to 16-year age group. The AUROC appeared to decrease when utilized to predict the need for hemostatic interventions including transcatheter arterial embolization or laparotomy for BLSI, in comparison to predicting the need for LSI (Table S2 & Figure S1). Conversely, the AUROC appeared to increase when predicting the necessity for blood transfusions within 24 h of hospital arrival, in comparison to predicting the need for LSI (Table S3 & Figure S2).
A sensitivity analysis is presented in Table S4 and Figure S3, where patients transferred to or from other hospitals were excluded. Another sensitivity analysis, which excluded patients with severe TBI, are detailed in Table S5 and Figure S4. Similar results were obtained: combined variables, categorized by age, improved predictive performance for LSI compared to either parameter alone or a combined assessment in the entire cohort.
In this multicenter retrospective cohort study, we found that the combined use of initial hemoglobin levels and the shock index, when tailored to specific age groups, consistently yielded a fair level of predictive performance. This was in contrast to the lower predictive accuracy observed when applying either parameter alone or a combined assessment to the entire cohort for determining the need for LSI in pediatric patients with BLSI. Specifically, the predictability of combined values significantly improved in the 1 to 6-year age group compared to the shock index alone. The utility of this approach is highlighted by our findings that hemoglobin levels and shock index cutoff values vary among different age groups. These variations enhance the predictability of the need for LSI in these diverse pediatric populations. Although normal hemoglobin levels vary among different races/ethnicities and regions , in general, pediatric populations tend to have lower normal hemoglobin values that increase with age: approximately 12.5 g/dL for ages 2 to 6 years; 13.5 g/dL for 6 to 12 years; and 14.5 g/dL for males and 14.0 g/dL for females between 12 to 18 years . The observation that the highest predictive performance for the need for LSI is found in the 1 to 6-year age group suggests that children in this age group may reach critical thresholds more rapidly in the event of trauma, likely due to their generally lower hemoglobin levels. The cut-off values we obtained for the shock index are in near-perfect alignment with those previously suggested by SIPA: > 1.2 for the 1 to 6-year-old age group, > 1.0 for the 7 to 12-year-old age group, and > 0.9 for the 13 to 16-year-old age group , , , , . In the 1 to 6-year age group, the potential reason that combined variables are statistically superior to shock index alone could be that tachycardia in younger children is more frequently induced by factors such as pain and agitation, rather than solely by hypovolemic shock . The variation in AUROC across different age groups may be in part attributed to the fact that injury patterns differ among these groups. Analysis of baseline characteristics reveals significant demographic variability across age groups, as demonstrated in previous studies , , which could markedly affect the AUROC. For instance, injury due to falls from height exhibit distinct patterns in various age cohorts. The proportion of pedestrian injuries is higher among younger children. In our secondary outcome analyses, we observed a variation in the AUROC when predicting different outcomes: the combined AUROC of hemoglobin and shock index appeared higher for predicting the necessity of blood transfusions within 24 h of hospital arrival compared to the need for hemostatic interventions across any age-specific group. This may be due to the direct relationship between hemoglobin levels and the decision to administer blood transfusions. Our findings remained consistent even after excluding patients who were transferred between hospitals or those with severe TBI, indicating the robustness and validity of our results. Our analysis revealed that the combination of initial hemoglobin levels and shock index, categorized by age, enhances predictive performance rather than applying either parameter alone or even a combined assessment in the entire cohort, aligning with our hypothesis. Although it is important to acknowledge that these two parameters alone cannot perfectly discriminate the need for LSI, their combined use considering age-specific groups and being readily and rapidly available, could aid physicians in making determinations and thereby enhance prompt management in pediatric BLSI patients. This study has several limitations. First, the study cohort is restricted to pediatric patients with BLSI. While liver and spleen injuries are common in blunt abdominal trauma, our findings cannot be directly applied to all patients presenting to the emergency department with blunt abdominal injuries. Second, the decision regarding the timing and choice of LSI, such as transcatheter arterial embolization or laparotomy, was entirely at the discretion of the attending physician or institution. This variability could introduce a degree of bias and inconsistency in the treatment approach, potentially affecting the generalizability of our findings. Third, although prehospital fluid resuscitation for pediatric trauma patients is relatively uncommon in Japan , we lacked data on whether the patients received fluid resuscitation in the prehospital setting. This could affect the initial hemoglobin levels and vital signs , which are crucial parameters in our study. Additionally, nearly 30% of the study cohort were transferred from other hospitals. Although we used the vital signs and laboratory data from the original facility where available, the exact proportion of patients with original versus transfer-time data is unknown. Such data could differ significantly due to initial treatments such as fluid administration. Nonetheless, our results remained robust even after excluding patients who were transferred between hospitals. Fourth, the number of patients registered varied significantly across institutions, which could affect robustness ( Figure S5 ). Moreover, this study included patients with BLSI of any severity, from minor to critical injuries, as identified through imaging or operative findings. However, the higher-than-expected rate of LSI may be explained by differences in practice patterns between Japan and the United States, where significantly fewer angiographic procedures are performed . Lastly, we did not perform external validation with other datasets, which may limit the applicability of our findings in other clinical settings. Despite these limitations, our study, using a large cohort, provides important insights into the specific needs for LSI in pediatric BLSI patients. It emphasizes the usefulness of utilizing a combined approach of initial hemoglobin levels and shock index, especially considering their variability across different age groups. While the identified SIPA and hemoglobin cut-off values provide objective reference points, they should be interpreted alongside clinical assessment rather than used in isolation. These values may help clinicians rapidly identify pediatric patients with BLSI at risk for LSI; however, real-time decision-making should remain multifactorial, incorporating additional clinical parameters and assessments.
This multicenter retrospective study revealed that an age-specific approach, combining initial hemoglobin levels with the shock index, consistently improves the predictability of LSI to a fair level in pediatric patients with BLSI, compared to using each parameter independently or a general assessment across the entire cohort. Notably, this combined approach showed statistical superiority over the shock index alone, particularly in the 1 to 6-year age group. These findings suggest that our method is a more useful tool for physicians, facilitating timely and accurate decision-making, and thus may enhance the management of pediatric BLSISupplementary Information 11.
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The effect of listening to preferred music after a stressful task on performance and psychophysiological responses in collegiate golfers | 01492c65-a256-4d93-b398-b03f71148aa7 | 9166680 | Physiology[mh] | Improving performances and self-motivational beliefs constitutes one of the major issues in the sports-exercises domain . However, mental stress and/or fatigue may exert disruption on task performance as well as other psychological ( e.g. , anxiety) and physiological ( e.g. , heart rate (HR)) responses among athletes ( e.g. , ; ; ; ; ). For example, it was found that pressured situations could decrease HR variability (HRV), which can reflect increased mental effort or increased HR, which can reflect an increase in anxiety and/or arousal . In the same vein, suggested that elevated competitive pressure elicits disruption of golf performance ( i.e., putting accuracy) through both psychological ( i.e., anxiety, effort) and physiological ( i.e., HR) pathways. Moreover, mental fatigue is associated with impaired physical activity, and technical and tactical performances during small-sided games ( e.g. , ; ). Furthermore, a recent systematic review revealed that mental fatigue could also impair a myriad of sport-specific psychomotor performances, including decision-making, reaction time, and accuracy outcomes. The current state of literature indicates that listening to music is a valuable strategy for improving task performance and certain psychological, physiological, or psychophysical responses . Due to its ability to act as a stimulant and/or relaxant , this auditory stimulus can promote greater athletic performance in different exercise settings ( e.g. , ; ; ). Moreover, listening to music is usually associated with better psychological states by increasing the level of motivation/enjoyment and counteracting the negative dimensions of mood such as anxiety and tension . Additionally, listening to music could guarantee better psychophysical states by reducing the perceived effort and fatigue . Furthermore, listening to music can have positive effects on physiological ( e.g. , HR; ) and biochemical parameters ( e.g. , blood lactate concentration; ). These presumed advantages of music intervention have been reported in different sports activities, such as swimming ( e.g. , ), running ( e.g. , ; ), cycling ( e.g. , ), soccer ( e.g. , ), and volleyball ( e.g. , ). The added value of listening to music is also attested in past scientific works within the sport of golf. Golf is a closed kinetic chain sport that requires fine and gross motor control skills. To achieve a successful golf shot ( i.e., hit the ball close to the target), practitioners should succeed in both “ swinging ” and “ putting ” performances. The full swing is a complex dynamic movement that requires excellent coordination of different body segments ( e.g. , head, upper/lower limbs, and trunk), and control of the velocity of swing performance during the golf swing cycle . The putting stroke is a sophisticated action that requires stable motor performance and mental status for the accuracy of shots . showed that listening to music (classical, country, rock, jazz, and hip hop/rap) during exercise improved golf-putting performance, where jazz was the most effective music type. Another qualitative study explored self-reported music use among amateur and semi-professional golfers . The results demonstrated that the use of music could help golfers to maintain attention, achieve optimal physiological arousal ( e.g. , through increased energy or calm nerves), regulate their mood (particularly following a poor shot or round), and improve psychological states ( e.g. , confidence, motivation, adherence, flow, and enjoyment). More recently, investigated the effects of listening to preferred music on the golf swing and putting performance, HR, HRV, and anxiety in collegiate golfers. The authors demonstrated that listening to self-selected music, either before or during exercise, could guarantee better cardiac-related responses and decrease anxiety levels. The current study was designed to extend the research base by examining the relationships between music and sports performance. More specifically, it explored whether listening to preferred music (before and during exercise) following mental stress/fatigue would affect golf performance ( i.e., putting and swinging), HR, HRV, and anxiety in amateur golfers. To the best of our knowledge, no previous research has addressed this issue in sports (in general) and golf (in particular), which is a significant contribution of the current study. Based on the literature, it was hypothesised that listening to preferred music following mental stress/fatigue would improve swinging and putting performance, and would result in better psychophysiological responses in collegiate golfers.
Experimental approach to the problem Participants Experimental procedure Music intervention Mental stress Golf performance assessments Cardiac-related response Anxiety assessment Statistical analyses Descriptive data of the measured variables were presented as mean and standard deviations (mean ± SD) or median (interquartile range [IQR]). The normal distribution of the study variables was evaluated using the Shapiro–Wilk test. A repeated measure ANOVA was used to compare putting performance, STAI-S, HR, and HRV. When a significant interaction or main effect was identified, a post-hoc analysis with Bonferroni contrast was used to identify the difference between the mean values. As a significant level of normality was identified in swinging and TFAI, nonparametric tests were performed for subsequent comparisons. The Friedman test with the Monte Carlo adjustment was performed to compare experimental conditions. The Wilcoxon rank test was used to identify differences between the baseline and post-intervention pairwise comparisons. The partial eta squared ( η p 2 ) was used for all repeated measure comparisons of effect size . Additionally, the percentage change in STAI-S and TFAI at baseline and post-intervention assessment was calculated as [12pt]{minimal}
}{}$variable=( _{post}-Variabl{e}_{baseline}}{Variabl{e}_{baseline}} ) 100$ % v a r i a b l e = V a r i a b l e p o s t − V a r i a b l e b a s e l i n e V a r i a b l e b a s e l i n e × 100 . An alpha value of p < 0.05 was set for significant differences between means or medians. All statistical analyses were performed using SPSS version 25.0 software for Windows (IBM, Armonk, NY, USA).
A counterbalanced measure and within-subjects design was used in this study. This laboratory-based experiment investigated the effects of pre-task and synchronised self-selected music interventions on golf performance, cardiac-related responses, and anxiety in collegiate golfers. The participants were a part of the control trial, pre-task music trial, and synchronised music trial in a randomised order, 48–72 hrs apart . A web-based program ( https://www.randomizer.org/ ) was used for randomisation. The golf swing and putting performances were evaluated in a virtual golf simulation environment. Additionally, baseline and post-performance measurements of cardiac-related responses and anxiety status were taken.
Sixteen male and four female collegiate golfers at the amateur level (age 20.1 ± 1.17 yrs., height = 173.8 ± 7.74 cm, body weight = 72.35 ± 12.67 kg) voluntarily participated in this study . All participants were healthy with no contraindication to participation. The inclusion criteria were as follows: (1) Collegiate golfers and (2) a training frequency of 4–5 times a week (weekly training time of 10–12 hrs). The exclusion criteria were as follows: (1) history of severe neuromuscular injury, (2) lower extremity or lower back injuries within six months, and (3) current diagnosis of cardiovascular or metabolic diseases. All participants signed informed consent forms and were familiarised with the experimental procedures one week before the experiment. Ethical approval was granted by the Institute Ethics Committee of the University of Taipei (UT-IRB-2021-010). This study was performed per the ethical standards of the Institutional Human Ethical Committee of the Declaration of Helsinki and its later amendments. The sample size was determined based on our recent study , using a setting of a priori compute required sample size: analysis of variance (ANOVA) repeated measures within factors and an α level of 0.05 (G*Power 3.1.9.4, Düsseldorf, Germany). The results revealed that 20 participants approached an actual power (1– β error probability) of 0.81.
Participants who met the inclusion and exclusion criteria first visited the laboratory for familiarisation and determination of individual preference in music and physical characteristics. Afterwards, the participants visited the laboratory on three separate occasions for control, pre-task music, and synchronised music trials with a 48–72 h interval between each trial. For each trial, a baseline measurement of resting HR and HRV was taken for 10 min in a seated position. The participants were then requested to answer the State-Trait Anxiety Inventory (STAI-S) and Three-Factor Anxiety Inventory (TFAI). Subsequently, the participants were exposed to a 10-min long pre-task music intervention or control condition (resting in a seated position for 10 min), followed by a 10 min Stroop Colour and Word Test (SCWT). During the assessment of golf performance, the participants performed five swings and twenty putts in a virtual golf environment . During the synchronised music trial, the participants carried out their swings and putts while listening to the self-selected music. Finally, HR, HRV, STAI-S, and TFAI were assessed again during the post-exercise measurement. All trials were conducted at the same time of day.
Personal preference in music was used as 15 min long music intervention. All participants selected pop music as their preferred music. The participants were instructed to sit on a comfortable chair and use their smartphones to play music via personal earphones. To prevent background noise a 3M protective earmuff (noise reduction rating of 27 dB, 1427, 3M, China) was used while listening to music. The music was defined as slow tempo music (<120 bpm) or fast tempo music (>120 bpm), per a study by . BPM analyser software was used to analyse the self-selected music tempo (Mixmeister, Cumberland, RI, USA).
A Chinese version of the SCWT was used to induce mental stress before the golf performance. The SCWT consisted of two subtasks: (1) naming Chinese colour words that were printed in the same colour as the word (simple identification) and (2) naming the colour of Chinese colour words printed in a different colour. The colours of the characters were black, blue, red, and yellow. The participants were asked to name the colours from the two subtasks left-to-right and top-to-bottom. They were then asked to reverse the naming order, i.e., from right-to-left, top-to-bottom, and repeat the entire process as quickly as possible in 10 min. The authors have permission to use this instrument from the copyright holders .
An indoor golf simulation system (Vision Compact, Golfzon, Korea) was used to assess golf performance. It was set to the driving range mode for swing performance, and the challenge mode, with a putting distance of 2.5 yards, for putting performance. Each participant used their seven-iron and putter and was asked to swing five times and putt twenty times. The performance of each shot was displayed on the screen using a Golfzon GDR automatic detector. The parameters of swing performance included flight distance with carry and roll distance (flight), flight distance, speed of the ball (speed), and launch angle (angle), while the parameters of putting performance included the number of successful trials (putting performance) and the error distance between the hole and ball in unsuccessful trials (putting distance). All participants were familiarised with golf practice in the Golfzon virtual environment during their routine training sessions.
Heart rate and HRV were used to evaluate autonomic nervous adaptation in response to music intervention. A portable HR monitor (Polar RS800CX, Polar Electro, Kempele, Finland) was used to record resting cardiac-related responses while seated for 10 min. The record of the first 5 min was not included in the analysis of HR and HRV indices to avoid the impact of orthostatic effects on autonomic nervous activity. All raw data were processed using professional HRV analysis software (Premium version 3.2.0, Kubios, Kuopio, Finland). A prior setting of moderate artefact correction and smoothing at 500 Lambda were used to process the data. The HRV indices included (1) time-domain analysis: standard deviation of normal R-R interval (SDNN) and mean sum of the squared differences between RR (RMSSD); (2) frequency domain analysis: low-frequency power spectrum (LF) and high-frequency power spectrum (HF); and (3) nonlinear analysis: standard deviation of the points perpendicular to the line of identity (SD1) and standard deviation along the line of identity (SD2). The spectrum of frequency bands for the LF and HF were set to 0.04–0.15 Hz and 0.15–0.4 Hz, respectively.
Two Chinese versions of leading anxiety measures were used to evaluate anxiety in this study: the STAI-S and TFAI. The STAI-S consists of 20 items related to individual awareness of anxiety , presented as a four-point Likert scale ranging from one (fully disagree) to four (fully agree). The sum of the scores for 20 items was used for the statistical analysis. The minimum STAI-S score was 20 points, while the maximum STAI-S score was 80 points. The development of the STAI-S scores was positively correlated with anxiety levels. The Cronbach’s alpha coefficients of the Chinese version of STAI-S were 0.90 and 0.81 for the State and Trait scales, respectively . The authors have permission to use this instrument from the copyright holders . The TFAI is a useful tool for quantifying the various psychological factors for anxiety and depression during exercise . Thus, we used this tool to evaluate the impact of anxiety levels on golf performance. The Chinese version of the TFAI consists of 21 items, divided into three components. Items 1–10 evaluate cognitive components (four items for anxiety and six items for self-awareness), 11–17 evaluate physiological components (four items for autonomic nervous responses and three items for somatosensory feedback), and 18–21 evaluate perceived components. A five-point Likert scale was used to quantify psychological feedback (from 1 = fully disagree to 5 = fully agree). The internal consistency of Cronbach’s alpha ranged from 0.85 to 0.86 . The authors have permission to use this instrument from the copyright holders .
Physical characteristics and golf level Golf performance Autonomic nervous function Anxiety status The STAI-S results showed no interaction ( F (2,38) = 0.471, p = 0.628, η p 2 = 0.024) and main effect (Trial: F (2,38) = 0.471, p = 0.673, η p 2 = 0.021; Time: F (2,38) = 3.379, p = 0.082, η p 2 = 0.151) . The pre- and pos t -test percentage change of the STAI-S had no main effect of trials ( F (2,38) = 0.213, p = 0.809, η p 2 = 0.011) in the ANOVA test . The TFAI results show that the Friedman test (inter-trial comparison) demonstrates a significant difference in the pre- and pos t -test percentage of physiological elements ( p = 0.012, pre-task trial = −1.92% <control trial = 0% <synchronised trial = 4.58%). However, no statistical differences were found in other comparisons ( p > 0.05) .
The physical characteristics and golf levels of the participants are listed in . Golf performance ranged from par +2 to +13.
demonstrates no significant difference in any golf swing variables (flight ( p = 0.116), flight distance ( p = 0.608), ball speed ( p = 0.819), and angle of ball travelling ( p = 0.550)) among the experimental trials. Additionally, no significant difference was found in the number of successful putts on target ( F (2,38) = 2.156, p = 0.130, η p 2 = 0.102) and distance error between the target and ball ( F (2,38) = 2.227, p = 0.122, η p 2 = 0.105) in statistical comparisons .
The results of a two-way repeated-measures ANOVA showed no interaction and main effect of trial or time on mean HR, SDNN, RMSSD, LF, HF, SD1, and SD2 (see ).
is study aimed to investigate the effects of self-selected music interventions on golf performance and psychophysiological responses after a mental stress test in collegiate golfers. The proposed hypotheses were rejected because there were no significant differences in golf performance, cardiac-related indices, and psychological responses among the experimental conditions ( i.e., control, pre-task, and synchronised music trials). Effects of music interventions on anxiety level Effects of music interventions on golf performance Effects of music interventions on autonomic nervous functions Limitations and directions for future research Functional implication The results of the present study showed that listening to preferred music (either before or during exercise) following mental stress/fatigue ( i.e., after performing the SCWT test) does not reduce anxiety in golfers. These results differ from those of previous sporting studies demonstrating the potential effect of music in counteracting the negative dimensions of mood ( i.e., anxiety and tension), and in improving positive attitudes such as motivation and feeling states ( e.g. , ; ). Moreover, the findings did not fit with the neuropsycho”-logical literature showing that music can modify brain function that can provoke positive effects on emotional regulation , and in reducing fatigue and its related symptoms . A plausible explanation for the non-positive effects of music on anxiety levels could be related to sleep loss. Indeed, the experimental protocol including the anxiety post-exercise assessment was carried out in the afternoon ( i.e., between 13:00 hrs and 15:00 hrs), referred to as the post-lunch dip . According to , the post-lunch dip is a period of sleepiness that occurs between 13:00 hrs and 16:00 hrs due in part to a slight reduction in core body temperature, which promotes a tendency to sleep (see also ; ). In this context, past scientific works showed that partial sleep deprivation appeared to be more vulnerable to negative mood sensations such as anxiety, fatigue and depression ( e.g. , ; ; ).
The current study results demonstrated that listening to preferred music following mental stress/fatigue does not affect swing and putting performances in amateur golfers. The findings are not in accordance with earlier research showing the benefits of using music before/during task either on physical performance ( e.g. , ; ) or on gross and fine motor skills ( e.g. , ). The non-positive effects of music on golf performance could be related to psychological effects. It is well known that psychological state is a determinant element before or during exercise . As mentioned in the section above, listening to music following mental fatigue failed to counteract negative mood ( i.e., anxiety) in golfers. In this context, recently demonstrated a causal link between mood state and performance. Another plausible explanation for the non-profit of listening to music following mental stress could be related to the study sample, where most of the participants were males. In this context, it was established that female athletes seemed to derive greater benefit from music during exercise, compared to male athletes . Further studies are needed to test whether our results would also apply if we tack into account such types of individual differences. Otherwise, to further explain the current findings, we could speculate that the non-change in golf performance could be related to the sleep loss during the execution of the experimental protocol ( i.e., post-lunch dip period), which led to modest reductions in muscle glycogen, which may signal fatigue to down-regulate muscle activation and prevent glycogen depletion .
It was found that listening to preferred music after performing the SCWT test does not affect HR and HRV among collegiate golfers. These results differ from those of previous sporting studies showing the positive effects of music on HR ( e.g. , ; ). Discrepancies between the findings could be related to the potential effect of a musical tempo. In the current study, based on the self-selection mode, we did not control music preference among participants. Indeed, 12 participants choose fast/motivational music with high tempo (>120 bpm), while 8 participants choose slow music with low tempo (<120 bpm) (for more on this point, see ). Some earlier research proved that musical tempo may differently influence diverse cardiovascular responses. For example, found that listening to motivational music (>120 bpm) has a more positive effect than listening to slow music (<120 bpm) on HR when performing treadmill endurance exercises. In the same vein, showed that listening to high musical tempo during steady-state treadmill exercise is more efficient than listening to low musical tempo for cardiovascular responses ( e.g. , oxygen consumption, stroke volume, frequency of breaths, cardiac output, and systemic vascular resistance). More recently, the relationship between different musical tempos and HR has been examined during endurance (walking for 10 min at 6.5 km/h on a treadmill) and high intensity (80% on 1 repetition maximum) exercises in active adult women . The authors found showed that HR increased with the increase in musical tempo either during endurance or high-intensity training. Therefore, it would be worthwhile in future studies to compare the effect of listening to different musical tempos following mental fatigue on cardiac-related responses in amateur golfers.
In addition to the above-mentioned limitations ( i.e., regarding the individual difference and musical tempo), other questions remain unanswered and require further research. This study is low in ecological validity as we used a computer-based virtual environment ( i.e., Golfzon golf simulator) to evaluate golf performance. It is unclear whether the current results would be replicated in a real putting green environment ( e.g. , ). Additionally, the intensity of mental stress/fatigue among the participants caused by the SCWT test was not evaluated in this study. As we highlighted above, another limitation is related to the potential effects of music tempo on golf performance and psychophysiological responses in this study. Lastly, this study does not take into account the potential effect of the time of day on performance and mood states. Indeed, the experimental protocol was carried out during the afternoon hours ( i.e., between 13:00 hrs and 15:00 hrs). It was established that there is an intraday variation in various exercise performances and mood states . It would be worthwhile to address this issue in future studies.
Golf is considered a sport based on closed-chain motor control and body coordination. However, psychological and mental aspects are key elements to success in professional competitions. During official tournaments, golfers had tight schedules on consecutive days. The ranking strategy and psychological and physiological strains during long periods of work can cause mental stress that affects golf performance. Music intervention and psychological skills are considered to be optimal strategies to overcome this issue. Despite positive reports of music intervention on psychophysiological modulation and exercise performance in literature, golf competition regulations may not permit the implementation of synchronised music intervention during play. However, golfers can use music tempos to facilitate rhythmic techniques to reduce competition stress occurs. This strategy is supported as an option for improving golf shot accuracy and decreasing the variability of swing performance .
In conclusion, the study found that there are no benefits of pre-task and synchronised music interventions on golf performance, cardiac responses, and reduction of anxiety levels in collegiate golfers after a mental stress task. Although the physical element of the TFAI showed significant differences in the pre- and post-test percentage of physiological elements, no change in cognitive and perceived elements indicated a minimal impact of music intervention on psychological adaptation after the mental stress task when the amateur collegiate golfers practice swing and putting skills in a virtual environment. Future studies should implement game-based environment and competition conditions to simulate real-world situations to explore this relationship.
10.7717/peerj.13557/supp-1 Supplemental Information 1 Swing performance raw data Click here for additional data file. 10.7717/peerj.13557/supp-2 Supplemental Information 2 Putting STAI-S TFAI raw data Click here for additional data file.
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Metabolic reprogramming in malignant A375 cells treated with a ruthenium (II) complex: insights from GCxGC-TOF/MS metabolomics | f3f528f8-6247-42c6-970b-8d6e1f0bd778 | 11825624 | Biochemistry[mh] | Melanoma remains one of the most aggressive and lethal forms of skin cancer, accounting for the majority of skin cancer-related deaths worldwide (Sadeq et al., ). While the advent of targeted therapies, along with immune checkpoint inhibitors, has marked significant progress in treating advanced melanoma, the long-term efficacy of these interventions remains limited (Sadeq et al., ; Ziogas et al., ). A substantial proportion of patients do not respond or eventually develop therapeutic resistance, leading to disease relapse and progression within a short period of remission (Rambow et al., ). This resistance, often driven by genetic heterogeneity and tumour plasticity, highlights the need for novel therapeutic approaches that can address the metabolic and adaptive mechanisms utilised by melanoma cells (Rambow et al., ). Ruthenium complexes have emerged as promising candidates in cancer treatment due to their unique chemical and biological properties (Thota et al., ). Unlike traditional platinum-based drugs, ruthenium complexes exhibit lower toxicity to non-malignant cells and greater selectivity for malignant cells (Lee et al., ; Sun et al., ). Ruthenium has various oxidation states, enabling it to participate in diverse biochemical interactions, and it can mimic iron, allowing it to exploit the iron transport pathways used by cancer cells (Lu et al., ). Therefore, researchers are actively exploring ruthenium-based compounds for their potential to offer effective and less toxic alternatives to conventional cancer therapies (Lin et al., ; Sun et al., ; Thota et al., ). The mechanism of action of ruthenium-based anticancer drugs is understood to some extent. However, to optimise drug design (enhance selectivity and efficacy), it is key to obtain comprehensive molecular signatures that inform how these anticancer drugs target unique metabolic processes in cancer cells. Cell culture metabolomics has gained traction and is currently being used to decipher the cellular metabolic alterations induced by metallodrugs in various cancer cell lines (De Castro et al., ; Castro et al., ; Galvez et al., ; Ghini, ; Halama, ; Zhang et al., ; Zong et al., ). Previously, our group synthesised, characterised, and tested the cytotoxicity of a series of ruthenium complexes containing a bis-amino phosphine ligand (Engelbrecht et al., ; Hussan et al., ). Among the complexes, GA113 was the most potent, with an IC 50 < 10 µM in malignant A753 melanoma cells. Using a nuclear magnetic resonance (NMR) metabolomics approach, Hussan et al. investigated the effect of GA113 on the metabolic profile of the A375 cells. In this study, we expand on this previous NMR investigation by applying a two-dimensional gas chromatography time-of-flight mass spectrometry (GCxGC-TOF/MS) metabolomics approach to obtain a more comprehensive molecular signature of A375 cells treated with GA113. Studies have reported on the application of GC-MS-based metabolomics to investigate the mechanism of action of metal-based anticancer drugs (Granit et al., ; Obrist et al., ; Pyo et al., ; Soares et al., ). However, only a limited number of studies have used GCxGC-TOF/MS. Although one-dimensional GC-MS can offer excellent resolution, some applications require increased resolution power. To this end, GCxGC-TOF/MS is increasingly being used in cutting-edge research as a powerful approach that offers enhanced separation, sensitivity, and resolution for complex biological samples (Winnike et al., ). In the context of anticancer drug design and development, GCxGC-TOF/MS metabolomics analysis can reveal off-target effects and potential biomarkers for response, which are valuable for predicting and minimising toxicity (Halama, ). Additionally, GCxGC-TOF/MS metabolomics can contribute to our understanding of how potential anticancer drugs interact with cellular redox systems, possibly allowing adjustments that fine-tune their oxidative stress-inducing effects and reduce adverse reactions in healthy cells (Halama, ). This study used a GCxGC-TOF/MS metabolomics approach to investigate the possible metabolic mechanism of action of GA113 in a human malignant A375 cell line. Complex synthesisCell cultureCytotoxicity assayCellular viabilityTreatment for GCxGC-TOF/MS metabolomicsQuality control samplesSample preparation for metabolite extraction and analysisGCxGC-TOF/MS analysisGCxGC-TOF/MS data processingChemometric and multivariate statistical modellingPathway mappingThe bimetallic complex (GA113; Fig. ) investigated here was synthesised according to previously reported methods and provided satisfactory analytical data (Engelbrecht et al., ; Hussan et al., ). Human malignant A375 melanoma cells were obtained from Prof. Kidzeru at the Hair and Skin Research Laboratory, Division of Dermatology, Department of Medicine, Faculty of Health Sciences, Groote Schuur Hospital, University of Cape Town, South Africa. Cells were cultured in Dulbecco’s modified Eagle medium (DMEM) (Hyclone™, GE Healthcare and Life Sciences, USA) with high glucose, supplemented with 10% foetal bovine serum (FBS) (Sigma, St. Louis, MO, USA) and 1% penicillin/streptomycin/amphotericin B solution (Sigma, St. Louis, MO, USA). Cultures were maintained in 75 cm² flasks within a sterile Heracell™ VIOS 160i incubator, providing a humidified atmosphere of 37 °C and 5% CO 2 . The culture medium was replaced with fresh-supplemented DMEM (20 mL) the following day. The cells were subcultured every two days. The treatment procedure and cytotoxicity evaluation of GA113 against the malignant A375 cells are reported in our previous publication (Engelbrecht et al., ). The IC 50 value (8.76 µM ± 1.37 µM) was calculated in Microsoft Excel from the dose-response curve using nonlinear regression analysis (Engelbrecht et al., ). The light microscopy and flow cytometry data generated from our previous study also showed that, after a 24-hour treatment exposure, GA113 induced obvious cell death in A375 cells (Engelbrecht et al., ; Hussan, ). The concentration of GA113 used in this study was selected based on the IC 50 value obtained from our previous publication (Engelbrecht et al., ). Initially, three concentrations were selected: low concentration (2.5 µM); middle concentration (5 µM); and IC 50 concentration. Before the GCxGC-TOF/MS analysis, the extent of cell death, following treatment with each concentration, was assessed using the Trypan blue exclusion assay (Thermofisher Scientific, USA). Based on the Trypan blue results (Table ), we selected a concentration of 5 µM for this study, ensuring minimal cytotoxicity (cell viability 80%). This concentration was selected to avoid metabolite changes resulting from reduced cell viability. A375 melanoma cells were cultured in six-well plates at a concentration of 5 × 10 5 cells/mL, with 5 mL per well, resulting in a total of 2.5 × 10 6 cells per well. Once the cells reached a confluency of 70–80%, the cells were treated with GA113 at 5 µM for 24 h. For the control cells, a fresh medium without treatment was added. Following treatment, the cells were washed three times with ice-cold phosphate-buffered saline (PBS) (1X). To quench cellular metabolism, 1 mL of ultra-pure ice-cold methanol (Romil Pure Chemistry, UK) was added to each well. The cells were subsequently harvested by scraping the bottoms of the plates using a cell scraper and transferred to Eppendorf tubes. The harvested cell samples were snap frozen in liquid nitrogen, labelled, and stored at -80 °C until shipped on dry ice to the Centre for Human Metabolomics at North-West University, South Africa, for further analysis. The same procedure was meticulously used for all samples to minimise experimental variability. Three independent assays were performed. A master pooled quality control (QC) sample was created by combining small volumes from all collected cell samples. Several smaller aliquots were prepared from this master QC sample. These QC aliquots were run as the first, middle, and last samples in each GCxGC-TOF/MS batch. To evaluate the quality of the obtained metabolomics data, the repeatability of the instrument and extraction process was assessed by evaluating these QCs. For metabolite extraction, all experimental samples and quality control (QC) samples were dried under a gentle stream of nitrogen gas to remove the methanol. A single-phase extraction method was used, where 50 µL of internal standard 3-phenylbutyric acid (Sigma-Aldrich, USA) was added to each sample vial, along with 1 mL of the extraction solvent, which was composed of chloroform, methanol, and water in a 1:3:1 ratio (Burdick and Jackson, Honeywell International Inc., USA). Additionally, a 3 mm tungsten bead (Retsch GmbH & Co. KG, Germany) was included in each tube. The samples were placed in a vibration mill and homogenised at 30 Hz for 5 min, followed by centrifugation at 10,000 × g for 5 min at 4 °C. The supernatant was transferred to glass GC vials and dried under a gentle stream of nitrogen at 40 °C for 20–30 min. For derivatization, each dried sample received 50 µL of methoxyamine hydrochloride (Sigma Aldrich, Germany) and was incubated at 50 °C for 90 min. Subsequently, 40 µL of N, O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane (TMCS) (Sigma Aldrich, USA) was added, and the samples were incubated at 60 °C for 60 min. The derivatized samples were then transferred to 0.25 mL inserts in clean GC sample vials and analysed using GCxGC-TOF/MS. Analysis was carried out using a Pegasus 4D GCxGC-TOF/MS (Leco Corporation, USA), which consisted of an Agilent 7890 A GC (Agilent, Georgia, USA) coupled to a time-of-flight mass spectrometer (TOF/MS) (Leco Corporation, USA) equipped with a Gerstel Multi-Purpose Sampler (MPS) (Gerstel GmbH & Co. KG, Germany). The system was equipped with a cryogenic cooler. The first separation was carried out on an Rxi-5Sil-MS primary column (30 m x 0.25 mm x 0.25 µM) (Restek, South Africa) and the second separation was done on an Rxi-17 secondary column (1.380 m x 0.25 mm x 0.25 µM) (Restek, South Africa). Helium was used as the carrier gas with a constant flow rate of 1 mL/min. One microlitre of each sample, in a random injection order, was injected with a split ratio of 1:10. The samples were injected with a constant inlet temperature of 270 °C. The primary GC oven temperature was initially programmed at 70 °C for 2 min, then increased at a rate of 4 °C/min to a final temperature of 300 °C, where it was held for 2 min. The secondary oven was initially programmed at 85 °C for 2 min, then increased at 4.5 °C/min to a final temperature of 300 °C, where it was held for 4.5 min. The modulator was programmed with an initial temperature of 100 °C for 2 min after which it was increased by 4 °C/min to a final temperature of 310 °C, where it was held for 12 min. Cryomodulation and a hot pulse of nitrogen gas of 0.5 s every 3 s were used to control the effluent that emerged from the column into the secondary column. There was a solvent delay for the first 350 s of each run. The transfer line temperature was held at 280 °C and the ion source temperature at 200 °C for the duration of the run. The acquisition voltage of the detector was 150 V, and electron ionisation spectra were recorded at 70 eV. Data were acquired at 200 spectra/sec. The MS covered a m/z range of 50–800. ChromaTOF software version 4.50 (Leco Corporation, USA) was used for peak identification, mass spectral deconvolution, and peak alignment at a signal ratio of 100, with a minimum of three apexing peaks. Compounds were identified by comparing their mass fragmentation patterns and their retention times with commercially available National Institute of Standards and Technology (NIST) spectral libraries (named ‘mainlib’ and ‘replib’) after using a level 3 identification method published by Schymanski et al. . Peaks identified in the system as blank samples were deleted from the dataset when these peaks had an intensity of one-third or more of that detected for the same peak in the analytical samples, as these were contaminants. The same experimental procedure was performed on the extraction blanks– an initially empty sample vial containing no biological specimen. Thereafter, the compound areas were normalised according to the total useful MS signal (TUS) detected for each sample. The resulting data set was formatted and exported to MetaboAnalyst version 6.0 software ( https://www.metaboanalyst.ca/ ). A batch effect correction, using the ComBat method, was applied using the ‘other utilities’ feature in MetaboAnalyst. The batch-corrected data set was then normalised to the sample median, log-transformed, and auto-scaled. Principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA) were applied to obtain grouping information and significant metabolites, respectively. PLS-DA was used to calculate the VIP values for each variable. These variables were then subjected to univariate analysis using a difference-of-means test (Wilcoxon test). The PLS-DA model was cross-validated to provide estimates of the specificity and sensitivity of the model (Q 2 and R 2 , respectively). The metabolites with VIP > 1 and p < 0.05 were selected as final differential metabolites. Additionally, fold changes (FCs) were calculated to evaluate differences between metabolites in treated compared to untreated cells. The identities of the statistically significant metabolites were confirmed through the Human Metabolome Database (HMDB) ( https://hmdb.ca/ ). MThe statistically significant metabolites were used to determine the biological pathways that were altered/disrupted under the given treatment conditions. The built-in eukaryote Homo sapiens (KEGG) pathway library was used as a reference and the hypergeometric test for over-representation analysis and the relative-betweenness centrality for pathway topology analysis were employed as the pathway mapping algorithms. Quality control– batch effect analysisMetabolite profiling of A375 cells after GA113 treatmentVariable selection– significantly altered metabolitesAltered metabolic pathways Based on the 19 significantly altered annotated metabolites, metabolic pathway analysis was performed using MetaboAnalyst version 6.0 software ( https://www.metaboanalyst.ca/ ). Metabolic pathways with influence values greater than 0.1 and/or p-value < 0.05 were used as the main metabolic pathways to analyse the potential mechanism of action. Based on the above criteria, the following pathways were considered relevant: pantothenate and coenzyme A (CoA) biosynthesis, glutathione metabolism, citrate cycle, cysteine and methionine metabolism, arginine and proline metabolism, and alanine, aspartate, and glutamate metabolism (Fig. ). The severity of any batch effect was assessed and summarised using principal component analyses (PCA). The batch effect assessment was performed on the quality control (QC) samples. No prominent batch effect was visible; however, batch correction was performed but proved insignificant (Supplementary Fig. ). PCA was done to analyse the intrinsic variation in the GCxGC-TOF/MS dataset (Fig. A). The PCA model was derived from principal component 1 (PC1), which accounted for 17.7% of the total variance, and PC2, which accounted for 12.1% of the total variance. To optimise the observed differences and extract relevant biological information, a supervised partial least squares-discriminant analysis (PLS-DA) model was generated for the treated 5 µM concentration compared to the untreated control (Fig. B). The PLS-DA score plots were further validated through cross-validation, yielding robust predictive parameters. The PLS-DA model showed a prediction accuracy of 90% (Q 2 ) and a spectral variation (R 2 X) of 99% (Supplementary Fig. ). A total of 255 metabolites were detected, but only 130 were successfully annotated by spectral comparison with a commercial NIST library. Although the remaining 125 could not be annotated, they were still included in all statistical analyses and classified as ‘unknown’. Because of the classification procedure, some compounds with similar molecular structures, and consequently similar mass spectra, were assigned the same annotation based on the best library match. Retention times and unique mass fragments were examined to distinguish whether these were separate compounds. If confirmed as distinct, the duplicate annotation was retained with an added code and the concentrations were analysed as independent compounds. Treatment with GA113 at 5 µM induced significant changes in 33 metabolites, 19 of which were successfully annotated (Table ). Notably, several fatty acids, including hexadecanoic acid, octanoic acid, and ethyl oleate, exhibited significant reduction in the treated cells. Conversely, citric acid, proline, cysteine, and phosphoric acid levels were significantly elevated in the A375 cells after GA113 treatment. A quantitative comparison, in the form of box plots of the significant metabolites, is shown in Fig. . We have previously shown that our synthesised bimetallic ruthenium complex (referred to as GA113) exerts an evident cytotoxic effect on the malignant A375 cell line (Engelbrecht et al., ; Hussan et al., ). In this study, we extended the analysis using a GCxGC-TOF/MS-based metabolomics approach to better understand the cellular response of the A375 cells to GA113. The findings of this study provide evidence of the metabolic reprogramming induced by GA113 treatment in A375 melanoma cells. The identification of 33 signatory metabolites (associated with GA113 treatment) highlights the potential of this bimetallic complex as a therapeutic agent. The pathway analysis revealed significant alterations in critical metabolic pathways, particularly those related to glutathione metabolism, pantothenate and CoA biosynthesis. GA113 disrupts oxidative metabolism and energy productionGA113 disrupts amino acid biosynthesisGA113 disrupts lipid metabolism and fatty acid synthesisSignificant changes in glutathione metabolism and cysteine and methionine metabolism were observed, with notable upregulation of cysteine and phosphate and downregulation of pyroglutamic acid. Glutathione metabolism is closely interconnected with cysteine and methionine metabolism, as these pathways share intermediates and enzymes, and influence cellular redox homeostasis, detoxification, and sulphur metabolism. In this regard, cysteine and pyroglutamic acid play a critical role in cancer metabolism by fuelling cancer cell proliferation, survival, and adaptation to metabolic stress (Lieu et al., ; Min et al., ; Wei et al., ). Both pyroglutamic acid and cysteine are critical components of the γ-glutamyl cycle. Pyroglutamic acid reflects the activity of the cycle, while cysteine availability limits glutathione synthesis (Bachhawat & Yadav, ; Stipanuk et al., ; Wu et al., ). Cancer cells with high oxidative stress can channel pyroglutamic acid directly into glutathione synthesis to maintain redox homeostasis (Gamarra et al., ; Tretter et al., ). Thus, decreased concentrations of pyroglutamic acid could suggest an imbalance in the cellular redox state, inferring the presence of oxidative damage. Furthermore, increased cysteine concentrations can bolster the cell’s capacity to combat oxidative stress, by increasing the pool of available precursors for glutathione (Wu et al., ). Therefore, GA113 could induce apoptosis in the A375 cells through oxidative stress. These observed changes in glutathione metabolism are consistent with our previous findings (Hussan et al., ). Unfortunately, we did not detect any changes in glutathione, which would further support the observed changes in glutathione metabolism. Compared to the untreated cells, the concentrations of pantothenic acid and citric acid (citrate) increased, while 2-butenedioic acid (also known as fumarate) decreased in the GA113-treated cells. Citrate is a precursor to acetyl-CoA, which is involved in energy production and lipid biosynthesis (Icard et al., ; Zhao et al., ). Increased pantothenic acid can lead to increased levels of coenzyme A (CoA), which supports the conversion of citrate to acetyl-CoA for fatty acid synthesis (Czumaj et al., ; Leonardi & Jackowski, ). Pantothenic acid is also a precursor of CoA, which is involved in several metabolic pathways, including the citrate cycle, fatty acid synthesis, and acetylation reactions (Czumaj et al., ; Leonardi & Jackowski, ). Cancer cells often exhibit altered metabolism, including increased lipid biosynthesis for membrane production and energy storage. Pantothenic acid, through its role in CoA synthesis, supports fatty acid synthesis by providing acetyl-CoA. Thus, increased pantothenic acid could be a response to the increased energy and biosynthetic demands placed on the treated A375 cells. Thus, the upregulation of pantothenic acid can suggest that the cells are trying to cope with oxidative stress, DNA damage, and disruption of the cell cycle induced by the treatment. In addition, the observed increase in citrate levels could be a sign of enhanced mitochondrial activity to support energy production (Martínez-Reyes & Chandel, ; Zara et al., ). Taken together, the combination of decreased 2-butenedioic acid (fumarate) and increased citrate suggests a disrupted citrate cycle, potentially caused by impaired downstream flux, while increased levels of pantothenic acid suggest a shift toward oxidative metabolism and energy production in response to GA113 treatment. Metabolic pathway analysis showed significant changes in arginine and proline metabolism, as well as alanine, aspartate, and glutamate metabolism. These pathways are crucial in cellular metabolism and are tightly linked in the context of amino acid biosynthesis and catabolism (Chandel, ; Reitzer, ). Previous studies have highlighted the role of amino acid biosynthesis in cancer, particularly in supporting nucleotide biosynthesis and maintaining redox homeostasis, which is vital for sustaining cell proliferation and combating oxidative stress (Wei et al., ). The observed shifts in amino acid levels (proline, aspartic acid, aminomalonic acid, and ketoleucine) further reinforce the hypothesis that GA113 exerts its anticancer effects by targeting metabolic networks fundamental for melanoma cell survival. To meet energy demands, cancer cells can redirect metabolic dependencies through increased lipid and fatty acid synthesis, which supports rapid proliferation (Butler et al., ). In addition, when cancer cells are treated with an apoptotic inducer, fatty acid levels can decrease due to several factors related to the ability of cancer cells to synthesise or acquire fatty acids (Gnocchi et al., ). In this study, the concentrations of several fatty acids, including methyl palmitate (a methyl ester of palmitic acid), methyl stearate (a methyl ester of stearic acid), ethyl oleate, octanoic acid, 1-monooleoylglycerol, and octanamide (a derivative of octanoic acid), were significantly decreased in the treated cells compared to the untreated cells (Table ). The decrease in the abovementioned metabolites suggests a substantial disruption in lipid metabolism and fatty acid biosynthesis, both of which are crucial for maintaining the structural integrity of cell membranes and supporting bioenergetic needs (Duarte et al., ; Lumaquin-Yin et al., ; Pellerin et al., ). Methyl esters can act as markers of lipid metabolism and oxidative stress. Thus, the observed decrease in methyl palmitate and methyl stearate can reflect altered lipid turnover or oxidative degradation. The decreased levels of octanoic acid and octanamide can suggest disrupted fatty acid β-oxidation, as octanoic acid is a key intermediate in mitochondrial energy metabolism (Altinoz et al., ; Rial et al., ). Ethyl oleate and 1-monooleoylglycerol are derivatives of oleic acid, which is often involved in lipid signalling and energy storage (Koundouros & Poulogiannis, ). Thus, the observed changes in ethyl oleate and 1-monooleoylglycerol suggest that the GA113 complex can induce apoptosis by influencing the biological processes related to membrane synthesis and structural integrity. Although our study successfully highlighted potential drug targets, some key limitations should be noted. For instance, we only analysed the treated A375 cells at a single time point (24 h after treatment). Metabolism is inherently time-dependent, with metabolites fluctuating in response to cellular processes, environmental changes, or treatment effects. By focussing on a single time point, the study could have missed critical phases of metabolic activity. This limitation restricts the ability to distinguish transient changes from sustained metabolic shifts, making it challenging to fully understand the mechanisms underlying observed effects or to identify causal relationships between treatment and metabolic outcomes. Nonetheless, the findings of this study highlight several crucial areas for future research. For example, to further validate our findings, studies on the metabolic effect of GA113 in non-cancerous cells (e.g., HEK293) are required to determine and compare the mechanism of action in both malignant and non-malignant cell lines. Such studies will contribute to the identification of specific targets of GA113 in both cell lines. To further establish the therapeutic potential of GA113, in vivo studies are essential to validate the metabolic effects observed in A375 melanoma cells and to assess the pharmacokinetics, pharmacodynamics, and toxicity profile in an animal model. In this study, we used GCxGC-TOF/MS to analyse metabolite alterations in A375 cells treated with GA113 using univariate and multivariate statistical methods. Our findings suggest that exposure to GA113 can potentially lead to oxidative stress at the cellular level and disrupt amino acid, lipid and energy metabolism. These findings confirm the value of metabolomics in elucidating the potential mode of action of anticancer agents. Given the dynamic nature of cancer metabolism, leveraging metabolomics approaches can facilitate the identification of biomarkers that could guide therapeutic decision making and patient stratification in clinical settings. Below is the link to the electronic supplementary material. Supplementary Material 1 |
Understanding how young African adults interact with peer-generated sexual health information on Facebook and uncovering strategies for successful organic engagement | dd382c90-3787-44d0-97c2-56fb102aab52 | 8611981 | Health Communication[mh] | Sexual behaviour among young African adults presents significant public health concerns, in part because young people continue to have high rates of sexually transmittable infections, including Human Immunodeficiency Virus (HIV) , an unmet need for family planning, and low efficacy for condom use . On the other hand, sexuality also forms an integral aspect of health and wellbeing, especially in the context of comprehensive sexuality education and unrestricted access to health services . As a result, there is an increasing need to provide young people with comprehensive sexual health education to enhance intimacy and the realization of relationship goals and empower them to make informed decisions about their sexual and reproductive health . Today, several African countries have scaled up the dissemination of sexual health information using mass media, bulk messaging platforms, and in-school training, among many others . There is increasing advocacy for experimenting with alternative and new methods to sexual health promotion, particularly those that provide safe spaces to engage and provide young people with comprehensive sexuality education . Accordingly, social media internet sites like Facebook, Twitter and YouTube are quickly replacing traditional forms of communication because they offer individual users rapid transference of ideas and opinions through a relatively low-cost and user-friendly network . More importantly, social media platforms offer new channels for health communication that, when matched to the needs and preferences of the target audience, can increase the chances of programming success . This could be because, unlike other methods of disseminating sexual health information, social media sites offer a multidirectional communication model in which audience members participate actively in discussions and share their knowledge and experiences, rather than being passive recipients of sexual health information . These multidirectional engagements ultimately offer a promising opportunity to understand young people’s perspectives and identify dominant stereotypes and misinformation while correcting misinformation and addressing concerns that emerge from such interactions . Furthermore, the extensive reach of social networking sites like Facebook and their interaction functions offer huge potentials in delivering health promotion messages . In fact, a recent scoping review demonstrated that sexual health education delivered via social media effectively increases health knowledge, awareness and ultimately motivates behavioural change . Young adults alike are receptive to this communication channel as multiple studies have shown that adolescents and young adults prefer to access sexual and reproductive health information from social media, particularly Facebook . As young people use these forms of new media, including social media, there may be new opportunities to listen to and engage with young African adults about sexual health issues . Nevertheless, health information is only effective if it reaches and engages with its target audience. Interactive health education strategies that leverage the full potential of social media by encouraging participation and engagement rather than providing a one-way flow of information are reported to have greater potential to enhance behaviour change . This is important because engagement metrics such as reactions (favourites), comments (replies) and shares (retweets) provide rudimentary markers of diffusion and are used by social media platforms, including Facebook’s algorithm to determine which social media content is shown to users and those in their network . As a result, increasing user engagement has become a primary objective of social media interventions. However, while many young adults interact with their friends and families on social media, many sexual health interventions have highlighted concerns about attrition and low engagement with sexual health information , while others have attempted to increase user engagement through the use of paid advertisements . Since paid advertisements are not cost-effective and not sustainable, there is an increasing need to understand the predictors of user organic (unpaid) engagement, especially for potentially sensitive topics like sexual health information. Despite this reality, it remains unclear whether and how young African adults use digital innovations like social media to access sexual health information. As a result, understanding the most successful tactics for reaching, engaging, and keeping young people in social media-based sexual health promotion, as well as the type of material that encourages user interaction or engagement, has become a higher priority. An awareness of these will definitely provide valuable insights for supporting successful online sexual health promotion campaigns and guide the design and development of context-specific social media content and the use of features that have a high appeal for young African adults . Predictors of engagement with health information on social media Current research The present study aimed to (1) characterize peer-generated sexual and reproductive health information on a peer-led Facebook group that facilitates discussions about sexuality and reproductive health; (2) identify post features and content associated with greater user engagement. In achieving these objects, this study extends scholarship primarily in two ways. First, analyzing the different engagement metrics enable us to delineate the predictors of different types of user engagement. In addition, leveraging a platform where young people consume, produce and simultaneously interact with sexual health information provides a unique opportunity to understand the features of sexual health information that resonates and probably be compatible with young adults’ social media engagement habits. Secondly, the use of human annotators enables us to identify additional features that predict user engagement while also considering the content and context of the messages. Furthermore, our analysis comes especially when there is a pressing need to digitize sexual health information and augment existing sexual health education strategies with digital innovations to minimize in-person contact. Therefore, the findings of this study will be of immense importance for the development of useful and engaging sexual health information to increase information dissemination and, ultimately, behavioural change.
An important consideration for effective sexual health education on social media is increasing user engagement, especially because more interactive social media content can increase exposure and ultimately better support behaviour change . Recent works looking at different social media users have found that most users tend to be passive, using social media to seek information or engage casually, keeping up with the online activities of others without sharing content or leaving an opinion . As a result, a vast number of studies have examined multiple pathways to increase organic (unpaid) user engagement. At the individual level, Olamijuwon & Odimegwu , using data from young African adults recruited via Facebook, highlight that young people were likely to access and interact with sexual health information on social media if such use improves their awareness of sexual and reproductive health and rights, is free of effort, and aligns with the way they interact with other information on social media. In their study, Andrade et al. (2018) also recommend that preventive messaging or other health promotion content should be strategically incorporated into habitual messages to keep participants connected with friends and ensure that the messages being conveyed resonate with their needs. Another study of top user profiles with high user engagement by Veale et al. found that regular post updates directly engaging with users through individual responses and acknowledgements and encouraging interaction and conversation by posing questions were keys to successful engagement. Direct invitation for user engagement, such as asking users to comment or share a post, have also been associated with increased user engagement for comments and shares but not liking a post in another study . On the other hand, asking a question was negatively associated with engagement . Furthermore, the use of multimedia content is significantly associated with higher levels of user engagement . This finding resonates with a prior study and some recent studies , all of which confirmed that multimedia content was positively increased engagement. Rus and Cameron , in their study of 10 diabetes-related Facebook pages, found that the use of imagery was the strongest predictor of user engagement, including liking and sharing a post. Goedel et al. also found that posts containing photos, links, and videos received higher cumulative engagement scores than posts containing status updates only. After disaggregating by specific user engagement metrics, evidence from their study suggested that posts with links and photos received fewer comments status updates while posts with videos received higher engagement on all metrics . Although combined with an advertisement, Pedrana et al. also found that the webisode format of video uploads and the combination of education with entertainment was an effective driver of success in delivering health information to gay men. Adolescents in Tanzania also reported that peer-to-peer sharing of sexual health information would be higher if they were humourous . Their findings altogether were consistent with the richness theory , suggesting that richer media content engages audiences better. Beyond activity-based efforts to increase user engagement and content types, the content of messages and the expressions conveyed have also been shown to be associated with user engagement. Posts with positive affect elicit higher engagement, perhaps reflecting the well-documented heuristic bias towards positive messaging . In a similar study of 20 Facebook health profiles, Kite et al. found that positive sentiments were significantly associated with high user engagement. Like the form of posts, the content of posts has also been documented to be associated with user engagement. A study of gay, bisexual and other men who have sex with men found that posts about pre-exposure prophylaxis and stigma exhibited greater engagement, while posts about dating exhibited lower levels of engagement . This finding was also substantiated by a more recent study of HIV-related messages on Twitter, highlighting that fear-related language was a strong predictor of retweet counts on Twitter . While many of the studies above offer an interesting yet diverse perspective on increasing user engagement, they are mostly limited in their approach and analysis. First is that most of the studies cited have used informatics-based methodology , which may not be very effective, as these approaches are subject to measurement error or obscure some data richness . In addition, the use of automated analysis may only provide a partial understanding of the meanings embedded in a corpus of data and does not provide a profound insight into the context and processes that generated such discourse . Other studies, on the contrary, have utilized paid advertisements to increase user engagement or focused on specific audiences such as the lesbian, gay, bisexual, and transgender or gay and bisexual men . In addition, many current studies have examined user engagement by creating an aggregate index that combines likes, shares and comments . This approach implies that interaction and engagement metrics are weighted equally. However, the relative merits of each engagement could vary according to educational objectives. For example, exposure to some messages could reap huge benefits, while others may need to promote interaction between educators and users . As a result, it is increasingly becoming important to evaluate different metrics separately to enable better targeting of different strategies based on education objectives (to increase reach, or interaction or both). In addition, by summing up all metrics, researchers may be obscuring important relationships; notably, since interaction metrics may indicate different levels of engagement. For example, liking a post requires little cognitive effort than leaving a comment or sharing a post with other people in a user’s network. As a result, the predictors of superficial forms of engagement, such as liking a post, might be different from those requiring more cognitive efforts, such as leaving a comment , as observed by Goedel et al. . In addition, most of the existing scholarship on the message properties that predicts organic user engagement are concentrated in developed countries, while scanty evidence abounds in developing countries, including Africa. The increasing adoption of social media platforms for sexual health education in African countries necessitates an urgent need to explore the predictors of engagement both from a global perspective and from the perspective of young adults since interventions addressing sensitive subjects and those targeting young people might be uniquely constrained by contexts and user willingness. Furthermore, the extant scholarship has focused mostly on user engagement from the professional perspective, while very few studies have assessed user engagement from young people’s perspectives . However, peer education may be more successful than adult-centred or didactic education in dealing with the sexual health challenges faced by young people. Given the huge gap in scholarship, there’s an urgent need for further analysis to document the type of participation that will best promote health awareness. This study builds on past research and leverages the latest technology on social media to understand the unique determinants of various types of engagement with peer-generated sexual health information.
Data source Text sample Metadata Coding Analysis Data for the study was obtained from a youth-led Facebook group that facilitates discussion about sexuality among young African adults. We focused on data from Facebook because Facebook is the most popular and frequently used social media platform by many people of various ages worldwide, including Africa . The group was created in 2016 by an individual and had over 25 content moderators, mostly young adults from different African countries. The group is also public, implying that anyone could find the group or join the group, see who is in the group and what is posted on the group. Facebook users who find the group can self-select to join the group or be invited by anyone already in the group. The group has 176,461 members, mostly from African countries and contains several messages on intimate relationships, dating and marriage. A summary description of the group demographics is presented in Table . About 91% of the group members live in Nigeria, with a lower percentage of participants from other African countries, including Ghana (0.6%), South Africa (0.4%), Kenya (0.3%), and the Republic of Benin (0.3%). About 51% of the group members are women, while adolescents and young adults account for about 71% of the group membership. The demographic diversity of participants in the group and the diversity of the messages posted on the group make it a valuable resource for studying how young African adults interact with peer-generated sexuality information.
A data scrapping application (Sociograph.io) was used to download public wall posts on the Facebook group. The application was installed directly on the Facebook group with administrative rights so that it could access all messages and interactions. Permission to access public wall posts on the group was obtained from the group creator after receiving a detailed description of the study and its potential for sexual and reproductive health intervention. As with other studies using social media data from existing groups, obtaining informed consent from all the group members was practically impossible . However, we made conscious efforts to mitigate risks against research subjects and the group. First, all the data extracted contained no personal identifying characteristics, such as the name of the content creator. However, since some content creators on the group included their information (such as mobile numbers, links to other groups, names and email addresses) on public wall posts shared on the group, we carefully examined each post and removed these during data coding. This strategy aligns with recommendations in the literature . We also rephrased quotes included in our study to minimize the identification of posts through a keyword search on the social media platform. A total of 62,986 member-generated posts and 897,967 comments made since 2017 were retrieved from the group. These wall posts include messages related or not related to sexuality since the members of the group could post any messages on the group regardless of whether they focus specifically on sexuality. Our data analysis and management followed a two-stage process. In the first stage, we developed a keyword-based dictionary comprising words related to several aspects of sexual and reproductive health such as “condom,” “contracept,” “rape,” “virgin,” “sex,” and “s*x,” among several others. A full list of the keywords is presented in Table . We included these words in the dictionary based on their popularity and potential to fully capture all relevant sexuality messages. The keyword-based filtering looped through each corpus of messages and retained only posts that included at least one of the keywords. We also did not find any relevant message that had already not been captured by any of the keywords during data validation. With the keyword-based filtering, we identified a total of 8497 posts that had at least one or more keywords. The 8497 posts identified were reviewed by seven research assistants who reviewed an average of 2550 Facebook posts each over two weeks. This implied that two research assistants reviewed each post. The first author reviewed the remainder of the posts. Each assistant was asked to classify the posts based on whether they related to a set of predefined sub-domains of sexual and reproductive health, including sexual abstinence and contraceptive use, among others. The assistants were trained extensively before the activity. About 54% of the posts were deleted if two assistants classified the posts as not being related to sexual and reproductive health. The final dataset during this phase comprised 3947 posts shared on the group between June 1, 2018, and May 31, 2019.
Metadata represents supplementary information—data about the data—that is embedded in each Facebook post on the group and included in the corpus of data collected. These metadata include a postID (a unique numerical identifier assigned to each Facebook post), the post date, post type (status/link/photo/video) and measures of interactions and propagation such as reactions (like/angry/haha/love), comments and shares. In addition to coded categories, these data were used to describe the interactions with sexual and reproductive health information on the group.
To develop an initial coding system, we used past definitions of sexuality education to identify four unique sexual and reproductive health topics: abuse, birth control and abortion, intimate relations, and sexual abstinence . After an extensive review of the literature , we further classified a group of messages about abstaining from all non-penetrative sexual activities (or (not) having sexual feelings such as self-pleasure touching, and kissing) as sexual purity while messages related to penetrative sexual activity (primarily before marriage) as sexual abstinence. This description is also consistent with what is known in literature and what young adults define as sex . The final codebook included coding categories, all with definitions, and examples are presented in Table . We further engaged six of the initial research assistants to review the 3947 Facebook messages classified to be related to several aspects of sexual and reproductive health. All six assistants were young adults aged 20–25 years and were university students. The assistants were re-trained and asked to classify the Facebook posts based on topic, strategy, and tone of communication. An average of 1315 messages was reviewed and classified by each of the six assistants during this phase. Krippendorff’s alpha for topic classification (α = 0.86), strategy (α = 0.91) and tone (α = 0.73) of communication was above the acceptable minimum levels of reliability . The first author also reviewed a random sample of 500 messages in the dataset to ensure consistency and accuracy in data coding.
Content analysis was used to examine the messages shared on the Facebook group to assess how young adults interacted with the different sub-domains/topics of sexual and reproductive health, type and tone of message communications. Quantitative content analysis involves the systemic analysis of texts or symbols of communication that have assigned numeric values based on a predefined coding scheme . It also involves analyzing relationships among the assigned numeric values using statistical methods to describe the communication and draw valid inferences from the texts to its context . In our investigation, we adopted several measures to answer our main research question. We also examined interactions and propagation of messages on the group using the three counts of engagement comprising of “reactions” (like, love, sad, and angry), “comments,” and “shares,” all of which are considered as aspects of “electronic word-of-mouth” and could stimulate different communication behaviours . We also assessed the bivariate relationships between the different topics, messaging strategy and the tone of communication. Finally, we fitted four negative binomial regression models using the glm.nb function from the MASS package to delineate statistically significant differences in young adults interacting with each of the messages by topic, type, strategy, and tone of communication. This model was chosen because the data were highly skewed and overdispersed . We ran four models, one for each measure of message interaction and propagation: Model 1 considered associations between the sum of all interactions and each of the message metadata. Model 2 considered the associations between the number of reactions and each of the message categories. Model 3 considered the association between the number of comments and each of the message categories. Finally, Model 4 considered the association between the number of comments and each of the message categories. Interpretation of the results was made using incidence rate ratios (IRR) and 95% confidence intervals. An incidence rate ratio greater than one (IRR > 1) implied a higher likelihood, while incidence rate ratios less than one (IRR < 1) implied a lower likelihood, and incidence rate ratios equal to one (IRR = 1) implied no difference in risks. All data analyses were conducted in R . De-identified data supporting this study’s findings and reproducible R codes for tables, all graphs and negative binomial model outputs are available online. Ethical approval for the study was obtained from the non-medical human research ethics committee at the University of the Witwatersrand.
Characterizing peer-communicated sexuality information by topic, strategy, type and tone of communication Interaction and propagation of peer-communicated sexuality information The level of interaction for all sexual health information shared on the group is presented in Fig. . The median level of interaction was highest for reactions (x̃ = 54) and comments (x̃ = 10) but lower for post shares (x̃ = 10). Table presents the results of the multiple negative binomial regression model testing associations between the measures of message interaction and propagation. The results showed that interactions with messages on abuse were significantly different from interactions with messages on intimate relations in terms of the number of reactions and shares. Members of the group were significantly more likely to react to [IRR: 1.14, 95% CI: 1.04–1.24], and comment on [IRR: 1.26, 95% CI: 1.12–1.43], sexual abstinence-based messages compared to messages on intimate relations. Compared to messages on intimate relations, those in the group were significantly less likely to share messages on abuse [IRR:0.34, 95% CI: 0.26–0.46], sexual abstinence [IRR: 0.56, 95% CI: 0.49–0.63], and sexual purity [IRR: 0.57, 95% CI: 0.48–0.69]. Messages that evoked fear [IRR:0.75, 95% CI: 0.66–0.86] or guilt [IRR:0.82, 95% CI: 0.72–0.92] received significantly fewer reactions compared to neutral messages. Messages that evoked fear [IRR: 0.81, 95% CI: 0.67–0.99] also received a significantly fewer number of comments, while stigma based messages [IRR: 1.55, 95% CI: 1.13–2.06] received a significantly higher number of comments compared to neutral messages. No statistically significant differences were observed in the propagation (number of shares) of messages by the tone of communication. Sharing an experience or telling a story were associated with a higher number of reactions and comments compared to counselling. Messages requesting an opinion also had a significantly higher number of comments [IRR: 4.25, 95% CI: 3.57–5.10] but a lower number of reactions [IRR: 0.48, 95% CI: 0.43–0.55] and shares [IRR: 0.13, 95% CI: 0.11–0.16] compared to counselling messages. Including a photo in messages was associated with a higher number of reactions [IRR: 1.76, 95% CI: 1.61–1.92], comments [IRR: 1.16, 95% CI: 1.02–1.32] and shares [IRR: 1.91, 95% CI: 1.68–2.16] compared to text-only messages. Including a link, on the other hand, was associated with a lower number of reactions [IRR: 0.17, 95% CI: 0.11–0.27], comments [IRR: 0.16, 95% CI: 0.09–0.31], and shares [IRR: 0.34, 95% CI: 0.19–0.67] compared to text-only messages.
Table summarises messages shared on the group by topic classification, strategy, and tone. Most of the messages on the group were about relationships and dating, while about 40% of the messages were on sexual abstinence or purity. Regarding messaging strategy, close to one-quarter of the messages were regular status updates, while about 10% of the messages requested opinions, and about 14% told a story. The tone of about one-quarter of the messages evoked fear, stigma, or guilt. Lastly, most messages on the group are text-only posts (does not include any multimedia or link), while about 22% of messages on the group included at least a photo or video. We examined significant differences in the tone and strategy for which different topic classifications were communicated in Fig. . The results showed that about 46% of messages on abuse were telling a story compared to less than one-quarter among birth control and abortion-related messages (21%), intimate relations (14%), sexual abstinence (11%), and sexual purity (12). Requests for opinions were more common among family planning messages (13%) and sexual abstinence (12%). Furthermore, topic classification across tone showed that more than half of family planning messages evoked fear (26%), stigma (5%), or guilt (21%). Similarly, 41 and 33% of sexual abstinence and purity messages evoked fear, stigma, or guilt.
his study is one of the first to document the actual use of Facebook as a resource for peer communication of sexual health information and intimate relations among young African adults. Some notable findings from our data highlighted three critical areas of consideration in designing effective and engaging sexual and reproductive health information for young African adults. First, assessing the levels of engagement based on the different metrics suggested that young people were more likely to engage superficially with peer-generated sexual health messages using reactions than leaving comments or sharing messages with those in their network. In addition, our analysis showed that compared to messages on intimate relations, young adults were significantly less likely to share sexual abstinence and purity-based messages with their network. Although our study did not explore in detail the content of the messages shared in this network, concerns about anonymity and privacy may have affected the propagation of these messages. This finding resonates with prior studies suggesting that young adults may be less likely to interact with sensitive topics such as sexual and reproductive health information to circumvent monitoring by parents and friends . Secondly, our analysis also revealed the use of visual content and multimedia in communicating sexual health information. Consistent with a recent study on HIV information dissemination on Twitter, we found that young people were more likely to interact with messages with visual or multimedia content but less likely to interact with messages with links . This finding also resonates with prior studies and the richness theory , all of which affirmed the positive association between the use of rich message features like multimedia and increased user engagement. Young adults likely find it easier to comprehend messages with multimedia content and may ignore messages including a link due to the financial effort and time required to visit the link and understand the messages . Interestingly, our data revealed that young adults were more likely to interact with messages telling a story or sharing an experience. When asked for their opinions, young adults were also more likely to comment. This approach may be useful in getting young adults’ perspectives about issues of importance. Furthermore, the preponderance of messages on intimate relationships, sexual abstinence particularly until marriage, and purity-based messages on the group highlight how sexuality messaging have been reduced to diseases, danger and a response to the HIV/AIDS epidemic— a pattern that is consistent with the literature . Precisely, there is a dominance of a precautionary voice and a language of consequence, in which young people on the group are advised to abstain from sexual practices or ‘face consequences’ of HIV infection, sexually transmitted infections (STIs) and pregnancy. This finding is further corroborated by the significant amount of fear-based, stigma, and guilt appeal messages. Precisely, we observed that close to one-quarter of the sexuality information on the group evoked fear, stigma or guilt, with variations as high as more than half among family planning messages and more than one-quarter of sexual abstinence and purity messages. This pattern of fear, shame and blame tactic is particularly of great concern given that young people who should be key for delivering comprehensive and less threatening sexuality information are observed to be reinforcing existing dominant binary gender roles, norms and moralistic positions on young people’s sexuality. Evidence of the use of fear appeals and “scare tactics” in sexual and reproductive health messages have been observed in the African literature . In one study of parent-led sexual health information, it emerged that parents deliberately misinform their children about sexuality using fear-based appeals, stigma and guilt focusing themes that depict young people’s sexual behaviours in terms of deviance, immorality, and waywardness as approaches to regulating young people’s awareness and knowledge of sexual and reproductive health . Sexuality education in South Africa and indeed much of Africa also appears to be dominated by negative connotations of sexuality through the narratives of sickness, danger, doomed futures, violence, and repercussions . For example, horrific depictions of sexually transmitted diseases and premarital childbearing have reportedly been used to frighten students into abstinence . These negative connations of sexuality are primarily because of the dominant misconception that sexual health education will inspire sexual imaginations in young people . These approaches of sexual health communication are further rooted in religious teachings, and cultural attitudes that promote sexual abstinence-only until marriage but which altogether does not address the sexual health challenges of young people nor are effective in motivating behavioural change . Precisely, Bhana et al. emphasized that sexual health interventions that emphasize ‘risk’ over ‘desire’ and ‘shame’ over ‘pleasure’ risk speak to no one, especially young people whose bodies and experiences tell them otherwise. Till today, the role of fear, guilt, and stigma appeals in behavioural change has yielded mixed findings and is contested both by researchers and practitioners . Some studies have shown that the use of fear appeals in messages generates strong responses and may indeed increase awareness of the hazards of unhealthy practices, influencing attitudes, intentions, and ultimately behavioural changes . For example, fear appeals in HIV prevention messages during the early period of the national response to the HIV epidemic are believed to have contributed to an approximate 66% decline in HIV prevalence in Uganda . The use of fear appeals in sexual health education has also been observed to have no adverse consequences or undesirable outcomes on the population they intend to target . However, other studies contest that even when fear appeals arouse the interest of those who are exposed to them, they are often not associated with health behaviour changes . Others argue that by inundating young adults with fear, they fail to seek out information on reducing their risk or finding out whether they are infected . Surprisingly, young adults on our peer-led Facebook group were less likely to interact with messages with fear and guilt appeals. However, this finding contrasts with prior literature on the dissemination of sexual health information on social media . Most of these studies were focused on HIV prevention messages on Twitter, and they had a different audience compared to the present study. Nonetheless, the low level of interaction with non-neutral messages is likely a sign of disapproval of these messages and the hesitance to propagate the same while also leaving comments to highlight these concerns. Finally, our study contributes to the literature in important ways, but it is not without limitations. The first is that we attempted to utilize data that was cross-cutting throughout Africa. However, about 90% of the members of the Facebook group are from Nigeria. As a result, the discussions on the group may have been dominated by voices from Nigeria, where religion and culture are known to play pivotal roles in sexual health communication. Nonetheless, the role of religion and culture in sexual health promotion has also been observed in other African countries like South Africa, Uganda, and several others. Furthermore, we retrieved wall posts and comments from only one Facebook group. As a result, the generalizability of our findings is limited. Nevertheless, we found similar associations as did previous studies and identified new predictors of user engagement. While we focus on data from Facebook, we believe that many of the findings, especially those involving the use of rich messages features, will extend to other platforms, including multimedia social media sites like Instagram. While our data set comprises a significant number of meaningful discourses about sexuality education for and by young African adults, these messages on the group did not represent all such discourse. Most significantly, we observed an abundance of sexual abstinence and purity-based messages. As a result, the findings should be interpreted with caution. It is also worthy of note that the level of interactions and propagation of messages on the group may be influenced by author popularity. For example, young adults may interact with messages from authors who are well known in the group. However, our data do not include author profile details, so we could not examine this association. Despite the limitations, our study provides insight that could be useful when considering social media for sexual health communication among young African adults.
We examined the use of Facebook for peer-communication of sexuality information as well as how young African adults interact with these. We found that young adults were more likely to superficially interact with peer-communicated sexual health information through likes than engage (comments) or propagate such messages. The use of storytelling and the inclusion of multimedia such as photos or videos was associated with higher levels of interaction with peer-communicated sexuality information. On the other hand, the use of fear appeals in sexuality information was associated with lower levels of interaction through comments and likes. These findings provide valuable insight and pave the way for the design of useful, engaging, and context-specific sexual health information that uses features that have a high appeal for young African adults and advance the sexual and reproductive health of young African adults. The data derived from our analysis can be used to complement policy design in developing tailored participatory sexual health promotion for young African adults. This opportunity may help empower the sexual and reproductive health rights of young African adults and reduce some of the misinformation that impedes progress. Since our analysis did not explore what young adults are doing with the information, particularly those that evoked fear, guilt, or stigma, future studies could examine this and how young adults respond to these messages. Most importantly, future studies are needed to explicate whether the level of engagement with fear-based sexual health information on social media are correlated with the perceived efficacy and if such messages motivate significant behavioural changes as demonstrated in the theories of fear-based .
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Enrichment of novel entomopathogenic | cbe13e7f-88a1-4f9f-a486-369a3ff0c8f6 | 11382411 | Microbiology[mh] | Plants face various environmental and biotic challenges during their lifetime. Among these challenges, insect herbivores represent a pervasive threat and are responsible for about 20% of the loss in total crop production worldwide annually . Plants have evolved versatile defense strategies that help them withstand insect attacks, including the establishment of physical barriers, production of toxic metabolites, and activation of phytohormone responses . Plants and their associated microbiomes have co-evolved for more than 400 million years and have formed a “holobiont” consisting of the plant host and its symbionts . Plant-associated microbiota are also referred to as the plant’s second genome and play fundamental roles in plant growth and stress tolerance . Accumulating evidence indicates that plants can recruit certain beneficial microbes to suppress the growth of soil-borne pathogens . For example, the roots of pathogen-infected wheat , sugar beet , and Arabidopsis plants can attract groups of beneficial microbes that protect the next generation of plants. Pseudomonas spp. are bacteria that play an important role in promoting plant growth and pathogen resistance by producing various antimicrobial metabolites . A greater richness of related Pseudomonas spp. has been demonstrated to be correlated with pathogen inhibition in plants and is required for plant health . Some strains of Pseudomonas fluorescens group, such as P. protegens and P. chlororaphis , also cause systemic infections and the eventual death of several Lepidopteran, Dipteran, Coleopteran, and Hemipteran insects . However, whether plant-associated bacteria contribute to plant defenses against insects in natural ecosystems is unknown. Plagiodera versicolora is one of the most damaging pest species to Salicaceae plants such as willow ( Salix ) and poplar ( Populus ). These insects are widely distributed across northern Africa, America, Europe, and Asia. Both P. versicolora larvae and adults feed on the leaves of willow and poplar, especially during the summer. During the winter season, adult P. versicolora individuals burrow into the soil to undergo overwintering, posing a continued threat to plants in the following year . The life cycle of P. versicolora consists of several stages, including an egg stage lasting 5–6 days, a larval stage lasting 14 days, a pupal stage lasting 4–5 days, and an adult stage that spans 3–4 weeks . Since Salicaceae plants are the natural hosts of P. versicolora , the Salix-P. versicolora association has been utilized as a model system to investigate the evolution adaptations of herbivorous insects in terms of their resource-utilization traits . In this study, using the weeping willow ( Salix babylonica ) -P. versicolora association as a model system, we revealed that insect-damaged willows can enrich their microbiota in specific microbes that enhance their resistance against further damage by the insect pests. We demonstrate that insect-damaged willows are more resistant to P. versicolora than healthy willows in the field. We further identified certain novel Pseudomonas strains with insecticidal activities that are enriched in the microbiota of willows that were damaged by P. versicolora . Our results suggest a mechanism by which plants exploit specific entomopathogenic bacteria for defense against insect pests in nature.
Sampling Processing of samples Preparation of axenic willow leaves DNA extraction Analysis of microbiota Genome sequencing and assembly Phylogenetic analysis Quantification of Insect rearing Insect bioassays on detached leaves Synthetic community of leaf Introduction of Insect bioassays on whole plants Statistical analysis of data Samples were collected from two willow ( Salix babylonica ) field trials. Field site 1 (S1) is located at the Shahu Lake (30° 34′ 17″ N, 114° 20′ 4″ E), and field site 2 (S2) is located at the Nanhu lake (30° 28′ 47″ N, 114° 21′ 33″ E) in Wuhan, Hubei province, China (SI Appendix, Fig. A and 1B). The two sampling sites are located in a temperate climate zone with the same annual mean temperature of 22 °C and annual mean precipitation of 1343.5 mm. The leaves, roots, and rhizosphere soil of willows were sampled on August 11–12, 2021, as previously described . The rhizosphere soil was defined as the soil particles adhering to the roots . At each site, willows without beetle damage were classified as healthy (SI Appendix, Fig. C). Willows damaged by beetles were classified as sick (SI Appendix, Fig. D). The healthy leaves of healthy willows will be abbreviated as HL- HW , the healthy leaves of sick willows as HL- SW , and the sick leaves of sick willows as SL. Ten replicates from healthy and sick willows were collected from ten adjacent plots at each site. The distance between healthy and sick willows was about 0.5 km (SI Appendix, Fig. E). The samples were transported with dry ice and stored at − 80 °C until analysis.
Willow leaves (three leaves per sample) were surface-sterilized with 75% (v/v) ethanol for 1 min and washed with sterile Millipore water three times . Root samples were rinsed with sterile water to wash off the attached soil, surface-sterilized with 2.5% NaClO (w/v) for 5 min and subsequently with 70% (v/v) ethanol for 1 min, and washed with sterile Millipore water three times . The treated plant samples (leaves and roots) were then chopped into small pieces using a sterile scalpel and homogenized with a sterile pestle under axenic conditions. Rhizosphere soil and homogenized plant samples were stored at − 80 °C prior to DNA extraction.
Preparation of axenic willow leaves was conducted as described previously . Briefly, sick leaves (SL) and healthy leaves (HL) from sample sites were washed under running tap water for 3 min to remove all the dirt particles and impurities, and subsequently soaked in 75% alcohol for 1 min. After rinsed with sterile water three times, the leaves were immersed in 0.1% HgCl 2 for 5 min, and rinsed with sterile water three times. Removal of bacteria was confirmed by a colony-forming unit assay and PCR analysis using conserved primers for the 16S rRNA gene of bacteria (Fig. S2).
Approximately 250 mg of rhizosphere soil was used for DNA extraction using a PowerSoil DNA Isolation Kit following the manufacturer’s instructions (MoBio, Carlsbad, CA, USA). DNA extraction from willow leaves and roots (100 mg per sample) was performed using a MoBioPowerPlant Pro DNA Isolation Kit (Mo Bio Laboratories) according to the manufacturer’s protocol.
The bacterial communities from all samples were determined based on their 16S rRNA amplicon profiles on an Illumina NovaSeq platform. Sequencing libraries from bacterial DNA were generated with a NEBNext® Ultra™ IIDNA Library Prep Kit (Cat No. E7645). Raw sequences were split according to their unique barcodes and trimmed off the adaptors and primer sequences using QIIME . Paired-end reads were merged using FLASH (Version 1.2.11, http://ccb.jhu.edu/software/FLASH/ ). Quality filtering of the raw tags was performed using fastp software (version 0.20.0) to obtain high-quality clean tags, which were compared to the reference database (Silva database https://www.arbsilva.de/ ) to detect and remove chimeric sequences, yielding final effective tags . Denoising was performed with DADA2 in QIIME2 software (Version QIIME2-202,006) to obtain initial amplicon sequence variants (ASVs). The sequences annotated as chloroplast were removed. ASVs with an abundance of < 5 reads were removed . Microbial diversity and community composition were analyzed using vegan packages in R (version 3.5.3). Non-metric multidimensional scaling (NMDS) based on Bray–Curtis dissimilarities was used to identify differences between microbial communities. Compositional differences in NMDS between healthy and diseased samples were assessed using ANOSIM based on the Bray–Curtis distance (Table S3–8). Linear discriminant analysis (LDA) of effect size (LEfSe) was applied to the ASV table to identify differentially abundant bacterial taxa from the phyllosphere, root, and rhizosphere soil samples between beetle-damaged and healthy willows. Absolute LDA scores (> 2) were used to analyze statistical significance.
Samples were sequenced using multiplexed libraries on a Novaseq 6000 instrument to produce paired-end reads with lengths of 150 bp. For each sample, read quality was assessed with the FastQC tool ( http://www.bioinformatics.babraham.ac.uk/projects /fastqc/ ) and low-quality reads were removed by Quake . All genome assemblies were performed with ABySS using a k-mer size of 21. The best assembly for each strain, with the largest scaffold N50, was annotated by Prokka (v1.14.6) . The final assembly genome information is shown in Table S9.
Phylogenetic trees were reconstructed using the maximum likelihood method in MEGA version 7.0 . Bootstrap support values were calculated from 1000 replicates. The phylogenetic tree was visualized with iTOL . The 14 reference strains were as follows: P. quercus (MT036111), P. coleopterorum (KM888184), P. koreensis (AF468452), P. neuropathica (LR797591), P. iridis (LR797554), P. piscium (LR797558), P. parafulva (FNYJ01000011), P. chlororaphis (BBQB01000031), P. alloputida (LT718459), P. flavescens (FNDG01000047), P. alcaligenes (BATI01000076), P. resinovorans (Z76668), P. aeruginosa (BAMA01000316), and P. psychrotolerans (FMWB01000061). Acetobacter ascendens LMG 1590 (CP015164) was selected as an outgroup. The accession numbers of the 16S rRNA sequences are provided within the parentheses.
Pseudomonas spp. colony-forming units, strain isolation, and identification The density of Pseudomonas spp. from the phyllosphere, root, and rhizosphere soil samples was tested by cultivation-based methods. Pseudomonas colony-forming units (CFUs) were determined via serial dilutions. Three aliquots (100 µL) per dilution were spread onto CFC (cephaloridine fucidin cetrimide) medium designed for Pseudomonas selection . The number of colonies was recorded after incubation at 28 °C for 3 days . Pseudomonas isolates were purified, and genomic DNA from each Pseudomonas culture was extracted using a MiniBEST Bacteria Genomic DNA Extraction kit (TaKaRa, China) following the manufacturer’s protocol. The 16S rRNA (1,466 bp) gene was amplified with primer pair 27F and 1492R (SI Appendix, Table ) and sequenced. The 16S rRNA gene sequence of Pseudomonas was searched against the EzBioCloud Database , a well-curated database of 16S rRNA sequences and bacterial genomes.
P. versicolora adults were collected from willow trees at Sha Lake Park in Wuhan, Hubei Province in China (30.35° N, 114.33 E) . P. versicolora was regularly reared by feeding with detached fresh willow leaves at 28 °C and 60 ± 5% relative humidity under a 16-h light/8-h dark photoperiod in transparent plastic boxes (40 cm × 24 cm × 10 cm). Fresh willow leaves were replaced daily in transparent plastic boxes to serve as food for P. versicolora . Newly laid eggs or hatched larvae were collected from willow leaves to conduct insect bioassays (at 28 °C, 60% ± 5% relative humidity, 16-h light/8-h dark photoperiod) . They have been maintained since 2022. The colony is refreshed with wild-type stock every 3 months.
The leaves of beetle-damaged and healthy willows were fed to. For each feeding experiment, synchronized larvae were selected, weighed individually, and divided into three groups (each group containing 10 larvae). To calculate the consumed leaf area, the leaves were photographed after feeding by P. versicolora larvae, and the consumed area was determined using ImageJ software ( https://imagej.nih.gov/ij/ ). Pupation rate and eclosion rate were monitored as described . The entire experiment was replicated three times. The pathogenicity test of Pseudomonas strains against insects employed the previously described methods of Vacheron et al. . First-instar larvae were fed with fresh willow leaves (8 cm 2 ) that had been painted with 5 µL of 5.0 × 10 6 bacterial cells in sterile phosphate-buffered saline (PBS). Willow leaves painted with the equivalent volume of PBS solution were used as a negative control. Larvae were divided into three groups (for three biological replicates), and each group had 10 larvae per treatment. Willow leaves painted with an equivalent volume of PBS were used as negative control. Treated leaves were exchanged daily, and insect survival was recorded daily for seven consecutive days. The entire experiment was replicated three times.
bacteria (SynCom) To generate SynCom, sick leaves (SL) and healthy leaves (HL) from sample sites were harvested (6 leaves were sampled from three individual plants), surface-sterilized with 75% ethanol for 1 min, and washed with sterile water three times. Leaves were ground in sterile water, and bacterial suspensions were diluted to 10 −2 and plated on R2A plates for 4 days at 22 °C . About 80 colonies from SL and HL were randomly picked to constitute SynCom- SL or SynCom- HL , respectively. SynCom- SL or SynCom- HL were prepared and the final OD 600 was adjusted to 0.06 (~ 2 × 10 7 CFU/mL), and 10 µL of the SynCom- SL or SynCom- HL suspension was painted to each 1 cm 2 of willow leaf.
Pseudomonas strains into willow plants Willow plants were grown in a growth chamber under controlled conditions (25 °C under a 16-h light/8-h dark photoperiod) . Bacterial re-introduction was conducted as described previously . Briefly, Pseudomonas strains were incubated overnight at 28 °C in an orbital shake. Bacterial cells were collected by centrifugation at 4000 rpm for 5 min to remove the supernatant, washed with sterile PBS twice, resuspended in sterile PBS to a cell density of 10 8 cells/ml, and then inoculated onto the leaves of willow plants. SynCom- mix7 is a mixture of PSE-30, PSE-32, PSE-34, PSE-37, PSE-38, PSE-39, and PSE-49 (1 × 10 8 cells/ml for total SynCom-7 mixtures; 1.4 × 10 7 cells/ml for each strain), and used to inoculate leaves of willow plants. Willow plants treated with PBS served as control. The CFU of Pseudomonas strains on the leaves was determined by a serial-dilution method on the CFC plate .
For bioassays with whole plants, first-instar P. versicolora larvae ( n = 30, divided into three experimental groups, with 10 larvae in each group) were allowed to feed on the healthy willow plants reintroduced with SynCom- mix7 or Pseudomonas strain PSE37/38. Larvae were divided into three groups, with 10 larvae in each group. Survival rates were recorded daily. The entire experiment was replicated three times.
Prior to statistical analysis, the normality of data distribution was assessed using the Shapiro–Wilk test ( P > 0.05). All statistical analyses were performed using SPSS version 19.0 software. Survival curves of first-instar P. versicolora larvae ( n = 30) were analyzed using the Kaplan–Meier method, and the differences in survival curves were determined using the log-rank test with a significance level at P < 0.05. The damaged leaf area, pupation rate, eclosion rate, and larval weight were analyzed using one-way analysis of variance (ANOVA). Data comprising two groups were analyzed using a Student’s t -test for unpaired comparisons, and data consisting of more than two groups were analyzed using one-way analysis of variance. Alpha diversity indices (e.g., Shannon index and Chao1 index) and beta-diversity were calculated using QIIME 2.
Beetle-induced activation of plant defense against Contribution of plant-associated microbiota in plant defense against Pseudomonas Enrichment of The Inoculation of a synthetic microbial community (SynCom) enhances willow resistance to beetles on whole plants P. versicolora To examine whether plants employ defensive tactics when repeatedly exposed to leaf beetles, we fed P. versicolora larvae with the sick leaves of insect-damaged sick willows (SL), the healthy leaves of healthy willows (HL- HW ), and sick willows (HL- SW ) that were sampled from two field sites in Wuhan, China (SI Appendix, Fig. ). We observed that P. versicolora larvae cause less damage to SL than HL- HW or HL- SW , based on the area consumed by the larvae (Fig. A and B). Moreover, compared to that with HL, feeding with SL significantly reduced the growth of P. versicolora larvae from day 2 onward (Fig. E) and substantially delayed pupation (Fig. C) and eclosion rate (Fig. D). These results suggest that beetle damage can activate plant defenses against further damage by pests.
P. versicolora To assess the contribution of plant-associated microbiota in the resistance of the beetle-damaged willows to P. versicolora , we assembled two synthetic microbial communities from sick leaves (SynCom- SL ) and healthy leaves (SynCom- HL ) of sick and healthy willow plants, respectively, and introduced them onto the axenic sick or healthy leaves that had been in vitro sterilized (Fig. and Fig. S3). Compared to the axenic healthy or sick leaves that were reintroduced with SynCom- HL , reintroduction of SynCom- SL significantly reduced the damage to willow leaves (Fig. A and B), and substantially delayed pupation (Fig. C), eclosion rate (Fig. D), and larval weight (Fig. E) of P. versicolora . These results demonstrated that plant-associated microbiota from the beetle-damaged willow contribute to the resistance of the beetle-damaged willow to P. versicolora .
spp. is enriched in the phyllosphere of beetle-damaged willows We next investigated the composition of the bacterial community within the phyllosphere of SL and HL by deep sequencing of amplified bacterial 16S rRNA sequences. Non-metric multidimensional scaling (NMDS) based on Bray–Curtis dissimilarity indicated that bacterial communities from SL and HL were significantly different in samples from both Shahu Lake and Nanhu Lake (Figs. A and A, B, ANOSIM, P < 0.01). We estimated alpha diversity based on the Shannon index and Simpson index and revealed a significant difference for the α-diversity indices between phyllosphere bacteria from SL and HL from Shahu Lake (Fig. B, C, P < 0.01). Linear discriminant analysis of effect size (LefSe) showed that the genus Pseudomonas was significantly more abundant in the phyllosphere of SL compared to HL (Fig. A and B). Similarly, we thus isolated Pseudomonas from SL and HL; indeed, more Pseudomonas species were present in SL samples than in HL (Fig. C). These results suggest that Pseudomonas spp. is enriched in willow leaves damaged by beetles.
Pseudomonas in the root and rhizosphere soil samples of beetle-damaged willows Several recent studies have indicated that infections with aboveground pathogens also alter the rhizosphere microbial community . We thus tested whether leaf damage by beetles also influenced the bacterial community in root and rhizosphere soil samples by 16S rRNA amplicon sequencing. NMDS based on Bray–Curtis dissimilarity revealed that bacterial communities from beetle-damaged sick root (SR) and healthy root (HR) were significantly different in samples from both Nanhu and Shahu Lake (Figs. A and C, D, ANOSIM, P < 0.01). Bacterial communities from beetle-damaged sick willows (SS) and healthy willows (HS) were significantly different in samples from both Nanhu and Shahu Lake (Figs. A and E, F, ANOSIM, P < 0.05). The alpha diversity of the bacterial community in the SR has no significant difference from that in the HR in terms of Shannon diversity index (Fig. B, C, P > 0.05). We observed no significant difference for α-diversity indices between rhizosphere soil bacteria from SS and those from HS (Fig. B, C, P > 0.05). LefSe showed that the genus Pseudomonas is was significantly more abundant in SR compared to HR samples (Fig. A and B). Similarly, Pseudomonas bacteria were significantly more abundant in SS compared to HS samples (Fig. C and D). We next isolated Pseudomonas from root and rhizosphere soil samples collected from beetle-damaged and healthy willows. We determined that the abundance of Pseudomonas isolated from SR or SS samples is significantly higher than that from HR or HS samples (Fig. E and F). These results suggest that Pseudomonas spp. is also enriched in the roots and rhizosphere soil of beetle-damaged willows, similar to the phyllosphere results above.
Pseudomonas enriched on beetle-damaged plants show insecticidal activity against beetles We isolated and identified 49 Pseudomonas strains from the phyllosphere, root, and rhizosphere soil samples collected from beetle-damaged and healthy willows. Based on a phylogenetic analysis of these strains, we established that they represent distinct phylogenetic groups based on 16S rRNA gene sequences. Importantly, we detected nine P. psychrotolerans (Pp) strains specifically on samples collected from SL (Fig. ). To test whether isolated P. psychrotolerans strains have insecticidal activity, we allowed P. versicolora larvae to feed on healthy willow leaves that had been painted with a cell suspension of these strains. We scored the survival rate of larvae. Two Pseudomonas strains, P. chlororaphis PcS1-2 isolated from SS and PcR3-3 isolated from SR, were used as positive control. We discovered that, of the nine P.psychrotolerans strains tested, seven resulted in a higher mortality rate for P. versicolora larvae (Fig. A). Moreover, P. versicolora larvae caused less damage to willow leaves painted with these P. psychrotolerans strains compared to those painted with phosphate-buffered saline (PBS) as control (Fig. B and C). These results demonstrate that some of the Pseudomonas strains that are enriched in the beetle-damaged willows exhibit insecticidal activity against P. versicolora . To identify potential genes associated with the observed insecticidal activities, we performed genome sequencing and assembly of P. psychrotolerans strains PSE37 or PSE38. Our analysis revealed the absence of genes encoding insecticidal proteins such as Fit toxin , IPD072 , Chitinase C, and Phospholipase C , which are commonly present in P. protegens and P. chlororaphis , in P. psychrotolerans strains PSE37 or PSE38 genome (SI Appendix, Table S9).
To measure the contribution of the enrichment of Pseudomonas strains in the beetle-damaged willows to the resistance to P. versicolora on whole plants, we reconstructed a synthetic microbial community (SynCom- mix7 ) composed of seven P. psychrotolerans (Pp) strains isolated from SL. Re-inoculation of SynCom- mix7 or Pseudomonas strain PSE37/38 to the healthy willow brought out 56% mortality of P. versicolora larvae compared to those inoculated with PBS control (Fig. ).
Our data show that Pseudomonas strains become enriched in the microbiota of willows damaged by insect herbivores. We further determined that some of these enriched Pseudomonas strains have insecticidal activity, thus contributing to the protection of willows from further attacks by P. versicolora . We were able to reconstruct a synthetic microbial community (SynCom) of Pseudomonas that could enhance willow resistance to P. versicolora . It has been reported that SynComs have been reconstructed for the purpose of biocontrol. An example of this is the SynCom consisting of Flavobacterium and Chitinophaga , which has been shown to enhance sugar beet resistance to fungal root disease . These findings provide new insights into the functional significance of insect damage-induced enrichment of Pseudomonas communities in willows. Consistent with this idea, plants inoculated with rhizobacterium decreased the growth of caterpillars after 4 days of feeding compared to non-inoculated control plants . Similarly, recent studies showed that fungal infection of plant roots can lead to the assemblage of bacterial groups with disease-suppressive functions . However, it should be noted that changes in plant nutrition, metabolite biosynthesis and secretion, and phytohormone contents can also significantly contribute to the plant resistance against insect pests. For example, introgression of the 7-epizingiberene biosynthetic pathway from wild tomato ( Solanum habrochaites ) to tomato ( Solanum lycopersicum ) cultivars produced plants that can synthesize this chemical that is toxic to spider mites while also making them less attractive to whiteflies . We showed that the aboveground insect damage can also induce the assemblage of an enriched community of Pseudomonas in the roots and rhizosphere soil of willows (Fig. ), suggesting that plant microbiome assembly and functions in the below and aboveground compartments under insect damage may be systemically linked. In agreement with this notion, several recent studies have also suggested that the pathogen infection of plants aboveground can induce the assemblage of a plant-beneficial bacterial consortium in the root or soil . For example, Berendsen et al. reported that upon foliar infection with the oomycete Hyaloperonospora arabidopsidis , three bacterial taxa are specifically enriched in the rhizosphere of Arabidopsis plants, induce systemic resistance against pathogens, and promote plant growth. It remains to be determined whether Pseudomonas spp. enriched in the rhizosphere soil also confer resistance against willow pathogens or increase the growth of willows. In this study, we identified several novel Pseudomonas strains from the phyllosphere with insecticidal activities against P. versicolora . However, the underlying mechanism and the genes responsible for these insecticidal activities are unknown. For example, P. psychrotolerans strains PSE37 or PSE38 were shown to be toxic to P. versicolora but lack the genes encoding insecticidal proteins (SI Appendix, Table S9). Therefore, the novel insecticidal Pseudomonas identified in this study could provide new insecticidal genes. Although a group of insecticidal Pseudomonas strains was shown to be enriched in the phyllosphere of willows (Figs. and ), their origin in the phyllosphere is unclear. It has been suggested that the rhizosphere soil could be a major source of bacteria detected in the phyllosphere . Whether insecticidal Pseudomonas strains can migrate from the rhizosphere soil to the phyllosphere needs to be investigated experimentally.
In summary, this study illuminates the complex interplay between plants, insects, and plant-associated microbiota in a natural system. By using willow, willow leaf beetle, and the associated microbiota as a model, we have uncovered a natural mechanism whereby upon damage by insect herbivory, plants can enrich their phyllosphere with insecticidal Pseudomonas to raise their defense against further damage by insect pests, providing new insight on plant defense against pests.
Additional file 1. Fig. S1–S3 and Table S1–S8.
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Frequent inappropriate implantable cardioverter defibrillator therapy was determined to be dual atrioventricular nodal non-reentrant tachycardia | dcb95b1b-1765-46ed-8e8b-95965ad61158 | 8036121 | Physiology[mh] | Introduction Implantation of an implantable cardiac defibrillator (ICD) is an effective method to protect against sudden cardiac death (SCD) in patients; however, inappropriate ICD therapy is common in the real world. Supraventricular tachycardia (SVT), such as atrial fibrillation, is a common cause. Here, we present the first case of inappropriate ICD therapy due to dual atrioventricular node non-reentrant tachycardia (DAVNNRT) in China. DAVNNRT is a rare type of SVT and is often identified as ventricular tachycardia by the supraventricular tachycardia-ventricular tachycardia (SVT-VT) discriminator of the ICD.
Case report A 73-year-old man with ischemic heart disease presented with palpitations accompanied by dyspnea and dizziness for almost 1 year. Ambulatory electrocardiography showed frequent multifocal premature ventricular beats and non-sustained VTs. He suffered from syncope and received direct current cardioversion before he came to our center; unfortunately, the electrocardiogram (ECG) was recorded by an external defibrillator and was not preserved. He received emergency percutaneous coronary intervention (PCI) therapy for acute myocardial infarction and received drug-eluting stents in the left circumflex (LCX) artery in another hospital 3 years earlier. Physical examination showed no positive signs on admission. Transthoracic echocardiography revealed an ejection fraction of 32%, with a hypokinetic inferior and inferolateral wall. Hypersensitive troponin I (HsTnI) and brain natriuretic peptide (BNP) levels increased to 0.463 μg/L and 499.18 ng/L, respectively. Subsequently, the patient underwent repeat PCI therapy, and coronary angiography (CAG) showed 80% to 85% stenosis of the left anterior descending (LAD) artery as well as the LCX artery. Consequently, drug-eluting stents were placed in the LAD artery and balloon inflation was successfully performed in the LCX artery. Considering the concurrence of ischemic heart disease (IHD), heart failure with reduced ejection fraction (HFrEF), and syncope due to VT, a single-chamber implantable cardioverter defibrillator (ICD) (Iforia7 VR-T DX Biotronik SE&Co. KG, Berlin, Germany) was implanted for secondary prevention of sudden death. ICD settings were as follows: mode = VDI; VT1/VT2/VF-detection-rate = 167/180/200 beats per minute (BPM); detection/redetection counter = VT1:40/30, and VT2:16/14. Unfortunately, he developed tachycardia. He underwent ICD therapy, including 173 rounds of anti-tachycardia pacing (ATP) and 5 rounds of shocks, in the 4 months following the ICD procedure (Fig. a−d). To avoid further inappropriate ICD therapy, we increased the VT1/VT2 detection rate and prolonged the VT detection time to avoid inappropriate ICD therapy. Additionally, amiodarone (600 mg per day) and metoprolol (23.75 mg per day) were prescribed, but they seemed to have no effect. The AV interval of the first 4 A and V waves in Figure b was approximately 570 ms, as shown in Figure e, which had a long PR interval. We found several series of narrow QRS complexes, which were automatically diagnosed as VT/ventricular fibrillation (VF) by the device, and we performed ATP therapy to terminate the VF (Fig. a). However, no ECG was recorded until the third admission. The ECG on the third admission (Fig. a) showed a narrow QRS complex tachycardia (NCT). The P waves in Figure a had the same morphology and axis as the P waves shown in Figure b, suggesting they were of sinus node origin, and each P wave occurred regularly with 2 QRS complexes following. It was noted that the R1R2 and R2R1 intervals showed the same regular pattern throughout the tracing, and the PR interval (PR1 and PR2) alternated between short and long intervals. Moreover, the second QRS presented with a relatively constant coupling interval after the short PR conducted beat. The morphology of the 2 QRS complexes was slightly different. This regularity indicated that the 2 related QRS complexes were generated by 1 given P wave, and the relationship was illustrated more clearly in an intracardiac electrogram (IEGM) from the ICD (Fig. c). The patient received EPS, and no VT was induced. The NCT mentioned above had a sudden and spontaneous onset with no programmed stimulation (Fig. ). The surface ECG shown at the top of Figures a and b is the same as that in Figure a. A CS 9, 10 catheter clearly recorded 1 high-amplitude atrial potential (marked A), along with 2 low-amplitude ventricular potentials (marked V1V2). As shown in Figure b, the radiofrequency ablation catheter (ABL) was positioned at the His bundle to mark his potential. We found 1 a wave followed by 2 H-V waves: H1-V1 and H2-V2. The AH1 and AH2 intervals were 120 ms and 510 ms, respectively. The latter had a tremendous prolongation compared with the former. The HV interval was fixed, and both H1V1 and H2V2 intervals were 60 ms. Atrial burst stimulation (cycle length = 340 ms) can terminate tachycardia. Atrial S1S2 programmed stimulation (450/400 ms, −10 ms) was applied until the AVN refractory period was reached (450/360 ms). No “jump” phenomena were observed. Right ventricular apex pacing (S 1 S 1 ) with multiple cycle lengths showed no V-A retrograde conduction (Fig. c). The difference between AH1 and AH2 established that atrial excitation conducts down to the ventricle with 2 divided pathways of the AVN, which had pronounced differences in conduction velocities when compared with each other. Retrograde conduction between the ventricle and atrium was absent. We called this type of tachycardia, dual atrioventricular node non-reentrant tachycardia (DAVNNRT). The middle panel of Figure d shows the electrophysiological mechanism of this tachycardia. Atrial beats being conducted to the ventricle along a slow pathway might explain the long AV and PR intervals shown in Fig. b and e because the AV intervals had approximately the same length as the AV 2 intervals, as indicated in the EPS (Fig. b). Radiofrequency ablation of the slow pathway (Fig. e) terminated this tachycardia successfully, and the double ventricular response disappeared. We performed the EPS experiment again: ventricular pacing showed no V-A retrograde conduction, and programmed stimulation at the high right atrium as well as the CS7, 8 catheter could not induce tachycardia, which was the same as an intravenous drip of isoprenaline. No tachycardia occurred spontaneously. There was no inappropriate ICD therapy or tachycardia during follow-up, and the IEGM from the ICD showed normal AV intervals (Supplemental file, http://links.lww.com/MD2/A47 , and Supplementary data for reviewer http://links.lww.com/MD2/A48 ).
Discussion and conclusion Here, we report a case of inappropriate ICD therapy due to DAVNNRT, which was undiagnosed before ICD implantation. ICDs are effective in protecting patients against sudden cardiac death (SCD), particularly in patients with VT and heart failure due to ischemic cardiomyopathy. However, inappropriate ICD therapy, especially inappropriate shock, is common in the real world. It has a significant morbidity rate and the potential to trigger ventricular arrhythmias, leading to cardiac decompensation and death. In the MADIT II experiment, these phenomena occurred in approximately 11.5% of patients. The most common cause of supraventricular tachycardia (SVT) is atrial fibrillation (44%), followed by SVT (36%). To our knowledge, this is the first case of inappropriate ICD therapy due to DAVNNRT in a patient with IHD and HFrEF in China. Many methods for minimizing inappropriate ICD therapy have been developed, such as the implantation of ICDs with an SVT-VT discrimination function, home monitoring, atrial sensing, and dual-chamber ICDs. However, in this case, the ICD identified DAVNNRT as VT because atrial sensing indicated a relationship between the atrium and ventricle as V > A, ultimately resulting in inappropriate ICD therapy. Common types of NCT include sinus tachycardia, atrial fibrillation/flutter/tachycardia, atrioventricular node reentrant tachycardia (AVNRT), and atrioventricular reentrant tachycardia (AVRT). DAVNNRT is not a common type of NCT, and it was first described by Wu et al. The presence of imbalanced electrophysiological properties of the slow/fast pathway in the AVN usually generates AVNRT. In rare situations, supraventricular beats can occur simultaneously with fast and slow pathways, which generates 1:2 AV conduction and causes DAVNNRT. The decisive conditions for this arrhythmia are as follows: changes in sinus excitation, occurrence of atrial and ventricular premature beats, and the need for the electrophysiological characteristics (including conduction velocity, refractory period, and backward conduction) of the 2 pathways and the distal common pathway to differ. The middle panel of Figure d shows the tachycardia-building process. These conditions are affected by the autonomic nervous system and various drugs in most cases; therefore, they rarely occur in the real world. Peiker et al have accounted for the electrophysiological characteristics of DAVNNRT in a review of 68 cases from 1995 to 2014. The authors indicated that the most significant indication of DAVNNT on ECG is a P wave followed by 2 narrow QRS complexes. Because it is not widely known, DAVNNRT may be diagnosed as atrial fibrillation, atrial premature beats, or other SVTs, especially since dual AV nodal conduction may be intermittent. Bigeminal junctional ectopy could be another arrhythmogenic mechanism for this, but it usually shows more irregular variations in the coupling interval with the previous sinus beat. DAVNNRT has a fixed H-V interval and a slight variation in the R1R2 interval. Due to the variable conduction of the slow and fast pathways, slight changes in QRS morphology may be detected. As recently described by De Ponti et al, the different inputs into the bundle of His from the fast and slow pathways, suggesting the longitudinal dissociation of the distal AVN extending to the bundle of His (referred to as Zhang's phenomenon (or His electrogram alternans)), potentially explains the different QRS complex morphologies. Cardiovascular diseases, such as IHD, could cause changes in the extent and heterogeneity of structural discontinuities. The variability of cardiac cycle time and ventricular wall tension between the 2 ventricular beats may also contribute to the minimal difference between the 2 QRS complexes in this patient. The difference between 2 AH intervals ranged from 265 to 520 ms (359 ± 46 ms), suggesting the different electrophysiological characteristics of the 2 pathways, and the slow pathway conduction was slow enough to allow the His-Purkinje system to recover excitability after being depolarized by the first excitation over the fast pathway. The retrograde conduction between the ventricle and atrium was weak or absent, as has been observed in previous studies. As described by Rivner et al article, supraventricular beats can only conduct down the fast pathway, both the fast and slow AV nodal pathways, and the slow pathway only. Of note, as with this patient, atrial beats at a rate of 100 BPM are conducted along the slow pathway (Fig. e). In contrast, as shown in Figure b, atrial beats at a rate of 50 BPM are conducted along the fast pathway, which is probably due to the different electrophysiological characteristics of dual AVN pathways, the presence of concealed retrograde conduction, and the refractory period of the distal bundle of His. This finding is illustrated in the left and right panels of Figure d. Interestingly, tachycardia and inappropriate ICD therapy mainly occur at an atrial rate between 60 and 100 BPM. It is certain that DAVNNRT can be cured by effective ablation of the slow pathway to suppress its forward conduction and has a good prognosis. In fact, the dual pathway of AVN is common, and it is not necessary to eliminate the slow pathway for all patients only if it generates tachycardia or for patients who undergo implantation of an ICD. In conclusion, DAVNNRT is a rare type of tachycardia that appears as an irregular narrow QRS complex with 1:2 AV conduction, especially discriminated as VT by the SVT-VT discriminator of the ICD. We present a rare reason for inappropriate ICD therapy. It is important for us to have a full understanding of this arrhythmia to avoid misdiagnosis and incorrect treatment.
We thank AJE ( https://www.aje.cn ) for its linguistic assistance during the preparation of this manuscript.
Clinical data collection and interpretation: Chengming Ma, Shiyu Dai, and Xiaohong Yu. Data curation: Chengming Ma, Shiyu Dai. Editing and revision: Xiaomeng Yin and Yunlong Xia. Figure drafting : Chengming Ma and Wenwen Li. Formal analysis: Xiaohong Yu. Software: Wenwen li. Supervision: Xiaomeng Yin, Yunlong Xia. Writing – original draft: Chengming Ma. Writing – review & editing: Xiaomeng Yin, Yunlong Xia, Lianjun Gao.
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Impact of the Human Leukocyte Antigen Complex on Idiopathic Pulmonary Fibrosis Development and Progression in the Sardinian Population | a6b1e0e7-c8a8-4862-baa3-8ee3950c74e0 | 11942992 | Pathologic Processes[mh] | Interstitial lung diseases (ILDs) consist of approximately 200 different diseases that may lead to inflammation and scarring of the lung tissue . A recent publication by the American Thoracic Society Consensus Statements classified ILDs into the following categories: idiopathic ILDs, autoimmune-related ILDs, exposure-related ILDs, interstitial lung diseases with cysts or airspace filling, ILDs related to distinct primary diseases, and other ILDs [ , , ]. Among the idiopathic categories, idiopathic pulmonary fibrosis (IPF) is the most prevalent and aggressive type of ILD. IPF is a chronic and progressive lung disease of unknown cause in which the alveolar and interstitial architecture is disrupted by the deposition of altered extracellular matrix. These modifications lead to restrictive lung disease, interfering with both gas exchange and lung compliance, and resulting in respiratory failure and death [ , , ]. IPF disease is characterized by a poor prognosis, with a median overall survival from 2.5 to 3.5 years from the time of diagnosis [ , , ]. The incidence of idiopathic pulmonary fibrosis has been increasing over time, and in Europe and North America, it is estimated to be between 2.8 and 18 cases per 100,000 people/year . Moreover, a recent study showed that IPF prevalence in the U.S. in patients over the age of 65 increased from 202 to 495 cases per 100,000 people . Several risk factors for IPF have been identified. Among the non-genetic ones, male gender, age, and tobacco use are the most prominent . Multiple types of environmental exposure have also been associated with IPF onset, including metal and wood dust, agriculture and farming, viruses, silica, and stone [ , , ]. Recently, a growing body of evidence has suggested that genetic susceptibility plays a crucial role in the development of idiopathic pulmonary fibrosis. IPF occurs both sporadically and in families, which is consistent with an underlying genetic predisposition . Several genetic variations have been associated with its pathogenic mechanisms, such as surfactant mutations, protein misfolding, ER stress, and telomere shortening associated with abnormalities in DNA repair [ , , ]. However, despite extensive investigations, the etiology of IPF remains unclear. Genetic variation in the human leukocyte antigen ( HLA ) region, through multiple and complex molecular mechanisms, is responsible for the diverse immune responses that can lead to persistent chronic inflammation, promote recurrent infections, and contribute to respiratory diseases . Recent studies have reported a higher frequency of certain HLA alleles in patients with IPF . The Sardinian population exhibits unique genetic characteristics due to its insularity and is recognized as a well-known outlier in the European genetic landscape . Several genetic peculiarities have been observed, including a high frequency of rare uniparental haplotypes, the extensive linkage disequilibrium of autosomal markers, and elevated levels of homozygosity, particularly in the HLA loci . These features make the Sardinian population an ideal model for studying genetic predisposition to complex diseases, as its reduced genetic variability allows for the identification of specific risk alleles with greater statistical power. Leveraging these genetic characteristics, this study aims to assess whether specific allelic and haplotypic variants in the HLA region contribute to both the susceptibility to and clinical progression of IPF.
2.1. Workflow of IPF Patients Selection 2.2. Clinical and Demographic Baseline Parameters of IPF Patients 2.3. Comparison of HLA Allele and Haplotype Frequencies Between IPF Patients and Healthy Donors 2.4. Comparison of HLA Allele and Haplotype Frequencies Between IPF Patients Based on Disease Outcomes 2.5. Impact of HLA on Overall Survival A multivariate analysis using a logistic regression model was conducted to determine the independence of factors associated with the outcome of idiopathic pulmonary fibrosis ( ). The multifactorial comparison of patients with slow and rapid progression forms of IPF included age ≤ 55 years, age ≥ 65 years, male gender, smoking history, antifibrotic therapy (Nintedanib, Pirfenidone, or no therapy), HLA alleles/haplotypes ( HLA-DRB1*04:05 ; HLA-A*01:01 , DQB1*03:01 ; HLA-A*02:01 , DQB1*02:01 ; HLA-B*49:01 , C*07:01 ; HLA-A*30:02 , B*18:01 , C*05:01 ; HLA-A*30:02 , B*18:01 , C*05:01 , DQA1*05:01 , DQB1*02:01 , DRB1*03:01 ). The analysis highlighted that the HLA-DRB1*04:05 allele and the extended haplotype HLA-A*30:02 , B*18:01 , C*05:01 , DQA1*05:01 , DQB1*02:01 , DRB1*03:01 represented independent genetic variables significantly associated with the stable or slow progression form of the disease (OR M = 0.11, P M = 0.010 and OR M = 0.065, P M = 0.010, respectively). The Kaplan–Meier curves ( ) show the overall survival (OS) over 60 months of the 103 patients, with a median follow-up of 43.3 months. At 60 months, the OS was 78.6% (81/103). A shows the OS curves of the 103 patients over 60 months, stratified into two groups based on the presence or absence of HLA-DRB1*04:05. The patients with HLA-DRB1*04:05 exhibited a significantly reduced risk of mortality (X 2 = 4.57; log-rank = 0.032) compared to the other patients. At 60 months, the OS was 95.2% (20/21) for patients with HLA-DRB1*04:05 , compared to 74.4% (61/82) for patients without this allele. The median follow-up was 46.4 months for patients with HLA-DRB1*04:05 and 42.5 months for patients without this allele. It is interesting to note that already, at 36 months of follow-up, patients with HLA-DRB1*04:05 had significantly better pulmonary function parameters ( B). The percentages of the predicted values for single-breath carbon monoxide diffusing capacity (DLCO%p) were significantly greater in these patients (67.6 ± 19.5% vs. 53.6 ± 21.3%, respectively, p = 0.005). No significant difference was observed between the two groups in relation to forced vital capacity as a percentage of the predicted values (FVC%p) (70.9 ± 22.9% vs. 72.3 ± 21.5%, respectively, p = 0.723). A shows the overall survival (OS)the extended haplotype HLA-A*30:02 , -B*18:01 , -C*05:01 , -DRB1* 03:01. Similarly to HLA-DRB1*04:05 , and independently of this allele, the presence of the extended haplotype also determined better survival for the patients who possessed it. In fact, after 60 months of follow-up, the OS was 95.5% (21/22) for patients with this haplotype, compared to 74.1% (60/81) for patients without it (X 2 = 6.44; log-rank = 0.011). The median follow-up was 57.1 months for patients with this extended HLA haplotype and 39.5 months for patients without it. Furthermore, the extended haplotype was associated not only with a lower risk of mortality but also with better pulmonary function values. In particular, t8.3 ± 26.4% vs. 53.7 ± 20.8%, respectively, p = 0.02). Furthermore, to explore any connections between HLA alleles and other clinical parameters, we analyzed the available blood test data, including the presence of autoantibodies, as reported in .
For our first analysis, we carefully evaluated various parameters to determine which patients were ideal candidates for enrollment in the present study. A total of 136 patients with interstitial lung diseases (ILDs) were recruited between January 2020 and July 2024 and follow-up was conducted at the Department of Pneumology of Binaghi Hospital (ASL, Cagliari). Since the aim of this study was to evaluate a potential association between HLA class I and II alleles/haplotypes and the onset of IPF and the rapidity of disease progression, patients were stratified into two subgroups: (i) IPF characterized by stable and/or slow progression and (ii) IPF characterized by rapid progression. To avoid confounding factors and biases in the analysis, thirty-one patients with pulmonary comorbidities were excluded: six patients with lung cancer, seventeen patients with pulmonary fibroelastosis, two patients with chronic obstructive pulmonary disease (COPD), and six patients affected by progressive pulmonary fibrosis (PPF). Two additional patients were excluded due to incomplete clinical and follow-up data. Finally, we included 103 patients, which were in turn divided into IPF patients affected by a slow progression form of disease ( n = 69) and those affected by a rapid progression form ( n = 34) ( ). All selected patients had idiopathic forms of the disease, which were not easily attributable to a known cause or secondary to an autoimmune disease, in contrast to ILD forms associated with environmental exposure. Notably, five patients (5/103; 5%) had occupations that, according to previous studies, have been identified as potential environmental risk factors for fibrosis due to fine particulate matter exposure. Among them, four (4/103; 4%) worked in construction, and one (1/103; 1%) was a baker.
A total of 103 IPF patients were studied ( ). The patients were divided into two subgroups based on their clinical presentation and disease outcome: 69 patients with a stable or slow progression (S group) form of IPF, and 34 patients with a rapid progression (R group) of the disease, and who had clinical characteristics that led to their inclusion on the lung transplant waiting list. The mean age at diagnosis was 70.0 ± 8.2 years, and 79 patients (76.7%) were male. More than 70% of the patients ( n = 74) were former smokers. A total of 59 patients (57.3%) were treated with Nintedanib, 24 patients (23.3%) with Pirfenidone, while 20 patients (19.4%) did not receive any antifibrotic therapy due to poor compliance or the onset of severe side effects. Similar and statistically insignificant percentages characterized both subgroups of patients. Significant differences were observed in the pulmonary function parameters at the time of diagnosis: the extent of pulmonary restriction, assessed by forced vital capacity (FVC) as a percentage of the predicted values (FVC%p), was less severe in the S group of IPF patients compared to the R group (85.0 ± 18.7% vs. 70.4 ± 18.5%, respectively, p = 0.0003). Similarly, the predicted values for single-breath carbon monoxide diffusing capacity (DLCO%p), an indicator of intrapulmonary gas exchange, were significantly greater in the S group of IPF patients (69.6 ± 17.0%) than in the R group of IPF patients (49.2 ± 16.8%) ( p < 0.0001). These differences persisted and became more pronounced at the 24- and 36-month follow-ups. The overall survival at 12, 36, and 60 months was significantly higher in the S group compared to the R group: 100.0% (69/69) vs. 85.3% (29/34) at 12 months, p = 0.003; 98.6% (68/69) vs. 61.8% (21/34) at 36 months, p < 0.0001; and 95.1% (66/69) vs. 47.1% (16/34) at 60 months, p < 0.0001.
Next, to explore potential correlations between HLA and susceptibility to or protection against IPF, we compared the allelic and haplotypic HLA frequencies between IPF patients ( n = 103) and healthy donors ( n = 303). Only a few differences in allelic frequencies were observed between the IPF patients and controls for both class I and class II HLA genes ( ). The frequencies of the HLA alleles and two-loci haplotypes that reached statistical significance ( p < 0.05) are presented in . summarizes the most statistically significant results ( p < 0.02) from the comparison of HLA allele and two-loci haplotype frequencies between IPF patients and the control population. A significant reduction in HLA-C*04:01:01 and HLA-DPB1*04:02:01 was observed in the IPF patients compared to the control group ( ). Notably, the HLA-DQB1*04:01:01 allele was present in the IPF patients but was completely absent in the control population [3/206 (1.46%) vs. 0/606 (0%); OR > 1.22; p = 0.016]. An analysis of the two-loci HLA haplotype frequencies revealed a significantly higher frequency of the HLA-A*02:01:01, -DRB1*04:05:01 haplotype in IPF patients, along with a strong increase in the HLA-A*32:01:01 , HLA-C*02:02:02 haplotype frequency (see ). Of particular note, the HLA-C*02:02:02 , DQA1*02:01:01 haplotype was found exclusively in IPF patients and was completely absent in the control population [4/206 (1.94%) vs. 0/606 (0%); OR > 1.96; p = 0.004]. Conversely, the HLA-A*11:01:01 , HLA-C*04:01:01 and HLA-C*04:01:01 , -DQB1*03:01:01 haplotypes were significantly underrepresented in the patients compared to the controls (see ). Finally, a comparison of the extended HLA haplotype frequencies between the IPF patients and the controls did not reveal any statistically significant differences ( ).
Regarding the frequency analysis of the two-loci haplotypes, our results highlight a few significant differences for both HLA class I and II ( ). The HLA alleles and two-loci haplotypes which reached statistical significance ( p < 0.05) are shown in . summarizes the most statistically significant results ( p ≤ 0.02) from the comparison of HLA allele and two-loci haplotype frequencies between the two patient groups (R vs. S group). Alleles and haplotypes with a frequency > 2% were considered. Notably, there was a significantly lower frequency of the HLA-DRB1*04:05:01 allele in the R group patients affected by the rapid progression form of IPF compared to S group patients [2/68 (2.94%) vs. 20/138 (14.49%); OR 0.18 (95% CI 0.02–0.78), p = 0.014]. Our analysis revealed that the HLA-A*01:01:01 , DQB1*03:01:01 haplotype frequency was higher in R group than S group patients [5/68 (7.35%) vs. 1/138 (0.72%), OR 10.9 (95% CI 1.23–95.0); p = 0.016]. The same trend was also observed for the HLA-A*02:01:01 , DQB1*02:01:01 haplotype frequency [5/68 (7.35%) vs. 1/138 (0.72%), OR 10.9 (95% CI 1.23–95.0); p = 0.016]. Conversely, the HLA-B*49:01:01 , HLA-C*07:01:01 haplotypes were detected only in S group patients who exhibited a slow disease progression [0/68 (0%) vs. 12/138 (8.70%), OR 0.13 (95% CI 0.00–1.18); p = 0.010]. Finally, we compared the frequencies of the three most common HLA extended haplotypes in the Sardinian population between the two groups of IPF patients exhibiting slow or rapid disease progression ( ). It is noteworthy that the extended HLA haplotype HLA-A*30:02 , B*18:01 , C*05:01 , DQA1*05:01 , DQB1*02:01 , DRB1* 03:01 was significantly associated with the stable/slow progression form of IPF. This haplotype was found in 21 of the 138 patients (15.22%) with stable or slow disease progression but in only 1 of the 68 patients (1.47%) with rapid disease progression [OR 0.08 (95% CI 0.01–0.63); p = 0.002]. These findings suggest a potential protective role of this extended haplotype in mitigating the aggressive course of IPF.
In this study, we analyzed genotype data from the HLA alleles in independent case–control cohorts to investigate their association with IPF susceptibility and disease progression. Previous studies have linked HLA alleles, such as HLA-DQB1*06:02 and HLA-DRB1*15:01 , to fibrotic lung diseases . Additionally, a recent study on the Sardinian population demonstrated a protective effect of the extended haplotype HLA-A*02:05 , B*58:01 , C*07:01 , DRB1*03:01 against severe pneumonia caused by SARS-CoV-2 infection . In contrast to previous findings, our study observed no significant differences in HLA allele frequencies when comparing patients with idiopathic pulmonary fibrosis (IPF) and the healthy control population. Only a few alleles, such as HLA-DQB1*04:01:01 , HLA-C*04:01:01 , and HLA-DPB1*04:02:01 , showed some differences in their frequency. However, these differences were only marginally significant and may have reflected random variation rather than a true association with IPF susceptibility. This lack of a strong association suggests that, although genetic predisposition likely plays a role in IPF pathogenesis, the contribution of individual HLA alleles may not be as prominent in the onset of the disease in this cohort. Moreover, the absence of substantial differences in HLA alleles between IPF patients and controls could be attributed to the limited genetic variability in the Sardinian population. Indeed, previous studies on HLA and IPF have demonstrated that, for instance, the allele DRB1*15:01 is overrepresented in patients with idiopathic pulmonary fibrosis . However, this finding was not replicated in our study, probably due to the unique allelic characteristics of the Sardinian population . Specifically, the DRB1*15:01 allele has an extremely low frequency (<0.05) among the over one thousand Sardinian HLA typing entries reported in the Allele Frequency Net database [URL: https://www.allelefrequencies.net/ (accessed on 15 January 2025)]. This rarity could explain the lack of correlation between this allele and the disease within the studied population. One of the most intriguing aspects of our findings is the potential protective effect of specific HLA variants on IPF progression. As described in the Materials and Methods, patients were stratified based on disease severity. Our data revealed that the presence of the HLA allele DRB1*04:05 was associated with greater survival in IPF patients (log-rank test, p = 0.032) over a 60-month follow-up period ( A). Indeed, this allele correlated with better gas exchange, as indicated by higher DL CO %p values at 36 months ( p = 0.005). Reductions in DL CO are commonly utilized as indicators of disease progression and bad prognosis . They may also serve as a supportive criteria for considering lung transplantation in patients with IPF . In the existing literature, HLA-DRB1*04:05 has been extensively linked to autoimmune diseases, including rheumatoid arthritis (RA) and autoimmune hepatitis (AIH) [ , , , ]. This allele features a distinctive amino acid sequence, known as the “shared epitope” (SE), which is thought to facilitate the presentation of self-antigens to T lymphocytes, thereby contributing to autoimmunity. Specifically, the S57-LLEQRRAA (67–74) sequence in the third hypervariable region of HLA-DRB104:05/*04:10 has been identified as a critical factor in autoimmune predisposition . Additionally, this allele has also been linked to drug-induced interstitial lung disease (DILD), a life-threatening adverse reaction . All of these studies highlight the association of HLA-DRB1*04:05 with a predisposition to autoimmune diseases and adverse drug reactions, often with pulmonary involvement, which appears to contrast with our findings. However, what emerges from our study is the potential for this allele to modulate immune reactivity. While excessive immune activation can lead to autoimmune diseases, a finely tuned immune response may confer protection against infections or environmental insults . Respiratory infections have been suggested as potential triggers for the development and progression of interstitial lung diseases, including idiopathic pulmonary fibrosis . While this disease is not traditionally classified as an immunological disorder, it is increasingly recognized to involve abnormal adaptive immune responses . Most IPF patients exhibit IgG autoantibodies against specific autoantigens, which are distinct from those associated with classic autoimmune diseases such as lupus or scleroderma . These autoantibody responses often correlate with clinical manifestations of IPF. The T cells in IPF patients show evidence of heightened prior activation, with the increased production of inflammatory and profibrotic mediators, including TGF-β1, and impaired regulatory function . Moreover, HLA class II molecules are also expressed in the alveolar epithelial cells from the lungs of patients with IPF . Additionally, activated dendritic cells with enhanced antigen-presenting capabilities accumulate in the lung parenchyma. CD4 T cells, both in the lungs and peripheral blood, exhibit oligoclonal proliferation, suggesting repeated stimulation by a limited set of antigens . These findings suggest ongoing immune activation potentially driven by environmental triggers. In this context, a potential mechanism underlying these protective effects could involve the regulation of immune responses in IPF. Specific HLA alleles/haplotypes, including DRB1*04:05 , may enhance the immune system’s ability to respond to environmental triggers, such as infections or irritants, thereby reducing the risk of exacerbation and progressive fibrosis. This allele may influence immune cell recruitment, cytokine production, or T cell responses, all of which play a crucial role in IPF progression. Additionally, the protective role of HLA-DRB1*04:05 could be linked to a decreased susceptibility to pulmonary infections—both viral and bacterial—which are known to exacerbate lung fibrosis and contribute to disease progression. This protective mechanism may explain the better clinical outcomes observed in IPF patients carrying this allele. Furthermore, HLA alleles are often in strong linkage disequilibrium (LD) with other alleles within the same haplotype. As a result, the observed associations may not be directly attributable to the specific allele but rather to another allele in LD . To rule out such an influence, a multivariate analysis was conducted, which confirmed that this possibility could be excluded ( p = 0.802). Similarly, another important finding of this study pertains to the extended haplotype HLA-A*30:02 , B*18:01 , C*05:01 , DQA1*05:01 , DQB1*02:01 , DRB1*03:01 . Notably, similar to HLA-DRB1*04:05, this extended haplotype was associated with improved survival outcomes in carriers (log-rank = 0.011). This extended haplotype, commonly found in Sardinia, has been previously associated with autoimmune diseases such as multiple sclerosis, autoimmune type I hepatitis, severe COVID-19, insulin-dependent diabetes mellitus, and celiac disease [ , , , , , , , , ]. Specifically, this haplotype exhibits the highest frequency (about 14%) in Sardinia and represents the strongest linkage disequilibrium observed globally . Interestingly, while this extended haplotype has been associated with increased susceptibility or worse disease outcomes in other autoimmune conditions, in the specific case of this disease, it appears to play a protective role, contributing to a better prognosis. One plausible hypothesis is that the protective effect of this haplotype may involve the regulation of immune responses, enhancing immune function. The immune responses from this extended haplotype could help to mitigate disease exacerbations and contribute to better long-term outcomes, suggesting a context-specific role of this haplotype in immune tolerance and regulation. In our study, this haplotype does not appear to be associated with the development of IPF but rather with a slower progression of the disease. This effect could be partially explained by the increased expression of HLA-G molecules observed in individuals carrying this extended haplotype . It is well established that there is a strong linkage disequilibrium between the extended haplotype HLA-A*30:02 , B*18:01 , C*05:01 , DQA1*05:01 , DQB1*02:01 , DRB1*03:01 and the HLA-G*01:01:01/UTR-1 haplotype, which is characterized by high levels of HLA-G molecule expression . The potent immunomodulatory effect of HLA-G molecules could mitigate the chronic inflammatory process, a key component in the pathogenesis of IPF, thereby contributing to a slower progression of the disease. A differential regulatory mechanism of the HLA system has been observed, in which specific HLA genotypes play contrasting roles in various diseases. For instance, the HLA-DRB1*03 , DRB1*07 , and DRB1*15 genotypes are predisposing risk factors for the development of sarcoidosis. However, these same genotypes exhibit a protective effect in tuberculosis (TB) . This complex interplay of HLA genotypes in disease susceptibility highlights the importance of genetic factors. Indeed, only a few patients (5%) included in the study reported potential occupational exposure due to working in the construction sector, and one patient worked as a baker. However, the limited number of cases excludes a significant correlation, and the pulmonologist’s assessment ruled out any environmental contribution as a causative factor.
4.1. Study Cohorts 4.2. Ethics Statement 4.3. DNA Extraction and HLA Genotyping 4.4. Statistical Methods Summary statistics were calculated for the clinical and genetic data of the IPF patients: interquartile ranges (IQR), medians, means, standard deviations (SD), and mean differences were calculated for all continuous variables; and percentages and odds ratios (OR) were calculated for the categorical data. p values and 95% confidence intervals (95% CI) were obtained using Student’s t -test or Fisher’s exact test, as appropriate. Specifically, only alleles/haplotypes with an overall frequency > 2% and/or a p value < 0.02 were included in the main tables. The adoption of a p value threshold of <0.02 was used to take a conservative approach to highlight more robust associations . All data with a p value < 0.05 were reported in the . A sample size calculation was carried out to determine the minimum proportional differences in the HLA alleles and two-loci haplotypes which were needed to obtain statistically significant results by comparing two groups of given sizes. By setting the statistical power at 90% and the p value < 0.05, we found that, in the comparison between n_1 = 103 IPF patients and n_2= 303 controls, or between n_1 = 34 IPF patients with a rapid progression of the disease and n_2 = 69 IPF patients with slow progression, significant results could be obtained if the minimum proportional difference in the HLA alleles and two-loci haplotypes increased up to 6.0% or up to 5.2%, respectively, as the proportion in each group increased. The significant proportional differences in the HLA alleles and two-loci haplotypes obtained from our data turned out to be all higher than the minimum proportional differences given by the sample size study. The frequencies of the HLA alleles and haplotypes were obtained by a programming code created with R version 4.4.2 [URL: https://www.R-project.org/ (accessed on 17 January 2025)] , which was used to perform all the statistical analyses. A multivariate analysis based on a logistic regression model was conducted to determine the independence between the factors associated with the different outcomes of idiopathic pulmonary fibrosis. The multifactorial comparison between patients with a slow and rapid progression of IPF included age ≤ 55 yr, age ≥ 65 yr, male gender, smoking history, antifibrotic therapy (Nintedanib, Pirfenidone, or no therapy), andRB1*03:01 ). In the comparisons between the slow and rapid group patients, the computation of p values (P M ), odds ratios (OR M ), and 95% confidence intervals (95% CI M ) for all the clinical and genetic variables was adjusted accordingly to the two factors with the strongest correlation to the different outcome of the disease: HLA-DRB1*04:05 and the extend haplotype HLA-A*30:02 , B*18:01 , C*05:01 , DQA1*05:01 , DQB1*02:01 , DRB1*03:01 . Kaplan–Meier curves were used to illustrate the overall survival (OS) from the date of diagnosis to the date of the last follow-up or death. IPF patients were stratified into several groups according to clinical and genetic ( HLA allele/haplotype) parameters. The log-rank test was used for comparisons of the different combinations. Forced vital capacities as a percentage of the predicted values (FVC%p), and the percentages of the predicted values for single-breath carbon monoxide diffusing capacities (DL CO %p) were evaluated at the time of diagnosis, and at the 24- and 36-month follow-up. The values were expressed using median values, 95% confidence intervals, and violin plots, which allowed for the visualization of the distribution of a variable, with its density represented by the width of the violin in each region. A boxplot was included in the violin to easily assess the median and interquartile range. The subgroups of patients were stratified according to the presence or absence of HLA alleles/haplotypes that were significantly associated with disease outcomes ( HLA-DRB1*04:05 and the extended HLA haplotype HLA-A*30:02 , B*18:01 , C*05:01 , DQA1*05:01 , DQB1*02:01 , DRB1*03:01 ). p values and 95% confidence intervals were computed using Student’s t -test.
A total of 103 IPF patients were recruited over a period of 26 months, from January 2022 to July 2024, and enrolled in the study at the Department of Pneumology of Binaghi Hospital (ASL, Cagliari, Italy). The diagnosis of IPF and PPF was made in agreement with the American Thoracic Society, European Respiratory Society, Japanese Respiratory Society, and the Asociación Latinoamericano de Tórax guidelines and recommendations . In particular, all clinical information, chest radiographs, computerized tomography (CT), and pulmonary function tests (PFT), which included forced vital capacity (FVC), expressed as a percentage of the predicted values (FVC%p), and single-breath carbon monoxide diffusing capacity (DLCO), expressed as a percentage of the predicted values (DLCO%p) were taken into account. The clinical and demographic characteristics of the patients were based on age, sex, body mass index (BMI), smoking history, and comorbidities at the time of diagnosis. The extent of pulmonary restriction deficit, determined by FVC%p, and intrapulmonary gas exchange, quantified by DLCO%p, were assessed at the time of diagnosis and during the progression of the disease (at 12, 24, 36, and 60 months). IPF patients were divided into two groups based on disease severity: the slow progression group (S) consisted of 69 patients with a stable clinical condition or slow disease progression, whereas the rapid progression (R) group was represented by 34 patients who met the criteria for being listed for lung transplant. These criteria included rapid clinical deterioration with an annual decline of >10% in ventilatory indices (FVC%p and DL CO %p) and/or the need for oxygen therapy . Three hundred and six healthy controls were selected from the Sardinian Voluntary Bone Marrow Donor Registry, which is highly representative of the genetically homogeneous population of the island of Sardinia, Italy . Three-hundred and three controls were chosen to appropriately reflect the male-to-female ratio and genetic frequencies of the population from the central and southern areas of Sardinia, where the IPF patients were recruited.
Patients were recruited and enrolled in the study protocol at the Department of Biomedical Sciences and Public Health of the University of Cagliari, the Department of Pneumology of Binaghi Hospital of the Sardinian Regional Company for the Protection of Health (ASL Cagliari). Written informed consent was obtained from all the patients and controls in accordance with the ethical standards (institutional and national) of the local human research committee. The study protocol, including informed consent procedures, conformed to the ethical guidelines of the Declaration of Helsinki and was approved by the responsible ethics committee (Ethics Committee of the Cagliari University Hospital; protocol number GT/2020/10894). Records of written informed consent were kept on file and were included in the clinical record of each patient.
Blood was collected into 3 mL EDTA tubes and stored at 4 °C for no more than 12 h prior to processing. Blood samples were incubated for 10 min with red cell lysis buffer (RCLB) to lyse erythrocytes and were centrifuged at 2500× g for 10 min at 4 °C to separate plasma from cellular components. The peripheral blood mononuclear cell (PBMC) pellet was resuspended in 200 µL of PBS buffer and processed to extract DNA using the QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany). Briefly, the resuspended sample was mixed with 50 µL of Qiagen protease, followed by 1 mL of AL buffer, and incubated at 56 °C for 10 min. After digestion, the sample was mixed with 1 mL of ethanol and loaded onto a spin column and centrifuged at 10,000× g . The column was washed sequentially with 500 µL of Buffer AW1 and 500 µL of Buffer AW2, followed by centrifugation for 1 min at 20,000× g to dry. Purified DNA was eluted from the column with 100 µL of Elution Buffer (Qiagen, Hilden, Germany). Class I and class II HLA alleles were genotyped in both the patient and control populations using the AlloSeq Tx17 early pooling protocol, targeting 17 HLA genes ( HLA-A , -B , -C , -E , -F , -G , -H , -DRB1 , -DRB3/4/5 , -DQA1 , -DQB1 , -DPA1 , -DPB1 , -MICA , -MICB ). Library preparation was performed using the AlloSeq Tx17 kit (CareDx, Brisbane, CA, USA), compatible with Illumina sequencing platforms. Sequencing was conducted on the MiSeq platform (Illumina, San Diego, CA, USA) using a 2 × 150 paired-end (PE) v2.0 flow cell. HLA genotypes were assigned using AlloSeq Assign analysis software v1.0.3 (CareDx, USA) and the IPD-IMGT/HLA database version 3.45.1.2.
In conclusion, while our study does not identify major differences in HLA allele frequencies between IPF patients and controls, it highlights the complex role of the HLA system in IPF progression. Our findings suggest that certain HLA allelic and haplotype variants, particularly those associated with slower disease progression, may play a protective role in IPF, potentially by modulating immune responses and reducing inflammation. The unique genetic characteristics of the Sardinian population, with its limited genetic diversity, provide an ideal model for studying genetic influences on the disease, and may serve as a basis for identifying predictive markers of disease progression and responses to therapy in IPF. Future studies should further investigate the role of specific HLA alleles and haplotypes in IPF, particularly in relation to disease progression and response to treatment. However, our study has certain limitations, including a relatively small cohort size. Nevertheless, this is balanced by the fact that, as a single-center study, the data have been thoroughly reviewed, and the cohort, although limited in size, was carefully selected to minimize potential confounding factors. Furthermore, this study provides new perspectives for future research. Functional studies on immune cells from IPF patients would be particularly useful for further investigating the mechanisms through which specific HLA alleles and haplotypes influence disease progression and immune regulation.
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Robot-assisted repair of ureteral stricture | 7ed96ed9-800a-4b88-a875-27d8573ebae8 | 11438720 | Robotic Surgical Procedures[mh] | The causes of ureteral stricture may be iatrogenic, traumatic, congenital, immunological, infection-related, inflammatory, or caused by stones. Among these, previous endoscopic procedures, such as endoscopic treatment of urologic stones and ureteroscopy, are the most common iatrogenic causes . Endoscopic intervention has a positive prognosis for patients with untreated, nonischemic, benign ureteral strictures that are smaller than 2 cm and affect functioning renal units . However, endoscopic treatment is often suboptimal for strictures longer than 2 cm . Surgical options for strictures longer than 2 cm in the distal, middle, or proximal segments of the urinary tract include ureter-to-ureter anastomosis (UU), ureteral reimplantation , psoas hitches , Boari flaps , appendiceal flaps , buccal mucosa graft (BMG) ureteroplasty , ileal replacement and renal autotransplantation . Prior to surgery, it is important to conduct a comprehensive evaluation that includes renal function tests and CT scans to identify the exact position and duration of the stricture accurately. Within the surgical setting, intravenous or retrograde pyelography, as well as flexible ureteroscopy with fluoroscopy, is available as options for precisely identifying the location of the stricture. The positioning of the trocar for distal ureter strictures is consistent with that of previous pelvic operations, such as radical cystectomy or robot-assisted prostatectomy. For mid- and proximal ureter strictures, it is preferable to use a 60-degree lateral decubitus position. This position helps reduce the need to adjust the operating table to extend the area excessively . Minimally invasive reconstruction of the urinary tract must adhere to the established principles of open surgery, including delicate handling of the urinary tract to avoid ureter damage, optimizing the protection of blood supply to the ureter, ensuring tension-free connections, and strengthening the reconstruction with additional flaps on the peritoneum or perinephric fat. This approach aids in providing additional blood circulation and serves to avoid leakage. The incorporation of near-infrared fluorescence (NIRF) with indocyanine green (ICG) is a widely adopted innovation that greatly streamlines surgical procedures. The use of ureteral injection of ICG followed by visualization under NIRF allows quick and accurate identification of the ureter, facilitating precise localization of both the proximal and distal segments. Consequently, this approach improves the clarity of the boundaries of RUR ureteral strictures . Following the administration of ICG, tissues with sufficient blood circulation exhibit discernible green fluorescence, but scar tissue does not display any fluorescence. ICG can be delivered via the opening of the renal pelvis , or it can be injected retrogradely into the urinary tract using a ureteral catheter. Both procedures can be employed concurrently. This approach facilitates accurate localization and comprehension of the anatomical features of the stricture during surgery, assisting in the identification of the most effective rebuilding approach. With the development of science and technology, robot technology has been widely used in the surgical treatment of ureteral stricture. Since it has outstanding results and many benefits, such as improved ergonomics, enhanced visual magnification, flexible joint movement, elimination of hand tremors, and the ability to incorporate other imaging techniques such as near-infrared fluorescence (NIRF) imaging, Indocyanine green (ICG) and FireflyTM imaging, patients can expect reduced surgical complications, accelerated recovery, and a good functional prognosis. This article reviews the various categories and the latest progress in robotic correction of ureteral stricture, which provides a valuable perspective for the surgical treatment of ureteral stricture.
A comprehensive literature search was carried out using PubMed. The search included the following as individual terms and in combination: “ureteral reconstruction”, “buccal graft”, “appendiceal interposition”, “ileal ureter”, “autotransplantation” “Boari flap”, “Psoas hitch”. No date criteria were utilized. All articles were considered for the review including case reports, single-center studies, review articles, and meta-analyses. 87 articles were assessed and analyzed for patient characteristics and procedural outcomes. This review article presents an interpretation of results from the articles assessed. A standardized meta-analysis was not conducted; however, this review article aims to provide a synthesis of ureteral reconstruction techniques and the advantages of robot-assisted over traditional laparoscopy to guide management decisions. Robotic ureterotomy Robotic ureteral reimplantation (with or without psoas hitch) Robot-assisted appendiceal covering flap Cheek mucosal graft urethroplasty Ileal replacement Robot-assisted autologous kidney transplantation Reconstruction of the ureter after uretero-anastomotic stricture Ureter-to-ureter anastomosis, also known as end-to-end anastomosis, is a highly effective method used to repair ureteral strictures in the proximal and mid-segments. These strictures are typically 2 to 3 cm long. Unlike ureteral reimplantation or a Boari flap, this method preserves the original structure of the bladder and prevents urine from flowing back into the ureter . In 1922, Nezhat and colleagues performed the first laparoscopic ureter-to-ureter anastomosis (LU), which resulted in functional outcomes similar to those of open surgery but with improved esthetics . The prognosis was favorable . However, the widespread use of LU is limited by the need for extensive laparoscopic expertise and a restricted operating area, making it difficult to achieve anatomical precision and precise suturing. In 2006, Yee performed the first recorded instance of robot-assisted ureter-to-ureter anastomosis (RAUU) . According to certain operators, RAUU is considered the most effective surgical method for removing short ureteral strictures (less than 3 cm) that do not respond to endoscopic therapy. When the patient is placed in a lateral posture, it is most effective to start the anatomical procedure from the unaffected section of the ureter and progressively advance toward the constricted area. ICG and NIRF are used to identify and outline strictures. For example, a 6-Fr ureteral catheter is inserted prior to surgery, and during the procedure, 10 mL of diluted ICG is injected above and below the narrowing using the catheter. According to the authors, the disappearance or decreased fluorescence of the affected ureter clearly indicates the upper and lower boundaries of the ureteral stricture . It is crucial to preserve the integrity of the outer layer and its blood supply. Incisions were made at both ends of the ureter, and the ends were sutured together using continuous single 4/0 or 5/0 sutures to create a surgical connection. Subsequently, a ureteral stent was placed and removed at the outpatient clinic 6 weeks after the operation. Repair posterior to the peritoneum reduces the risk of fistula development. Hemal et al. reported on a series of seven patients in which retrograde antegrade ureteral stenting (RAUU) was used to treat strictures in the proximal ureter. The average operating room time (ORT) was 110 min, ranging from 85 to 140 min. The average estimated blood loss (EBL) was 50 mL, ranging from less than 50 to 75 mL. The average length of stay (LOS) was 3 days, ranging from 2 to 6 days. The mean duration of follow-up was 28 months, during which there were no complications during or after surgery and no recurrences of ureteral stricture . Sun et al. conducted a study comparing 65 robotic procedures with 61 laparoscopic surgeries in adult patients. The results showed that the robotic group had shorter operation times, suture times, and lengths of hospital stay than did the conventional group. In addition, the robotic group exhibited lower levels of inflammation . Maria Buffi conducted a survey involving 17 patients who underwent RAUU treatment. The median operative duration was 150 min, ranging from 100 to 420 min. The postoperative complication rate was 17.6%, with 3 patients experiencing complications. Specifically, 2 patients had Clavien–Dindo class I complications, and 1 patient had a grade II complication. The percentage of recurrence-free patients 2 years after RAUU treatment was 94.1% (ranging from 65.0% to 99.1%) .
Robot-assisted ureter repair, also known as ureter bladder anastomosis, is suitable for distal ureter strictures that are within 5 cm of the entrance of the urethra. The benefit of pelvic muscle ligation lies in its ability to maintain the integrity of the ureter epithelium and protect the functionality of the unaffected contralateral ureter. In addition, this method reduces the risks associated with persistent urinary tract infections and electrolyte imbalances. Inaugural laparoscopic ureter bladder anastomosis was conducted in 1994 by Reddy and colleagues . Yohannes and colleagues later performed a procedure in 2003 in which robots were used to assist in the restoration of the urethra for patients with distal ureteral strictures . Subsequent studies have outlined other methods . The positioning of the trocar is similar to that of robot-assisted radical prostatectomy (RALP). The urethra was cut at the constricted front end and divided at the back edge. A bladder flap is utilized to aid in direct reimplantation of the urethra by suturing the mucosa of the bladder and urethra together. The posterior plate was secured with continuous 4/0 sutures, followed by the placement of a double-J stent and finalization of the anterior anastomosis. The bladder catheter was kept in place for 5–7 days. The rectus fascia sling operation is used to repair gaps ranging from 6 to 10 cm in length. In this procedure, the Retzius space is enlarged, and the anterior branch of the bladder on the opposite side is surgically removed to realign the bladder. The lateral bladder wall is stitched using 3–4 nonabsorbable 2/0 sutures that travel through the rectus muscle tendons in a longitudinal manner to prevent harm to the inguinal nerve. Alternatively, the bladder can be secured to the peritoneal sidewall to achieve a tension-free connection. The bifurcated urethra is inserted into a submucosal passageway, with the length of the passageway adjusted to accommodate greater ureter expansion. A double-J stent was then placed, and the bladder was sealed [ – ]. Patil et al. conducted a study on robot-assisted distal ureteral reconstructions for benign diseases in adults and reported no complications during or after surgery, demonstrating the viability of robotic technology for treating distal ureteral strictures . Casale and colleagues conducted a study on the largest collection of robot-assisted bladder extravesical reimplantation cases, effectively treating strictures without observing any significant complications . However, comprehensive research comparing open versus robot-assisted ureter repair is lacking. Kozinn et al. performed a comparative analysis that included 24 open reconstructions and 10 cases of robot-assisted reconstruction. The results showed a reduced duration of hospitalization and potentially decreased blood loss in the group that received assistance from robots. No stricture recurrence occurred in either group during the 2-year follow-up period . Elsamra examined a total of 125 cases of ureter reconstruction, comprising 20 cases of open surgery, 20 cases of laparoscopic surgery, and 85 cases of robot-assisted surgery. The present study revealed no disparities in terms of stricture recurrence rates or surgery duration across the three groups. However, patients who underwent minimally invasive procedures had shorter hospital stays and less blood loss (P < 0.02) . Schomburg et al. conducted a comparative study between 20 instances of open surgery and 20 instances of robot-assisted ureter repair and found no substantial disparities in estimated blood loss (EBL) or length of hospital stay (LOH), with comparable occurrences of surgical complications. The group that used robot assistance had a longer duration of surgery, but the amount of opioids used post-surgery was significantly lower than that used in the group that did not utilize robots (robo: 0.14 mg/kg, open: 0.25 mg/kg, p < 0.021). The severity of complications in the open group was frequently milder than that in the robot-assisted ureter reconstruction (RALUR) group . Schiavina et al. compared 16 cases of laparoscopic ureter reconstruction with 12 cases of robot-assisted ureter reconstruction, including rectus fascia sling procedures. No significant disparities were observed in the incidence of stricture recurrence between the two groups. However, the laparoscopic group experienced a slightly short duration of hospitalization ( P < 0.006). The robot-assisted group showed a decrease in blood loss that was statistically significant ( P < 0.004) . Future research investigating cost-effectiveness will be crucial for identifying the most effective way to treat this condition. The Boari flap is an alternative method used to treat abnormalities in the middle part of the urethra when a narrow removal results in major abnormalities due to a long damaged segment, making it impractical to connect the urethra and bladder. The Boari flap was initially documented in 1947 . In 2001, Fugita et al. introduced the laparoscopic Boari flap operation for treating lengthy ureteral blockage. No patients underwent successful surgery without complications, and no stricture recurred during an average follow-up period of 11 months . Yohannes and colleagues utilized the Boari flap to reimplant distal strictures in 2003, marking the initial implementation of robotic ureteral stricture repair technology . The robotic method uses a conventional trocar arrangement frequently employed in pelvic urological surgery, mobilizing the bladder on both sides before tying and separating the urachus and the pedicle of the opposite bladder. The superior vesical artery is used as a reference point for locating the base of the flap, which is then moved diagonally over the front wall of the bladder to the desired length. The incised ureter was repaired to the posterior wall using absorbable stitches. Double-J stents were implanted, and the front part was reshaped into a tube and sealed with uninterrupted sutures. The bladder is finally secured to the psoas muscle using nonabsorbable stitches. Indocyanine green (ICG) can be injected intravenously at any stage of the anastomosis procedure to verify vascular supply adequacy . Due to the infrequency of this clinical issue and the complexity of the surgical procedure, there is a lack of extensive expertise. Castillo et al. reported a success rate of 96.6% in a group of 30 patients with positive short-term and long-term results . In 2016, Stolzenburg et al. conducted a study recording the largest series of patient comprising 11 patients with benign ureteral strictures who underwent robot-assisted laparoscopic (RAL) surgery combined with Boari flap reimplantation. The success rate was comparable, with a complication rate of 9% .
Utilizing the appendix as a replacement for tissue for treating proximal and mid-ureter lesions 2–6 cm in length is a favorable alternative, eliminating the need to employ other sections of the intestine, such as the ileum . The initial case report dates back to 1912 and was produced by Melnikoff. Melnikoff employed the appendix in a tubular manner for this purpose . The following authors have embraced this method and achieved favorable results. Both individual case reports from single institutions and case series from several institutions indicate that interventions involving ureteral appendiceal surgery and ureteroplasty with an appendiceal covering flap are both safe and feasible, with a success rate of 92%. Nevertheless, these operations also have problems, such as fistulas and recurring strictures . It is mainly suitable for treating ureter strictures on the right side, but it can also be used to reconstruct ureter strictures on the left side. In 2009, Reggio et al. reported the initial instance of laparoscopic appendiceal ureterostomy for the purpose of rebuilding ureter strictures. The study included an 8-month follow-up period . This surgery involves cutting the appendix at its base while keeping its blood supply intact and then making it into a tube shape by making a lengthy incision on the side opposite to where the blood vessels are located. The stricture was detected, and excised, and its length was assessed using a ureter catheter. Afterwards, the appendix is stitched in a consistent and coordinated manner to the front side. The results of this method show promise; however, the proof is limited due to the small number of documented cases. Duty et al. documented laparoscopic extravesical repair in six patients, for a success rate of 66% in four out of six patients . A case series investigation conducted by Burns et al. showed that nine patients experienced positive long-term outcomes . In their latest publication, Wang et al. presented their first robotic experience utilizing an augmented anastomotic repair technique, which achieved a 100% success rate at the 6-month follow-up . Due to technological developments, this approach may soon become a compelling choice.
Cheek mucosal graft urethroplasty, also known as BMG urethroplasty, is a very efficient method specifically designed for treating ureter strictures longer than 3 cm that cannot be repaired through urethra-to-urethra connections. This technique is particularly advantageous for repairing the ureter because it reduces damage to the fragile blood supply of the ureter and makes it easier to create seamless connection without any stress. BMG ureteroplasty is especially beneficial for patients who have long ureteral strictures that cannot be treated with ureteroureterostomy and for those who have recurrent strictures after unsuccessful prior reconstruction. The utilization of cheek mucosa in the field of urology offers several benefits. It is conveniently reachable, has a low rate of illness or death [ – ], has characteristics resembling the lining of the urinary tract , and is well suited for a damp setting . In 1983, Naude and Somerville conducted initial studies on baboons to explore the use of buccal mucosa transplant ureteroplasty . In 1999, Naude documented the first human series with six patients who were treated using three distinct procedures. In total, four patients underwent BMG ureteroplasty, which involved removing the damaged section of the ureter and repairing it using a BMG graft. A different patient underwent a surgical procedure called enhanced anastomosis BMG ureteroplasty, which involved removing the damaged section of the ureter, connecting the healthy part of the ureter, and repairing it using a BMG graft. A single patient underwent tubular BMG ureteral replacement. Throughout the 24-month observation period, there was no stricture recurrence. Previous studies have shown that the recurrence rates for buccal mucosa externalized or tubularized ureter repair range from 71 to 100% . Kroepfl and colleagues conducted a case series in which they used open surgical surgery with BMG to treat long-segment ureter strictures. The success rate of this treatment was 71.4%, with a median follow-up of 18 months (ranging from 10 to 85 months). A single patient experienced repeated narrowing of the ureter 39 months after surgery and received temporary implantation of a stent in the renal ureter as a treatment. Another patient experienced recurring narrowing of the ureter 17 months after surgery and was treated by the insertion of a long-term renal ureteral stent . In 2015, Zhao et al. introduced the concept of robot-assisted BGM ureteroplasty by demonstrating the approach in four patients with proximal ureteral strictures. The operation, intended for proximal ureter strictures with a median length of 4 cm (ranging from 1.5 to 6 cm), yielded positive results throughout the 15.5-month monitoring period. There were no instances of reoccurring conditions, and none of the patients required further surgical intervention. The BGM ureteroplasty procedure was performed with robot assistance, with the patient positioned on their side. The ureter was carefully identified and dissected during surgery. The use of fluoroscopy, ureteroscopy, intravenous injection, and near-infrared fluorescence imaging allowed accurate identification of the stricture either antegrade or retrograde ICG administration. A linear cut was made at the location of the narrowing in the urethra. The decision to use either buccal mucosa exteriorization or buccal mucosa-enhanced anastomosis was based on the scar’s length and severity, in addition to the support provided by the omentum or perinephric fat . In 2017, Lee et al. published the most extensive collection of cases documented thus far, which included 12 patients who had robot-assisted BGM ureteral repair. Indocyanine green (ICG) was administered in a backward and/or forward direction into the inner lining of the ureter, and near-infrared fluorescence (NIRF) was used to visually detect ICG to pinpoint the beginning and end points of narrowing in the ureter. The strictures had a median length of 3 cm, with a range of 2–5 cm. The larger omentum was used to enclose the rebuilt ureters in all instances. A success rate of 83.3% (10 out of 12 patients) was reported . In 2018, Zhao et al. conducted an inaugural multicenter study to evaluate the reproducibility of the approach. This study included 19 individuals from 3 distinct medical centers, with a median stricture length of 4 cm. The overall success rate reached a commendable 90% . Lee, Zhao, and colleagues evaluated three robotic procedures specifically developed for treating moderate to long-segment (≥ 4 cm) strictures in the proximal urethra. The techniques mentioned are robot-assisted procedures for connecting the urethra to the urethra via downward renal fixation, connecting the urethra to the renal pelvis via downward renal fixation, and using a buccal mucosal graft for urethroplasty with the assistance of a robot. Based on a 24-month median follow-up, their data showed that 92.9% (13 out of 14 patients) of those who had RU-BMG had a successful outcome, with an interquartile range of 14–39 [ , , ]. Ultimately, BMG urethroplasty emerges as a compelling option for treating strictures located in the proximal and mid-segments of the urethra. It can be regarded as a primary or secondary choice and may also demonstrate potential in the treatment of distal ureter strictures. However, additional multi-institutional research and longer term follow-up are necessary to confirm the complete array of benefits and drawbacks of these methods.
Intestinal anastomosis is a viable alternative for replacing the ureter in patients with long-lasting ureteral strictures where endoscopic therapy or retrograde ureteroscopy is not suitable. It can also be considered salvage therapy after failed previous treatments and may be used in individuals with recurring kidney stone formation. However, this approach is not recommended for individuals with inflammatory bowel disease, radiation enteritis, neurogenic bladder, bladder outlet obstruction, or liver or kidney dysfunction . In 1906, Shoemaker was the first to use the ileum as a replacement for the ureter. This method gained popularity when Goodwin popularized it in 1959 . Gill and colleagues performed the first laparoscopic surgery in the year 2000 using extracorporeal ileal anastomosis . Wagner and colleagues conducted the inaugural machine-assisted ileal anastomosis for ureteral reconstruction surgery in 2008 . In 2014, Brandao and colleagues reported the initial case of intracorporeal ureteral ileal replacement surgery. Regardless of the patient’s condition, the surgical procedure lasted for more than 6 h. However, the patients did not experience any significant issues, and there were no instances of the condition recurring . Kocot conducted a comprehensive case series study to evaluate the long-term outcomes of 157 patients who underwent ileal ureteral insertion surgery; this is the largest study of its kind reported in the literature. The results indicated that 19.5% of the patients suffered from metabolic acidosis, while 6.3% had recurrent pyelonephritis. A significant proportion of patients, specifically 93.6%, experienced a reduction or maintenance of blood creatinine levels. In summary, remedial surgery was required in only 4.2% of patients . The operation involved two main steps: excising the constricted portion of the ureter, followed by isolating a sufficiently long and well-supplied segment of the ileum. A stapler is used to restore the intestinal connection while placing the ileal ureter behind the peritoneum. Subsequent procedures include connecting the renal pelvis and the branch of the ileal bladder, as well as inserting a ureter stent to prevent leakage and maintain normal intestinal movement . In 2016, Chopra and colleagues published a study on a group of 4 patients, with an average surgical duration of 450 min (ranging from 420 to 540 min) . Stein et al. (2009) showed that laparoscopic ileal ureteral surgery has advantages over open surgery in terms of recovery time and length of hospital stay . In 2019, Grosso et al. performed a surgical procedure called robot-assisted ileal ureteral replacement. The median duration of the operation was 270 min, with a range of 240 to 300 min. No problems, according to the Clavien–Dindo classification, were reported . Soyster and colleagues conducted a retrospective study on a cohort of 160 patients who underwent ileal ureteral (IU) surgery. Researchers have shown that ileal ureteral repair is feasible, preserves renal function, and reduces long-term complications . However, additional multi-institutional investigations are essential. Robot-assisted techniques offer improved surgical visualization and accuracy but require time-consuming readjustment and repositioning. However, Ubrig et al. presented a robot-assisted method that allows the completion of the process without requiring the patient to be repositioned .
Robot-assisted autologous kidney transplantation (RATx) is a viable therapeutic option for disorders such as lengthy ureteral or panureteral strictures, retroperitoneal fibrosis, loin pain-hematuria syndrome, and renal vascular problems (aneurysms, thrombosis, stenosis, and vascular damage). Although this approach produces positive functional results, it is associated with a rather high morbidity rate of 46% . An autologous transplantation procedure was initially conducted in 1963 using an open surgical technique involving a midline incision from the xiphoid process to the pubic symphysis. This surgery was performed to address serious damage in the proximal ureter . Open autologous transplantation after laparoscopic kidney removal has been reported to be a viable and effective choice, with success rates varying from 68 to 90% . Robot-assisted autologous renal transplantation (R-RATx) has demonstrated efficacy and achieved favorable outcomes. Gordon et al. documented the first total intracorporeal R-RATx, which required a total surgical duration of 425 min. The duration of warm ischemia was 2.3 min, while the duration of cold ischemia was 95.5 min. After the robotic removal of the donor kidney and cold perfusion, the kidney was placed in the iliac fossa, and the robot was deactivated. Afterwards, the patient was placed in a steep Trendelenburg posture, and the robot was positioned between the legs. Subsequently, the anastomosis between the iliac artery and ureter was prepared. Venous anastomosis required 17.3 min, while arterial anastomosis took 21.3 min. The duration required for the kidney to warm up (from the moment when cold perfusion stopped to the point where the kidney was released and reperfused) was 28.8 min. The ureterovesical anastomosis was successfully performed in a duration of 26.6 min. The amount of blood loss recorded was 50 ml . Lee et al. made various alterations to R-RATx with the objective of diminishing kidney ischemia time. The modifications involved employing a Vicryl inner ring device to fasten the catheter and implementing cold HTK perfusion with saline. The surgical procedure lasted 390 min, during which warm ischemia time and cold ischemia time were measured at 4 and 48 min, respectively . Sood et al. conducted the initial instance of robot-assisted R-RATx , whereas Araki et al. carried out the first instance outside of the United States . Decaestecker et al. conducted the initial series investigation, comprising 7 patients, which involved the combination of hand-assisted and total intracorporeal approaches. The durations of surgery, warm ischemia, and cold ischemia were 370 min, 2 min, and 178 min, respectively . R-RATx is deemed viable for treating intricate ureteral strictures in the future . Nevertheless, it is crucial to regard this approach as a viable alternative solely because of the possession of exceptionally skilled robotic surgeons and subsequent completion of transplantation-specific training.
The robot-assisted repair of ureteral stenosis remains applicable for cases where stenosis recurs following endoscopic or surgical interventions. Redo repair after ureteral anastomotic stenosis may become complicated due to the risk of significant periureteral fibrosis, changes in the dissecting plane, and further impairment of the fragile ureteral blood supply. Historically, open surgery has been commonly utilized for such cases. Current research indicates that robot-assisted surgery for recurrent ureteral stenosis post-reconstruction is equally effective as open surgery. Furthermore, robot-assisted technology demonstrates a reduction in blood loss and surgical time, thereby offering the advantages of minimally invasive surgery . For the treatment of recurrent UPJO, options include pyeloplasty revision with a pelvis flap, cup incision of the ureter, and downward nephropexy (DN) or robotic-assisted ureteropelvic junction reconstruction (RU-BMG) . For the treatment of recurrent proximal or mid-ureteral strictures, options include ureteroureterostomy, with or without DN or RU-BMG. In patients with localized fibrosis around the ureter and recurrent strictures in the proximal and mid-ureter (≤ 2 cm), ureteroureterostomy may be performed if feasible; otherwise, RU-BMG with a high-implant or augmented anastomosis is likely in most other cases . For the treatment of recurrent distal ureteral strictures, we employ ureteroneocystostomy, ureteroureterostomy, ureteroureterostomy, or appendiceal interposition . Therefore, for patients with recurrent strictures following failed endoscopic or surgical treatments, robotic ureteral reconstruction (RUR) can be performed, with studies indicating favorable midterm outcomes . While outcomes of using robotic reconstruction for re-stenosed uretero-anastomotic are promising, there is a lack of long-term follow-up data. In addition, the possibility of recurrent strictures despite robotic-assisted treatment of uretero-anastomotic stricture presents a challenge that needs to be addressed.
The existing body of research on robot-assisted ureteral repair techniques is primarily limited by the prevalence of small-scale studies conducted within a single institution. These studies also lack well-defined criteria for evaluating the effectiveness of the procedures. However, robot-assisted ureteral repair appears to be a viable and efficient alternative for treating this disease, yielding significantly more positive results and fewer complications than open procedures.
Ureteral strictures present a significant challenge for surgeons, especially when they are of considerable length or unresponsive to endoscopic treatment and thus require surgical intervention. Robot-assisted surgery provides surgeons with several advantages, such as an extended visual range, enhanced accuracy, reduced invasiveness, lower complication rates, quicker recovery, favorable functional outcomes, and improved cosmetic results.
Robot-assisted approaches for repairing ureteral strictures have emerged as a viable option, yielding positive functional outcomes and demonstrating lower complication rates than conventional open surgeries. However, extensive and long-term research studies are needed. Given the infrequent occurrence of this complex condition, there is currently a scarcity of scientific information available.
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Finite element analysis of the influence of perioral force on alveolar ridge healing in areas missing maxillary anterior teeth | f8c8c0f2-9e2f-4af3-b15e-37b124cb10a1 | 11700444 | Dentistry[mh] | Implants have long been used for the restoration of missing teeth in the aesthetic zone because of their association with good aesthetic and functional outcomes. However, it is still difficult for doctors to compensate for the effect of alveolar ridge absorption despite extensive experience in implant placement and the use of technology. Most patients whose maxillary anterior teeth are missing have different degrees of alveolar bone loss, which seriously affects patient prognosis and red–white aesthetics. In most cases, the decrease in alveolar ridge height occurs within 90 days after tooth extraction. Six months after extraction, the horizontal and vertical bone loss rates ranged from 29 to 63% and from 11 to 22%, respectively, and the width of the alveolar bone decreased by approximately 50% in the first year after tooth extraction . The causes of alveolar bone resorption after tooth extraction include trauma, blood supply, bacteria, inflammation, age, adverse stress and systemic disease. The loss of bundle bone after tooth extraction is considered an unavoidable and important factor that leads to horizontal bone resorption. The loss of Sharpey fibers connecting the cementum and alveolar bone and the interruption of the blood supply to the periodontal ligament (PDL) after tooth extraction lead to the resorption of bundle bone . In addition, compared with thick-walled bone, thinner facial bone wall is associated with more severe bone plate resorption . Jiang et al. reported that a thin buccal bone plate entirely composed of fascicular bone does not disrupt resorption regardless of the treatment method used. Owing to the weak bony plate on the labial side of the anterior tooth root and the presence of several fasciculate bones, bone remodeling is more active, and bone resorption is more prominent than that on the palatal side. Compared with that before extraction, the position of the alveolar ridge of the anterior tooth after extraction is more palatine, resulting in a defect in the contour of the lip . Farmer et al. observed the range of bone resorption in the maxillary extraction socket. They reported that at 6–8 weeks after healing, the bone wall of the extraction socket was an “inverted V shape”, and 42% of the patients lost at least 4 mm of buccal bone at the midpoint of the extraction socket. MacBeth et al. reported that the equal area of alveolar bone on CBCT images decreased by 11% and that the rate of labial bone cracking was 85% at 4 months after anterior tooth extraction. As an alveolar augmentation technology with definite clinical efficacy and long-term predictability, guided bone regeneration (GBR) has been widely accepted as a means to treat hard tissue defects in the aesthetic zone . However, when used as an alveolar ridge preservation technology, GBR cannot completely prevent alveolar bone resorption, and the results at different sites receiving the same treatment differ . There have been no reports of the obvious effect of soft tissue pressure on anterior alveolar healing. Jiang et al. hypothesized that the collapse of labial soft tissue in areas with missing anterior teeth affects alveolar remodeling and demonstrated that the application of labial pressure-bearing devices results in better alveolar ridge preservation than does natural healing. They also reported that even the relatively rigid microtitanium scaffold became slightly deformed during the healing process and that the middle part of the scaffold shifted palatally. These findings suggest that pressure is exerted on the alveolar soft tissue during the healing process. Mir-Mari et al. used GBR technology to study the volume stability of the enhanced area during mucosal flap suturing and reported that the suturing of the mucosal flap caused considerable pressure in the coronal part of the enhanced site, resulting in the displacement of granular graft materials and partial collapse of the collagen membrane. All the above studies suggest that long-term pressure on soft tissue may affect the healing process of the alveolar ridge. At present, there is still an insufficient theoretical basis on whether excessive stress is generated on the alveolar ridge in the missing tooth area. On the basis of the above background, the aim of this biomechanics study was to determine the influence of upper lip pressure on the distribution of stress in the area of missing maxillary anterior teeth via FEA to ultimately determine whether upper lip pressure affects the healing process of the alveolar ridge.
Establishment of a 3D finite element model of the maxillary anterior tooth region (Fig. Material properties, mesh division, contact conditions, boundary conditions and loading conditions Mechanical analysis Observation indicators In this study, we observed the overall stress distribution and displacement changes of the model in the presence and absence of teeth. The absolute values of the von Mises stress distribution and maximum displacement in each direction of models A and B were calculated.
) The research participant was a healthy adult male who provided informed consent and underwent CBCT of his head in accordance with ethical review by the Ethics Committee of Jinan Stomatology Hospital. The CBCT data were imported into Mimics 21.0 (Materialise, Belgium), a medical image modeling software, in DICOM format for 3D reconstruction. The maxillary anterior teeth (including the anterior maxilla and maxillary anterior teeth) were extracted, and the generated 3D model was output in stereolithography (STL) format. Geomagic Wrap 2021 (Geomagic, USA) was used to optimize the 3D model. In accordance with previous studies , the maxillary bone was shifted 0.9 mm inward to obtain the cancellous bone model, and the bone cortex model was obtained via Boolean subtraction with the original maxillary model. The same method was used to obtain a 1.2 mm gingival model and six 0.2 mm PDL models . The solid model was combined via the engineering modeling software SOLIDWORKS 2021 (Dassault Systèmes, France). The various parts of the model are assembled into Model A (Fig. ). The left maxillary central incisor was removed from Model A to obtain Model B (Fig. ). The output entity model is in “x_t” format. Then imported into the FEA software ANSYS 17.0 (ANSYS, USA) for solution.
With reference to previous studies , the model properties are set as homogeneous, continuous and isotropic linear elastomers. The material properties are shown in Table . Automatically mesh the model in ANSYS 17.0. The numbers of nodes and units are shown in Tables and . The bonding contact between the gingiva, PDL, cancellous bone, cortical bone, and teeth was set in the software, and the contact position did not change. The boundary conditions for the connection between the model and the maxilla, namely, the upper and distal surfaces, were set. With reference to previous studies, the resting upper lip pressure of normal people was 2 g/cm 2 . For the convenience of the study, the perioral force on the anterior alveolar ridge and teeth was set as the ideal state of internal and external balance ; that is, the palatal pressure was equal to the labial pressure. The pressure was applied to the gingiva and tooth surface of models A and B, and the stress areas were the labial surface of the anterior maxillary tooth and gingiva and the palatal surface of the maxillary gingiva. The direction was perpendicular to the surface of the stressed tissue.
After the resting perioral force was loaded, the stress cloud map and displacement cloud map of the model were obtained. The stress distribution and displacement changes in the maxillary gingiva, cortical bone and teeth were observed. Our FEA results were represented visually as the stress distribution and displacement via an ANSYS 17.0 predefined progressive visual color scale (ANSYS, USA).
von Mises stress distribution results of the model after loading Changes in displacement in each model after loading The von Mises stress distributions of the two models after loading are as follows (Fig. A and B). In the labial view, the von Mises stress distribution of Model A was more uniform, and there was no obvious stress concentration area. There was also no obvious stress concentration area on the gingival surface of Model B. However, Model B presented maximum von Mises stress at the crest of the labial alveolar ridge in the missing tooth area, with stress gradually decreasing from the crest of the alveolar ridge to the root area, forming an inverted V-shaped distribution. The maximum von Mises stress at the crest of the labial alveolar ridge in the missing tooth area in Model B was approximately 1.4 times greater than the stress at the same observed site in Model A (Fig. A). In addition. In the palatal view, neither Model A nor Model B had significant areas of stress concentration.
Figure A and B show the change in displacement in each model after loading. Model A showed maximum displacement in the incisors and a gradual decrease to the distal and root sides. In Model A, the gingival displacement toward the palatal side was significant, and the trend also showed maximum displacement at the top of the gingiva, gradually decreasing toward the distal and root sides. The displacement of alveolar bone in Model A also tends to gradually decrease to the distal and root sides. In Model B, the displacement of the gingiva at most positions is similar to that in Model A. But the displacement at the top of the gingiva in the missing tooth area was approximately 1.3 times greater than that at the same observation site in Model A. The displacement of the alveolar bone surface in Model B was similar to that in Model A.
FEA is widely used in the field of oral biomechanics . In this study, FEA was adopted to explore clinical problems, and force analysis was conducted with finite element software; thus, there was no need for an operation or long-term follow-up. Models can be used repeatedly, and their construction is simple and noninvasive. In the anterior maxillary region, the loss of alveolar ridge width after missing teeth is the main factor restricting implant restoration. This study is based on the hypothesis that a lip shield can slow alveolar ridge absorption and that the inhibitory effect of perioral force on the growth and development of alveolar bone in the three-dimensional direction affects the practical demand for preservation of the alveolar ridge in the aesthetic zone. The aim of this study was to explore the biomechanical effects of perioral force on the alveolar ridge in the anterior maxillary region. Changes in von Mises stress and displacement in the maxillary anterior tooth area under perioral force Role of resistance to upper lip pressure in the preservation of the alveolar ridge in the anterior deficient area of maxillary teeth Prospects and limitations This study revealed the existence of undue stress on the alveolar ridge in the missing area of the maxillary anterior teeth, and additional studies on the treatment methods used to resist this undue stress will be conducted in the future. The concentration of stress in the alveolar bone in the missing tooth area was analyzed only from the perspective of biomechanics, without considering other influencing factors, and the extent of the influence of adverse stress on alveolar bone reconstruction was not clear. FEA requires ideal research conditions. In this study, situations related to missing teeth were discussed. But factors such as the different material properties of different bones, the varying thickness of the labial bone plate, individual differences in the magnitude and direction of perioral forces, and the influence of dynamic forces under functional conditions have not been explicitly discussed. In future research, if one or several of the above factors can be discussed in depth, it will be highly beneficial for improving the research results.
Compared with Model A with complete dentition, Model B with missing teeth presented a stress concentration zone, and the stress was concentrated mainly at the crest of the labial alveolar ridge in the missing tooth area; that is, the soft tissue of the lips exerted adverse stress on the alveolar ridge in the missing tooth area of the maxillary anterior teeth. The gradual reduction in stress from the incisal end to the root is consistent with the “inverted V-shaped” absorption of the bone wall of the extraction socket observed by Farmer et al. . The changes in von Mises stress were mainly reflected in the sagittal direction, suggesting that the stress on the alveolar ridge caused by perioral force was mainly exerted in the sagittal direction, which may have resulted in a reduction in the width of the alveolar ridge. This finding was also confirmed by a recent study. Pelegrine et al. reported that the bone width in the anterior maxillary area was reduced by 31.35 ± 11.88% within 6 months after healing was completed without transplant surgery. The displacement changes are reflected mainly in the labial gingiva at the missing tooth area, indicating deformation of the gingival soft tissue (Fig. C). However, stress concentration still exists on the alveolar ridge in the missing tooth area (Fig. A), indicating that the gingiva cannot fully cushion against the adverse stress formed by the upper lip at that location. Importantly, the stress on the adjacent labial alveolar ridge in the missing tooth area of Model B was greatly reduced (Fig. A). We speculate that the stress on the adjacent alveolar ridge is partially borne by the alveolar ridge in the missing tooth area. That is, a portion of the adverse stress at the alveolar ridge in the missing tooth area comes from the adjacent alveolar ridge. The alveolar bone morphology of the maxillary anterior tooth region varies greatly across different regions and races. In the model built in this study, the buccal bone cortex of the maxillary anterior teeth was intact, but many patients had thin buccal bone walls or even different degrees of absence due to bone loss caused by congenital thin bone, tooth extraction trauma or original periodontal inflammation. Gakonyo et al. reported that 26% of buccal bone walls were missing among 1104 teeth, meaning that approximately 1 in every 4 anterior maxillary teeth were missing buccal walls. The thinner the buccal bone wall is, the more likely the alveolar ridge and soft tissue are to undergo more prominent changes in size. Existing evidence suggests that this more prominent change in size is the result of a series of changes, such as osteoblast death and osteoclast activity , caused by interruption of the blood supply. Since the resting pressure of the upper lip can cause slight deformation of the alveolar ridge with normal bone thickness, when the buccal bone wall is missing or thin, the alveolar ridge in the missing tooth area will inevitably have difficulty maintaining spatial stability because of the persistent adverse stress of the upper lip, which ultimately affects the healing of the alveolar bone. In people with different occlusal relationships, the magnitude of perioral force varies greatly. Studies have shown that the upper lip pressure of patients with Class II malocclusion is greater than that of Class I malocclusion patients . The upper lip pressure of patients with Class III malocclusion is the lowest. The position of the anterior teeth determines the upper lip pressure at rest. The lower upper lip pressure in patients with Class III malocclusion may be caused by the spatial relationship of the jaws . Meanwhile, studies have shown that patients with Class III malocclusion have lower tongue positions . This study adopted the ideal situation of internal and external muscle strength balance. In patients with different types of malocclusion, the stress on the alveolar ridge in the missing tooth area may change due to an imbalance in lip and tongue muscle strength.
The PASS principle of GBR emphasizes the importance of space maintenance and blood clot stabilization. When GBR is performed with membrane materials, the soft tissue pressure from above the barrier membrane can collapse the barrier membrane, resulting in reduced areas of new bone . The titanium mesh technique and the tent technique achieve better bone augmentation effects than the barrier membrane alone by preserving the space for new bone formation and stabilizing the bone graft material, autogenous bone particles and blood clots below it. The results of this study suggest that some means to counteract adverse stress on the alveolar ridge in the missing tooth area are conducive to providing a stable physical environment for the healing of the alveolar socket, which has been confirmed in clinical studies. Jiang et al. reported that both intra-alveolar transplantation and microtitanium scaffolds maintain space for new bone formation, which can be achieved by “supporting” the soft tissue of the lip externally or “occupying” the space internally with bone graft materials. Some studies also suggest that if the bone regeneration space can be maintained appropriately, ideal new bone formation may be achieved without bone graft materials. These findings suggest that new bone formation space may be a key factor in alveolar bone regeneration or hard tissue preservation in the fossa at the site of tooth extraction. In addition, the deformation of rigid materials such as titanium mesh or microtitanium scaffolds also indicates that stress on the alveolar ridge in the missing tooth area has adverse effects on the alveolar ridge.
In the resting state, the labial soft tissue exerts adverse stress on the alveolar ridge in the area of missing maxillary anterior teeth. The stress concentrated at the top of the alveolar ridge and gradually decreased toward the root. This stress may affect alveolar bone healing, causing a reduction in alveolar bone width. The measures taken to resist the pressure of the upper lip may have a positive effect on slowing alveolar ridge absorption in the anterior maxillary region.
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A patient-centred care model for patients with complicated multimorbidity: Protocol for a pilot cluster randomised trial in general practice, municipalities, and hospitals | 715b48ec-e018-4ba8-bb06-c0833e9c7fe6 | 11616888 | Patient-Centered Care[mh] | The increasing prevalence of multimorbidity–driven by the ageing population, and advanced diagnostic health technologies—represents a major challenge for the healthcare system and society [ – ]. Multimorbidity is commonly defined as the coexistence of two or more chronic conditions in the same person . In the Capital Region of Denmark and Region Zealand, approximately 21% and 37% of the citizens aged 16 years and older suffer from multimorbidity . Around 10% of people in Denmark with multimorbidity have complicated multimorbidity characterized by a severe symptom complex caused by more concomitant chronic conditions . Other researchers have proposed different definitions of complicated multimorbidity such as the severity of conditions and perception of illness . Patients with complicated multimorbidity often experience reduced health-related quality of life [ – ], high treatment burden, polypharmacy, reduced ability to work, and increased mortality . In this study, we define multimorbidity as complicated when the patient has two or more of three chronic conditions (diabetes, chronic obstructive pulmonary disease, or chronic heart conditions) , has been hospitalised, or visited an outpatient clinic due to their chronic condition(s) during the previous year , and take at least five different prescription drugs assessed from the Shared Medicine Record (SMR) in general practice . Care pathways for patients with complicated multimorbidity are often complex with multiple appointments, frequent ambulatory visits, hospital admission, and use of other healthcare services. The siloed organization of healthcare around single diseases, the lack of guidelines and incompatible IT systems can be a barrier to effective care for patients with complicated multimorbidity. . While the above challenges described are widely recognized and some consensus exists regarding key components for enhancing care , knowledge of the most effective organizational structure for healthcare services to deliver patient-centred, high-quality integrated care for patients with complicated multimorbidity remains limited . In Denmark, general practice is the key organisational setting in terms of offering people with complicated multimorbidity integrated, patient-centred care. To improve care for patients with complicated multimorbidity in general practice, we developed a complex intervention care model, “A patient-centred complex intervention for multimorbidity” (CIM) . The model was developed based on the Chronic Care Model , models of care for multimorbidity [ , , ] and results from our studies in multimorbidity [ , , ]. Based on the results from the feasibility study, we developed an improved version of the CIM model named “A patient-centred complex intervention for multimorbidity version 2” (CIM2) . The new CIM2 model includes improved training of healthcare professionals, strengthened identification of patients with complicated multimorbidity, adjustment of the extended consultation according to the consultation model of The Danish College of General Practitioners (DSAM), improved medical treatment, and strengthened integration of care services between healthcare organisations . The development of the CIM2 model relies on the Medical Research Council (MRC) framework for complex interventions . The MRC framework consists of four phases: 1) development or identification of an intervention, 2) assessment of the feasibility of the intervention and evaluation design, 3) evaluation of the interventions, and 4) impactful implementation. The development of the extended overview consultation in the feasibility study of CIM model caught the attention of the Danish Regions Organisation and The Danish Organization of General Practitioners. The collective agreement accepted in 2022 for general practice introduced a fee-for-service covering an extended overview consultation for people with complex multimorbidity . The CIM2 pilot study is expected to improve and strengthen the CIM2 model, and the results will be used to inform further interventions. Objective to determine the effectiveness of a general practice-based intervention aimed at improving treatment and care for patients with complicated multimorbidity.
Study design Study setting Sample size Randomization and blinding Eligibility criteria Control general practices Intervention general practices Ethics This study is a 1:1 pilot cluster RCT ( ). The study will include 14 general practices, of which seven will be randomized to the intervention group and seven to the control group, providing usual care. Each practice will recruit 25 patients, amounting to 350 patients in total. At the time of recruitment, and respectively at 6 and 12 months of the intervention period, the patient’s assessment of perceived patient-centred integrated care, health-related quality of life, and treatment burden will be measured in both groups. The patients in the intervention group will receive an extended overview consultation after recruitment and again after 12 months.
The study will take place in Region Zealand and the Capital Region of Denmark, in general practices, healthcare centres in municipalities, and hospital outpatient clinics during the 3 rd quarter of 2022 through the 4 th quarter of 2024. The general practices should have a minimum of 4,500 patients registered to ensure that the number of patients with complicated multimorbidity reaches the needed number of 25 patients per practice. The municipalities will be selected based on the rank of sociodemographic groups I-IV. Groups III and IV, the lowest sociodemographic groups, will be chosen as the prevalence of patients with complicated multimorbidity is expected to be higher . The possible benefits of the CIM2 model are expected to be larger in patients from municipalities characterized as belonging to lower sociodemographic groups.
The primary outcome measure of the study is the Patients Assessment Chronic Illness Care (PACIC) questionnaire . The difference of interest between the intervention and the control group is Δ = 0.36, with a mean value of 2.86, and with an expected standard deviation SD = 1, based on results from earlier studies . The power calculations take a potential Intraclass Correlation Coefficient (ICC) of 0.1 into account. With patients being allocated to clinics with 25 patients per clinic, and a random loss to follow up on 30% , we base ourselves on the following random effects model for those not lost to follow up: Y i = α G r o u p ( i ) + Z C l i n i c ( i ) + ε i , i = 1,2 … , n (1) so that the outcome Y consists of a group-specific level α (intervention/control), a clinic-specific random effect Z , allowing for intra-clinic correlation, and an individual noise term ε . Simulating the above model for 10,000 times per choice of the number of clinics in each group, with individually evaluated random loss to follow up, yields a power of 78%, and 84% for 6 and 7 clinics in each group, respectively, based on a two-sided test at the level α = 0.05. The uncertainty of these powers is found to be less the 1 percentage point, using the binomial formula. Thus, to obtain a power of 80% for detecting the specified difference Δ from the random effects modelling, 7 clinics of 25 patients, i.e., 175 patients are needed in the intervention and control group. As a consequence of an anticipated attrition rate (or drop-out rate) of approximately 30% due to mortality or other unforeseen circumstances, we plan to include 183 patients in each group.
General practices in the designated municipalities are contacted with an invitation to participate in the study. If they accept the invitation, they will receive detailed study information. The 14 participating general practices will be randomised into either the intervention group providing care as described in the CIM2 model or the control group, providing usual care. The general practices will be randomly allocated, at an allocation rate of 1:1, by a computer program. To ensure the concealment of allocation, a data manager from another organisation will provide the information on the randomization to the general practice and will be responsible for a randomization list, which will be available to the investigator. Due to the nature of the study, the general practices and the patients cannot be blinded.
Healthcare professionals in general practice included in the study identify eligible patients. We use the following inclusion criteria: The patient has two or more of three common chronic conditions (diabetes, chronic obstructive pulmonary disease, chronic heart condition) . The patient has been hospitalised or visited an outpatient clinic due to their chronic conditions during the previous year . The patient takes at least five different prescription drugs assessed from SMR in the general practice . Patients accepting to become part of the study get an invitation with a link to the project database in the Research Electronic Data Capture (REDCap) , a web-based application developed to capture data for clinical research. In the invitation from REDCap patients receive information about the project and are asked to 1) provide informed consent to participate in the study and 2) complete the baseline questionnaires. Patients will receive up to 2 reminders if they do not fill in the questionnaires. The patients will receive the questionnaires again at 6 and 12 months after inclusion and again up to 2 reminders for non-responders. Patients, who are unable to speak Danish, have a life expectancy of less than 12 months, or cannot give their informed consent, for example people with dementia, will be excluded from the study.
Healthcare professionals working in general practices allocated to the control group will receive the same information about the project concerning the recruitment of patients, collection of patient data including informed consent and patient questionnaires and the use of REDCap. Patients will receive usual care according to the Danish Health Authority’s National Clinical Guideline. Any changes in the intervention period affecting the usual care or activities for the control group will be recorded.
The elements of the CIM2 model are described in the following and is illustrated in . The teaching program will be developed in collaboration with The Danish College of General Practitioners. Healthcare professionals from general practice, nurses and physiotherapists from the municipalities, and healthcare professionals from the outpatient clinics participate in the training program. It will include topics on multimorbidity, project content, and methods to recruit and include patients (including informed consent). Further, the training program will offer training in the collection of patient data comprising the use of REDCap software for patient questionnaires. The patient intervention in general practice starts with an extended consultation, lasting 45 minutes, with the GP, the patient and possibly a relative, and the nurse care coordinator. The consultation is based on the guidance for a patient-centred overview status for patients with multimorbidity published by The Danish College of General Practitioners . The aim is to obtain an overview of the patient’s conditions, problems, needs, and potential beneficial changes in treatment. The patient’s goals, preferences, and needs are identified, and the medical treatment of the patient’s conditions is prioritized. An individual care plan is developed, covering activities in the three sectors that will take place within the 12-month intervention period. The individual care plans include 6 themes : 1) Listing of important diagnoses, 2) Overview of the patient’s prescription drugs, 3) Prioritising the patients’ treatment goals (using shared decision-making), 4) Development of a coordinated individual care plan with telephone follow-up and future visits, 5) To lower burden of treatment, the GP will identify outpatient visits at hospitals which could be reduced or replaced by a GP visit if the patient and the specialist agree, and 6) Referral to community-based rehabilitation programs if there is clinical indication and the patient is motivated . The individual care plan is printed for the patient and can be shared with the municipality and outpatient clinic based on an IT standard (MedCom). The GPs is reimbursed with 137 US dollars for the extended overview consultations. General practice coordinates the planned patient care between general practice, the municipality, and the hospital, and follow-up on the execution of planned healthcare activities. The care coordinator function might be undertaken by the GP or the nurse in the practice. The practice plans the division of responsibilities and tasks in the project between the GP and the nurse. The follow-up activities include telephone calls to the patient by the care coordinator at relevant time intervals according to the severity of conditions and planned activities. A second extended overview consultation takes place after 12 months. As mentioned earlier, the individual care plan is shared electronically with the healthcare centre in the municipality and with the outpatient clinics using the national standard IT communication tool MedCom.
The study was notified to the Danish Data Protection Agency (protocol: REG-161-2020) and the National Committee on Health Research Ethics in Region Zealand (protocol no.: EMN-2020-37129). The study is registered at ClinicalTrials.gov, identifier: NCT05406193. The study will be conducted in line with ethical principles for medical research as described in the Declaration of Helsinki . Personal identification is encrypted, and data will be kept in accordance with the requirements of the Danish Data Protection Agency. All results will be reported in the anonymity of respondents and participating general practices. Informed consent will be obtained from all participants before entering the study. Before signing the consent forms, participants will be informed that participation is voluntary and that they can withdraw anytime.
Primary outcome is the quality of care assessed by the PACIC questionnaire. The twenty-item PACIC questionnaire with five dimensions considers whether patients receive patient-centred care measuring aspects of care most important to patients; patient activation, organisation of treatment/decision-support, goal setting/individual adaptation, problem-solving/context, and follow-up/coordination. The PACIC questionnaire has been validated and translated into Danish [ – ]. Secondary outcomes include the patients’ health-related quality of life, measured by the EuroQol-5 Domain questionnaire (EQ-5D-5L). EQ-5D-5L is a generic instrument comprised of five questions covering mobility, self-care, everyday activities, pain, discomfort, anxiety, and depression, each item on a five-point scale from no problems to extreme problems. The EQ-5D-5L has been validated and translated into Danish . The treatment burden is measured by the Multimorbidity Treatment Burden Questionnaire (MTBQ), which has been validated and translated into Danish . The utilization of health care services (number of admissions), bed days, visits to emergency departments, outpatient visits, GP visits, out-of-hour GP visits, yearly control visits in general practice and specialist visits are assessed from national register data . The cost-effectiveness and Incremental Cost Effectiveness Ratio (ICER) of the CIM2 will be calculated based on Diagnose Related Group rates .
The qualitative evaluation consists of semi-structured interviews with health professionals representing all three sectors, as well as patients. Semi-structured interviews will be conducted with: General practitioners and nurses from the seven intervention practices. Health professionals from municipalities and hospitals involved in cross-sectoral collaboration. patients with a cross-sectorial course of treatment. The semi-structured interviews with health professionals will be guided by an interview guide, which covers the following subjects: the content and relevance of the extended consultation, patient-centeredness of the care pathway, the individual care plan, referral to rehabilitation, and the content of the rehabilitation program in the community. Furthermore, semi-structured interviews with patients will explore how patients experience the extended overview consultations. The interviews will assess the interviewees’ experiences with the model, their assessment of potentials and challenges applying the model and their potential input for improving the model. One focus will be the sharing of the individual care plan and communication with patients. Interviews will be conducted once participants are familiarised with the model. All interviews will be audio-recorded, transcribed and analysed using Nvivo software. Semi-structured interviews will be complemented with cross-sectorial focus group interviews with patients in the intervention group to assess how the CIM2, and the cross-sectorial elements associated with it, are experienced by the patients in the study. Additionally, focus group interviews will be conducted with healthcare professionals from the three sectors to gather their perspectives on the CIM2. The interviews are supported by an interview guide, which covers the following topics: Teaching program for healthcare professionals: Assessing the effectiveness and comprehensiveness of the training provided to healthcare professionals prior to implementing the CIM2. Structure of Extended Consultations: Evaluating the structure and organization of extended consultations in general practice settings, including their duration, format, and participant roles. Integration of Care: Exploring the extent to which the CIM2 facilitates and promotes integrated care, examining collaboration and communication among healthcare providers from different sectors. Information Sharing: Assessing the effectiveness of information sharing mechanisms between the three sectors, identifying potential barriers and facilitators to seamless information exchange. Referral to Rehabilitation: Evaluating the process of identifying patients who require rehabilitation services and the appropriateness of referral criteria. Content of Community Rehabilitation Programs: Assessing the relevance and effectiveness of community-based rehabilitation programs, ensuring alignment with patient needs and goals.
As the content of the intervention does not increase risks of adverse effects on the participants, a data monitoring committee is not needed nor is a plan for stopping the intervention in case of adverse events. Data management All data will be secured in a closed folder on the Region Zealand server with personal login access authorized by the primary investigator. The primary investigator has access to the full data set and the conversion key. Co-investigators will be given access when needed. The quantitative data is secured in the project database in REDCap during the intervention . A data management agreement and a collaboration contract have been mutually accepted by all involved parties.
Descriptive data are reported as mean (SD) or median (Interquartile range) for continuous variables depending on the distribution, and as numbers and percentages for categorical variables. Descriptive statistics for demographic data will be used to describe the population in the intervention and the control groups, as well as for the populations in each of the general practices. The primary analysis will investigate the effectiveness of the CIM2 model from the PACIC scores in the two groups. To our knowledge, no minimal important difference, which provides a ‘measure of the smallest change in the patient-reported outcome of interest that patients perceive as important, either beneficial or harmful, and that would lead the patient or clinician to consider a change in management’ , has been defined for the PACIC score. We expect a relevant change in the PACIC score to be 0.36 . The analysis will be based on an intention-to-treat focus, including all participants regardless of study adherence and drop-out. To create a dataset with maximum information, missing values in the PACIC scheme will be imputed using multiple imputations if the study subject has answered at least 50% of the PACIC scheme. A linear mixed model, see Eq ( ), will be used for the analysis as this model can handle the clustered observations within general practice. The variance structure will be chosen as the most suitable structure concerning the data assessed. If there is a difference between the groups at the time of randomisation, a sensitivity analysis including factors that vary between the two groups will be conducted. A comparative analysis without multiple imputations will be performed as another element of the sensitivity analysis. In addition to the intention-to-treat analysis, we will also perform a per-protocol analysis. To handle possible issues with clustered observations, the project will work with differences in the PACIC score post and before intervention. It is presumed however that this effect will be small, 0,01 to negligible in the survey population. The secondary analyses will compare scores of the EQ-5D-5L and MTBQ in the intervention group and the control group using the same methods as described. Cost-effectiveness will be assessed by the estimation of an ICER. The ICER defines the price for an increase in quality of life (QALY) when investing in CIM2 . The cost will constitute training costs, cost for the extended visits as well as the costs associated with all health care utilization in both primary and secondary care. The latter will reveal any potential effect of CIM2 on the use of health care in other sectors. QALY will be based on EQ-5D-5L measures. The time horizon will be the twelve months that the intervention lasts–hence no time discounting will be needed. The perspective will be that of the healthcare sector and the sensitivity of the ICER will be based on 95% confidence intervals estimated by probabilistic sensitivity analyses and illustrated by Cost-Effectiveness Acceptability Curves (CEACs) . The qualitative interviews with patients will be analysed thematically with a focus on themes related to the outcomes of the intervention, i.e., health-related quality of life and everyday experiences of living with multimorbidity. The analysis will include a focus on patients’ experiences with the new consultation in general practice and will focus on the patient’s experiences of integrated care. Thematic analysis of qualitative interviews with health professionals in all three sectors will identify 1) the acceptability of the intervention and 2) their experiences of possibilities and hindrances in cross-sectorial collaboration. The analyses will add to the quantitative analyses and contribute to an integral assessment of the CIM2 models’ success in providing improved integrated care to patients with multimorbidity.
This paper describes the protocol for a pilot cluster randomised controlled trial designed to evaluate the integrated care intervention, with a focus on general practice, aimed at improving care for patients with complicated multimorbidity. The CIM2 study will contribute novel and valuable insights into multicomponent interventions targeting an expanding population. Earlier studies have shown that GPs provided slightly fewer chronic care services than expected in practices where many patients with multimorbidity and low socioeconomic status were clustered, suggesting the inverse care law mechanisms . Furthermore, providing care to patients with complicated multimorbidity in general practice in geographical areas with higher rates of patients with multimorbidity seems to increase the risk of burnout among GPs . If the CIM2 model was integrated as part of usual care, it could contribute to all patients with multimorbidity were offered and received all the chronic care services they could benefit from. Furthermore, an implementation of CIM2 could also benefit general practitioners by possibly reducing the risk of burnout . A Danish study showed that a high crude rate of patient with multimorbidity increase GP’s likelihood of burnout. The study concluded that general practices with a high number of patients with multimorbidity need support to prevent suboptimal care and GP burnout . By implementing an extended consultation overview for patients with multimorbidity it is possible to compensate GPs economically in the existing combined fee-for-service and capitation reward system and supposedly decrease the stress levels GPs often experience caring for patients with complex care needs .
Studies on multimorbidity define multimorbidity as very heterogeneous and rarely include the severity of the conditions [ , , , ]. One study reported that a majority of authors using "multimorbidity" fail to provide a clear definition in their publications . This makes it difficult to compare different multimorbidity studies. As mentioned earlier in this paper, we defined multimorbidity as the coexistence of two or more chronic conditions in the same person, which is one of the more common definitions . We anticipate that this approach should facilitate comparison with previous studies. Firstly, the study design, being a pilot cluster RCT, may have inherent limitations in terms of generalizability to broader populations. The inclusion of specific geographical regions and general practices may introduce selections bias and limit the extrapolation of findings to different healthcare settings. Additionally, the use of specific chronic conditions (diabetes, chronic obstructive pulmonary disease, chronic heart condition) as criteria for complicated multimorbidity might not cover the full spectrum of conditions affecting patients, possibly influencing the generalizability of our results. Furthermore, the nature of the intervention, although evidence-based and informed by prior feasibility studies, may present challenges in implementation across diverse healthcare context. Factors such as variations in healthcare infrastructure, healthcare professionals, and patient populations could impact the effectiveness of the CIM2 model in different settings. From a clinical perspective, the PACIC questionnaire, though validated, might not fully capture the complexity of patient experiences in the context of complicated multimorbidity. We use a qualitative evaluation, such as in-depth interviews, to gain a deeper understanding of patient experiences.
A recurring challenge when conducting pilot cluster RCT studies is recruitment. There is a risk of bias during both the recruitment of general practices and patients. General practices with a high patient burden may be less likely to participate. To minimize the differences in patient burden between the general practices, we will solely recruit from municipalities with similarly low socioeconomic status. Patients recruited by their GP be more likely to participate when asked by a familiar healthcare professional whom they trust. However, there might still be a potential bias since the most vulnerable and sick patients will be less likely to participate in the study.
The scientific dissemination will consist of publication in peer-reviewed scientific journals and presented at national and international conferences. The results from the study will be presented at a seminar, inviting patients, relatives, and professionals from all three sectors included in the study. Short reports on recommendations for organisational cross-sectoral and cross-disciplinary collaboration on complicated multimorbidity will be made to the ensure dissemination of key findings and implications for practice to relevant stakeholders at the regional and national levels.
A successful pilot cluster RCT will yield valuable insights into recruitment strategies, how to get CIM2 implemented in general practice, municipalities, and out-patient clinics, how to improve the effectiveness and quality of the CIM2, sample size estimates for future studies and cross-sectorial collaboration. The qualitative findings will shed light on the experiences of patients and healthcare professionals with the CIM2, potentially identifying areas for improvement and informing its application in future studiProtocol (PDF)
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Incorporating Risk Stratification Into the Practice of Pediatric Preventive Cardiology | 6ea19fa0-bf9e-4ce8-96ff-d9179f256989 | 7358764 | Preventive Medicine[mh] | CVRFs, including obesity, dyslipidemia, hypertension, and at-risk behaviours including eating habits, physical inactivity, screen time, and smoking and electronic cigarette use are common in youth . Autopsy studies have demonstrated that the presence and intensity of such CVRFs are associated with atherosclerotic development and burden in childhood and young adulthood. , Moreover, modifiable CVRFs—such as obesity, dyslipidemia, and hypertension— track from childhood to adulthood; for example, an overweight teen has a 75% likelihood of becoming an obese adult. Thus, the timely management of CVRFs in youth has the potential to reduce or delay atherosclerotic cardiovascular disease in adulthood, particularly for youth with conditions predisposing them to increased CV risk. Prospectively evaluating the impact of management of CVRF in youth on the occurrence of manifest atherosclerotic cardiovascular disease in adulthood in a randomized controlled manner would be logistically very difficult and likely unethical, given the weight of evidence to date. On this basis, the United States Preventive Services Task Force (USPSTF) has indicated that insufficient evidence exists to recommend in favour of or against screening of lipid disorders and blood pressure in youth. , Despite this, as outlined here, indirect evidence suggests that the early and effective management of CVRFs can result in improvements in manifest cardiovascular disease in adulthood and noninvasive measures of atherosclerotic burden and vascular dysfunction in youth. For example, early initiation of statins in youth with FH recently has been shown to result in significant reductions in cardiovascular disease in adulthood compared with their FH-affected parents who had not started statin therapy until adulthood. Obesity is a particularly important CVRF to consider in childhood. Along with insulin resistance, it often provides the metabolic milieu for the development of a specific form of CVRF clustering known as cardiometabolic syndrome. , Although cardiometabolic syndrome is difficult to diagnose in childhood—owing, in part, to natural age-related fluctuations in body habitus and lipid and blood pressure normative values —the presence of CVRF clustering in youth is associated with endothelial dysfunction and is predictive of future cardiovascular disease in adulthood. Moreover, landmark autopsy studies have demonstrated that increasing CVRF clustering is associated with increased atherosclerotic burden in children and young adults. , As obesity tracks relatively strongly from childhood to adulthood, , the risk of CVRF clustering increases over time. Unfortunately, long-term effective first-line therapies for managing pediatric obesity remain elusive, as are strategies for sustained improvements in nutritional quality and physical activity. The first-line strategy in obesity management remains the use of multidisciplinary subspecialty obesity programs (when available). Pediatric dyslipidemia and elevations in lipoprotein(a) have gained increasing attention in recent decades. A clear correlation exists between the presence and severity of dyslipidemia in childhood and atherosclerotic burden at autopsy. , Pediatric dyslipidemias are also associated with vascular dysfunction and increased markers of early atherosclerosis from noninvasive assessment. , , FH, the most common inherited lipid disorder, is present in its heterozygous form in approximately 1:250 people, with an increased prevalence in specific populations, including French Canadians. It is characterized by lifelong marked elevations in low-density lipoprotein cholesterol (LDL-C) from birth, resulting in accelerated atherosclerosis and premature cardiovascular disease. Etiologic mutations typically involve the LDL receptor, apolipoprotein B, or proprotein convertase subtulisin/kexin type 9 (PCSK9). As heterozygous FH is clinically silent in youth, diagnostic criteria are needed to facilitate diagnosis, as indicated in a recently published Canadian definition. In the pre-statin era, the median age of first myocardial infarction was significantly earlier in the FH population than the general population: approximately 50 years in men and 60 years in women, , , with an estimated LDL-C burden in an untreated 35-year-old with FH equivalent to that of a typical 55-year-old. , FH serves as a model for not only the impact of severe elevations of isolated CVRFs but also for the impact of CVRF clustering on youth already at increased risk. For example, a recent systematic review and meta-analysis identified the presence of CVRFs—such as hypertension, diabetes mellitus, elevated lipoprotein(a), and smoking—as strongly associated with an increased risk of cardiovascular disease events in patients with FH. A large pediatric FH study showed that not only were more extreme levels of LDL-C in the affected child associated with more cardiovascular disease events in the family but that risk of events was further increased if the affected child also had low high-density lipoprotein cholesterol (HDL-C) or high lipoprotein(a). To this end, risk scores to predict cardiovascular disease events in the FH population have been developed, as traditional risk stratifying tools—such as the Framingham Risk Score—are not applicable to the FH population. , , Fortunately, a number of clinical trials have repeatedly shown that early use of statins in the pediatric FH population can slow or even reverse atherosclerotic progression , , , and may effectively normalize cardiovascular risk in this high-risk population. , Hypertension is another established risk factor for accelerated atherosclerosis. Pediatric hypertension can either be primary (essential) or secondary to an underlying at-risk disorder. Hypertension in youth is associated with evidence of cardiac (increased left-ventricular mass, systolic and diastolic dysfunction) and vascular (endothelial dysfunction, increased vascular stiffness, and increased carotid intima-media thickness) target organ damage that, in turn, is associated with progression to manifest cardiovascular disease in adulthood. , Moreover, blood pressure abnormalities track from childhood to adulthood, and blood pressure trajectories in childhood are associated with hypertension in adulthood. Fortunately, antihypertensive therapy can improve the left-ventricular hypertrophy (LVH) noted in pediatric , and adult , , , , persons with hypertension, and these improvements in LVH are independently associated with improved CV outcomes. Pediatric medical conditions identified as increased risk are categorized by the underlying disease profile and the degree of associated risk in . To demonstrate an approach to evaluation in clinical settings like these in childhood, the factors that confer added risk in 2 conditions—a medical diagnosis (diabetes mellitus) and a coronary artery diagnosis (Kawasaki disease)—are described. In addition to these established diagnoses, which are extensively reviewed in the AHA scientific statement referenced here, there are other recognized conditions in which evidence of associated accelerated atherosclerosis in childhood or very early adult life is emerging or under-recognized. One of these is the association between psychiatric disorders—specifically, major depressive disorders and bipolar disorder—and increased pediatric cardiovascular risk. Another risk condition is cystic fibrosis, and the emerging evidence for accelerated atherosclerosis in that population is described as an introduction to integrating CV risk assessment into management of these complex young patients. Clinicians and researchers must consider the long-term cardiovascular risk implications of various pediatric chronic diseases as their treatments and long-term prognoses continue to improve. This is particularly important and relevant for youth who have developed the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-associated pediatric inflammatory multisystem syndrome. The long-term vascular implications and similarities to Kawasaki disease, discussed here, remain to be determined. High-risk medical diagnosis: diabetes mellitus type 1 and type 2High-risk coronary artery condition: Kawasaki diseaseAt-risk condition,emerging evidence: cystic fibrosisype 1 diabetes (T1DM) is a condition of absolute, lifelong insulin deficiency caused by T-cell–mediated autoimmune destruction of pancreatic β-cells. This predominant form of diabetes mellites diagnosed in childhood and early adolescence leads to chronic, recurrent hyperglycemia, even with insulin-replacement therapy. Overall, the age-adjusted relative risk for atherosclerotic cardiovascular disease in T1DM is 10 times that of the general population. Subclinical vascular abnormalities are present in youths with T1DM, even in the first decade after diagnosis. When T1DM is diagnosed in childhood, cardiovascular disease is the leading cause of death beginning at age 20, at rates > 3% per year. Cardiovascular disease in patients with T1DM is the result of an accelerated atherosclerotic process. Hyperglycemia is the primary mediator and intensive glycemic control reduces cardiovascular disease events in adults and improves vascular abnormalities in adolescents. , Nephropathy complicates 30% of T1DM cases and accelerates atherosclerosis through chronic inflammation and uremia-related factors. Traditional CVRFs are prevalent with elevated total and LDL-C levels inversely correlated with glycemic control and significantly elevated apolipoprotein B and small, dense LDL particle levels, regardless of glycemic control. Hypertension is also common, found in 6% of T1DM youth. Obesity is increasingly prevalent, present in ∼40% of children with T1DM. Risk clustering mediates a profoundly atherogenic state and 14% to 45% of children with T1DM have 2 or more major CVRFs. By contrast with T1DM, type 2 diabetes mellitus (T2DM) is a condition of insulin resistance secondary to excessive weight gain as visceral fat, with the resultant inability of skeletal muscle, liver, and fat to respond to normal insulin levels. T2DM is increasingly diagnosed in youth, in parallel with rising obesity rates: > 85% of adolescents with T2DM are obese. In Canada, incidence of T2DM in youth ranges from 1.54 to 12.5 per 100,000 per year, compared with a mean of 11.8 per 100,000 per year in American teenagers. There is a strong genetic component; in adolescents who develop T2DM, 45% to 80% have at least 1 parent with T2DM. Native American and Canadian indigenous people plus South Asians, Pacific Islanders and Latinos are at increased risk for development of T2DM. In adults, both T1DM and T2DM are powerful predictors of future cardiovascular disease, equivalent to a history of previous coronary events. In youth, T2DM is associated with anatomic and histologic vascular changes at autopsy, structural and functional vascular changes in vivo , and early cardiovascular disease events. Multiple CVRFs accelerate the atherosclerotic process in youths with T2DM. Of primary importance is diabetic nephropathy, often present at the time of diagnosis of T2DM. Hyperglycemia increases cardiovascular disease risk with an 18% increase in events per 1% increase in hemoglobin A1c in adults. Up to 40% of Canadian and American adolescents with T2DM have elevated triglycerides and low HDL-C, the atherogenic pattern of combined dyslipidemia. , Over a 4-year follow-up, one-third of youths with T2DM in a long-term observational study developed hypertension. The cardiometabolic syndrome cluster of CVRFs described in the previous section is highly prevalent; its detection at a mean age of 12 years is an independent predictor of CV disease before 50 years of age. In adults with diabetes mellitus, management of CVRF definitively reduces cardiovascular disease events. An approach to reduction of CVRF in youth with high-risk conditions (such as T1DM and T2DM) is outlined in and in the treatment algorithm , as well as in the current Canadian guidelines. In morbidly obese adolescents with T2DM, gastric bypass has been shown to significantly reduce metabolic and cardiac abnormalities. This acute, self-limited arteritis of unknown etiology occurs primarily in children < 5 years of age. The incidence is highest in Asian (particularly Japenese) children and Pacific Islanders. The incidence in Canada and the United States is similar at 25 to 30 per 100,000 children < 5 years of age per year, compared with > 250 per 100,000 in Japan. , The acute illness is marked by dramatic inflammation of all medium-sized arteries, multiple organs, and tissues leading to a range of striking signs and symptoms. The critical problem is development of coronary artery (CA) aneurysms in 20% to 25% of untreated patients. The incidence of CA involvement is significantly lower at < 5% for children treated with intravenous immunoglobulin in the acute stage. The CA pathology involves a 3-stage arteriopathy: acute necrotizing arteritis, followed by chronic inflammation and vasculitis, and luminal myofibroblastic proliferation. Outcomes depend on the severity of CA involvement, which ranges from none to dilation to aneurysms of different sizes and characteristics. Myocardial infarctions occur because of acute thrombosis of aneurysms, or stenoses caused by progressive luminal occlusion from myofibroblastic proliferation. CA aneurysms from Kawasaki disease (KD) account for 5% of acute CA syndromes in adults <40 years of age. Children with CA aneurysms require specific long-term thromboprophylaxis beginning in the acute stage, when large aneurysms are at highest risk for thrombosis, and later with the concomitant development of stenoses. CA thrombosis with actual or impending lumen occlusion is an acute emergency requiring immediate thrombolysis. With large CA aneurysms, sustained thromboprophylaxis with anticoagulation is recommended; with small CA aneurysms, only a single or dual antiplatelet agent is recommended. Children with no CA involvement are thought to have no ongoing sequelae and are typically discharged from cardiology care. Atherosclerosis is not thought to be part of the specific vasculopathic process associated with KD, but CVRF optimization is recommended empirically in all children who have had CA aneurysms of any size. Statins are being evaluated in acute KD and in children with CA aneurysms in the convalescent stage. With persistent CA aneurysms, statin therapy is recommended for empiric use for its presumed pleotropic effects: specifically, its anti-inflammatory properties. Cystic fibrosis (CF) is the most common autosomal recessive disease seen in the Caucasian population. A mutation in the CF transmembrane conductance regulator gene disrupts regulation of chloride and sodium ions across epithelial cell membranes, resulting in buildup of thick mucus throughout the body. Although CF is a multiorgan disease, its effects on the pulmonary system are the leading cause of patient morbidity and mortality. Decreased right-ventricular function, paralleling the presence of pulmonary hypertension, is a well-established late complication, but, with increasing survival, there is emerging concern about CV disease. Certainly, known CVRFs for accelerated atherosclerosis are common in the CF population, including chronic inflammation, dyslipidemia (primarily low HDL-C), and nephropathy. , CF-related diabetes is the most common comorbidity, occurring in ∼20% of adolescents and 40% to 50% of adults. Although studies of left-ventricular function in patients with CF have been inconclusive, recent echocardiographic-strain analysis revealed significant biatrial enlargement, impaired left-atrial conduit and reservoir functions, and abnormal atrial volume indexes, which were significant predictors of mortality. Evaluation of arterial flow-mediated dilation has revealed both microvascular and conduit artery endothelial dysfunction in patients with CF, potentially secondary to oxidative stress. By contrast, coronary angiography performed in preparation for lung transplantation in a small series of middle-aged Canadian patients with CF revealed no evidence of luminal narrowing or focal stenosis despite the presence of diabetes in 64% and dyslipidemia in 78% of these patients. Clearly, further prospective evaluation is needed, as life expectancy continues to increase for this important group of patients. The goal for detecting, evaluating, and managing high-risk youth is to achieve primordial and primary prevention of cardiovascular disease. To accomplish this, the clinician must ensure that any of the underlying risk conditions are optimally managed and that other contributing CVRFs are either prevented from occurring or identified early and appropriately managed. There are a number of Canadian and international guidelines providing guidance on the management of at-risk conditions such as KD, diabetes mellitus, and heart transplantation and CVRFs such as dyslipidemia, , hypertension, , and obesity. These recommendations should guide the evaluation and management of specific CVRFs and conditions, with a general theme of pursuing more aggressive management, including lower thresholds for initiating pharmacotherapy and more aggressive treatment targets in patients with higher cardiovascular risk categories ( and ). All patients with identified CVRF or high-risk conditions should undergo thorough evaluations for other CVRFs, behaviours, and risk conditions , beginning with a detailed history and examination with subsequent investigations, as necessary, to exclude secondary causes and consequences of various CVRFs. All medications and supplements should be reviewed for their potential to exacerbate a CVRF or interact with a potential pharmacological strategy. A detailed family history of CVRFs and cardiovascular disease should be undertaken. A family history of premature cardiovascular disease (< 55 years old in men and < 65 years old in women) in extended first-degree family members (including grandparents, aunts, and uncles) is an important consideration, both in youth at risk for autosomal dominant-codominant conditions, such as FH, and in the general population. A strong family history may indicate a polygenic predisposition toward increased risk of cardiovascular disease. To this end, a cardiovascular risk score to quantify cardiovascular disease risk in a person's family history was recently formulated and applied to youths with bipolar disorder. A detailed dietary and physical activity history should be performed. Involvement of key allied health professionals, such as dieticians and exercise physiologists, should be undertaken. Access to these resources, among others, may be limited, particularly among low-socioeconomic status communities and people in geographically remote communities. To help improve access for vulnerable or at-risk communities, we encourage the exploration of unique strategies that have recently been increasingly incorporated, such as telephone or virtual assessments. These strategies, developed to help provide necessary care during the SARS-CoV-2 pandemic, should become mainstays to help widely scale the provision of comprehensive care for all Canadian youth. The initial physical examination should be complete and systematic to evaluate both for secondary causes and complications of the CVRFs and conditions in question. Height, weight, body mass index (BMI), and blood pressure should be measured in a standardized manner and then converted to age- and sex- (for height, weight, and BMI) or age-, sex-, and height-specific normative values (for blood pressure) to allow for appropriate categorization. , Waist circumference, particularly when indexed to height (waist-to-height ratio) is a useful additional measure that is more indicative of central adiposity and associated with CVRF clustering. , A subsequent laboratory assessment should involve the confirmation of the CVRF in question (for example, ambulatory blood pressure monitoring for hypertension), evaluation of associated CVRFs or conditions (for example, fasting glucose, lipid panel, liver enzymes for nonalcoholic fatty liver disease, lipoprotein(a) for patients with FH), consequences of the CVRFs or conditions (for example, renal function and echocardiography for patients with hypertension), and secondary causes of the CVRFs or conditions. Details of recommended investigations are found in relevant guidelines. , , and and the algorithm in demonstrate the approach to risk stratification and management for children with conditions predisposing to early cardiovascular disease. Management begins with stratification by disease process as indicated by . Assessment of all CVRFs allows further stratification: If > 2 RFs are identified, the patient is reclassified into the next highest tier . Tier-specific goals for CVRFs are indicated in step 3, with defined lower thresholds for initiating pharmacotherapy, and more aggressive treatment targets in patients with higher cardiovascular risk categories . Rigourous, age-appropriate education in diet, activity, and smoking cessation is indicated for all risk tiers. For high-risk children, intensive therapeutic lifestyle change (TLC) like this is combined with condition-specific management, as described in the following section. For children in the moderate-risk category, intensive TLC alone is indicated before initiating pharmacotherapy for up to 6 months. Children in at risk categories are managed expectantly with standard guidelines. For high-risk conditions, specific therapy is indicated to achieve blood pressure, LDL-C, glucose, and hemoglobin A1c goals. Management of the underlying condition should be optimized in conjunction with the managing pediatric subspecialist. As an example, in patients with T1DM, hyperglycemia should be minimized with frequent glucose checks and assessment of HbA1c and insulin levels per pediatric endocrinology. The management of dyslipidemia and the indications for pharmacotherapy are based on the patient’s risk category . , In high-risk patients with an LDL-C ≥ 3.4 mmol/L, consideration of concurrent statin therapy at the onset of lifestyle changes is warranted. Otherwise, the timing and thresholds for initiation of statins depend on the patient’s underlying cardiovascular risk categorization . The management of hypertriglyceridemia is typically reserved for those with marked elevations in triglyceride levels in an effort to reduce the risk for pancreatitis. Lifestyle modifications, including reduced intake of simple carbohydrates and added sugars and increased moderate to vigourous physical activity (≥ 5 hours per week), with weight-loss counselling as appropriate, should be undertaken. , If fasting triglyceride levels remain ≥ 4.5 mmol/L despite lifestyle changes, medications (fenofibrate, omega-3 fatty-acid supplements, or statins if there are also elevations in non–HDL-C) can be considered with consultation with a lipid specialist. A diagnosis of elevated blood pressure as defined by the AAP Clinical Practice Guideline does not generally require pharmacotherapy. Rather, TLC should be undertaken, including incorporation of a diet rich in fruits, vegetables, other sources of fiber, and lean protein diet with reductions in salt and added sugars (for example, the Dietary Approaches to Stop Hypertension [DASH] diet). Blood pressure should then be routinely assessed every 4 to 6 months. For patients with diagnosed hypertension, management is dependent on the severity of the hypertension (stage 1 vs stage 2 hypertension) and the patient’s cardiovascular risk category . The presence of target organ damage (such as elevations in indexed left-ventricular mass) may also prompt initiation of pharmacotherapy. First-line medications typically include angiotensin-converting enzyme inhibitors (ACEis), angiotensin receptor blockers (ARBs), long-acting calcium channel blockers, and thiazide diuretics. Unfortunately, head-to-head clinical trials comparing antihypertensives in pediatric hypertension do not exist, so choice of medication is largely based on clinician preference, with consideration of specific clinical circumstances (such as ACEis or ARBs in patients with chronic kidney disease or diabetes mellitus). Further details outlining the use of antihypertensive pharmacotherapy in youths are provided in the AAP Clinical Practice Guideline. A 14-year-old male patient was referred for evaluation and management of dyslipidemia. He had a lipid profile checked by his primary care provider as part of a clinical evaluation of his worsening (now severe) obesity ( , Step 1: Moderate Risk). He has no other health issues, is on no medications or supplements, and his past medical history is unremarkable. Review of systems was remarkable for snoring. His family history is remarkable in that his father is obese and is on medication for hypertension, dyslipidemia, and T2DM, and he had a myocardial infarction at 43 years of age. The paternal grandfather had a stroke at 72 years of age. The mother is obese and is on medication for T2DM, but her family history is negative for cardiovascular disease. The patient has a younger brother, 10 years of age, who has a normal BMI and is described as healthy and very active. The family history thus demonstrates the presence of premature cardiovascular disease on the paternal side and CVRFs on both the paternal and maternal sides. Physical examination showed his height to be at the 50th percentile, weight above the 95th percentile, with BMI above 120% of the 95th percentile. His waist-to-height ratio was 0.65 (elevated). His blood pressure by auscultation was 142/94 mm Hg, confirmed on repeated assessments. Acanthosis nigricans was evident around his neck and axilla. Abdominal striae were present. There were no other findings on examination. His lipid profile prompting the referral showed total cholesterol of 5.85 mmol/L, LDL-C 3.78 mmol/L, HDL-C 0.92 mmol/L, triglycerides 2.54 mmol/L, and non–HDL-C 4.93 mmol/L. His fasting glucose was borderline (6.2 mmol/L), hemoglobin A1c was at the upper end of normal (5.9%), and liver enzymes were mildly elevated. Lifestyle assessment showed high levels of several risk behaviours, including physical inactivity, likely driving his increasing adiposity. At first glance, this patient has several CVRFs, likely related to his severe obesity. He likely has at least stage 1 hypertension, a significant combined dyslipidemia, a positive family history for premature cardiovascular disease, and is at risk for the development of T2DM, nonalcoholic fatty liver disease, and obstructive sleep apnea. Given that his hypertension is likely related to his obesity, a limited evaluation for secondary causes was performed and was negative. Ambulatory blood pressure monitoring confirmed a diagnosis of sustained hypertension, showing mean daytime and night-time systolic and diastolic blood pressures above the 95th percentile, the majority of measures above the 95th percentile, and abnormal night-time dipping (percentage drop from the mean daytime to mean nighttime levels. Less than 10% is considered abnormal and indicates abnormal circadian variation in blood pressure ). An echocardiogram showed normal anatomy and ventricular function but evidence of increased left-ventricular mass secondary to his obesity and hypertension. A sleep study showed evidence of obstructive sleep apnea. A repeat fasting lipid profile showed similar findings to the values from referral, and it was thought that although his combined dyslipidemia was primarily related to his obesity, given the higher than expected LDL-C, there was likely also a familial component. His fasting glucose and hemoglobin A1c levels were borderline high; therefore, an oral glucose tolerance test was performed, which suggested insulin resistance but not T2DM. A liver ultrasound confirmed the presence of nonalcoholic steatohepatitis. Hence, the evaluation confirmed the presence of multiple CVRFs in the context of a positive family history. Although severe obesity placed the patient at baseline in a moderate-risk category , the presence of multiple CVRFs escalated his risk status to high risk ( , Step 2). Decision making regarding coordinated management of his multiple CVRFs started with an aggressive focus on improving his risk behaviours. He was referred to an exercise medicine program for evaluation and implementation of exercise prescription with targets of achieving an accumulation of 60 minutes of moderate to vigourous physical activity per day, while cutting screen time to no more than 1 hour per day ( , Step 4). Working with them, he developed a planned exercise program: 30 minutes per day after school using an exercise bike already in the home, walking the family dog for 15 to 20 minutes each day, walking to school, and using an exercise app each day for 15 minutes with body-weight strength-training exercises. His mother agreed to participate with him and to encourage him. A dietician worked with the family to eliminate sugar-sweetened beverages and limit junk food and eating out to no more than once a week and to emphasize selection of healthier food choices in every setting. He was to limit his intake of refined carbohydrates (white bread was his preferred snack food) and to increase his intake of vegetables. He would start preparing a healthier lunch to take to school, rather than his preferred lunch of cafeteria French fries. Given that he had evidence of target organ damage secondary to hypertension, a decision was made to start him on an ACEi, and he achieved target blood pressure control with improvements noted in subsequent ambulatory blood pressure monitoring ( , Step 5). His lipid profile remained unchanged after about 3 months of lifestyle changes (minimal improvement in his triglyceride level, no change in level of adiposity). Given his multiple CVRFs, a decision was made to start him on a low dose of a statin, which improved his LDL-C and non-HDL-C levels markedly to near normal. The family receives ongoing support from the exercise medicine program and dietician, and his insulin resistance has not worsened. Atherosclerosis begins in youth, and its progression is accelerated by the presence of CVRFs and risk conditions. In particular, the presence of multiple CVRFs and risk conditions can increase the rate of atherosclerotic progression geometrically, reducing the time to manifest CV disease events. Preventing or slowing atherosclerosis across the lifespan, beginning in youth, is necessary to achieve primordial and primary prevention goals. The total risk profile of children should be assessed and considered in management decisions and treatment goals, beginning with identification of those with multiple CVRFs or associated risk conditions. The authors report no funding sources relevant to the contents of this paper. Dr McCrindle is an investigator and consultant for Janssen Pharmaceutica and an investigator for Mezzion Pharmaceuticals. The other authors have no conflicts of interest to disclose. |
Feasibility Study of Endoscopic Surgery for Spontaneous Intracerebral Hemorrhage with Large Hematoma: a Comparison with Craniotomy Using Propensity Score Matching Analysis | 219a636c-19e1-46f9-afa2-7169e023df40 | 11950029 | Surgery[mh] | Spontaneous intracerebral hemorrhage (ICH) is a common subtype of stroke associated with high disability and mortality rates, imposing a substantial burden on families and society . The mortality rate is particularly elevated for patients with large hematomas. Although current clinical research suggests limited benefits of surgical treatment for patients with ICH, surgical intervention is widely considered an effective lifesaving measure and is extensively employed in clinical practice . Craniotomy with or without decompressive craniectomy is a conventional surgical approach that effectively evacuate hematomas, alleviates mass effect, and reduces intracranial pressure (ICP), thereby offering a chance for patient survival. However, craniotomy is associated with huge trauma, a high incidence of complications, and the need for subsequent cranioplasty . Endoscopy has been widely used in the treatment of ICH, with studies indicating superior outcomes compared to craniotomy for small to moderate hematomas [ – ]. However, research on the use of endoscopy for treating large hematomas is insufficient. Therefore, this study aims to compare endoscopic surgery and craniotomy for the treatment of large hematomas ICH to evaluate the feasibility and safety of endoscopic surgery.
Patient Data Surgical Treatment Endoscopic Surgery Craniotomy Statistical Analysis To mitigate selection bias and other confounding factors, a 1:1 propensity score matching analysis (caliper 0.2) was performed based on the following baseline characteristics: hematoma volume, ventricular hemorrhage, GCS score, hematoma location, and the time to surgery. Continuous variables with a normal distribution were presented as mean ± standard deviation and analyzed using the t -test. Nonnormally distributed variables were presented as the medians with interquartile ranges and analyzed using the Mann–Whitney U -test. Categorical data were represented as numbers and percentages and analyzed using the χ 2 test. A p value less than 0.05 was considered statistically significant.
A retrospective analysis was conducted on data from patients with spontaneous ICH admitted to the Chongqing Emergency Medical Center from January 2019 to June 2023. Prior to surgery, all patients or family members signed a surgical consent form. This study was approved by the hospital’s ethics committee. All patients underwent computer tomography (CT) angiography to exclude conditions such as arteriovenous malformations and aneurysms. Inclusion criteria for the study were the following: (1) spontaneous supratentorial ICH, (2) hematoma volume larger than 50 mL or evidence of unilateral cerebral herniation based on CT or clinical presentation, (3) Glasgow Coma Scale (GCS) score of 4 or higher, (4) admission within 24 h of onset, and (5) age over 18 years. Exclusion criteria were the following: (1) hemorrhage caused by trauma, tumors, aneurysms, or cerebrovascular malformations; (2) coagulation disorders; (3) multiple ICHs; (4) significant organ failure, such as cardiac or pulmonary dysfunction; and (5) history of stroke. On admission, all patients received tailored symptomatic treatment, including hemostasis, blood pressure and blood glucose control, dehydration, and nutritional support. The choice of surgical approach was based on various factors, including the patient’s condition, the surgeon’s expertise in the surgical technique, and the preferences of the patient’s family.
Endoscopic surgery was conducted under general anesthesia using endoscopy. The surgical approach was determined preoperatively based on the long axis of the hematoma as seen on CT scans. The procedure involved a linear incision, creation of a small bone flap, and insertion of a transparent plastic dilator following a cruciate durotomy. Hematoma evacuation and hemostasis were achieved using a rigid neuroendoscope. The bone flap was replaced after evacuation (Figs. a–d and ).
Craniotomy surgery was performed under general anesthesia following a preoperative design of the surgical incision based on CT scans. The procedure involved scalp incision, cranial drilling, and bone flap creation, followed by opening the dura mater and incising the cortex to access the hematoma cavity without disrupting nerves and blood vessels. Hematoma evacuation was conducted under direct vision or with the assistance of a microscope to ensure hemostasis. Decompression was achieved by suturing the dura mater without replacing the bone flap, followed by scalp closure to conclude the surgery (Figs. e–h and ). Drainage tubes were routinely placed in the hematoma cavity or lateral ventricle, and an ICP probe was placed in the ventricle or subcortical area at the end of the surgery for continuously monitoring for 5–7 days postoperatively. All patients were then transferred to the neurological intensive care unit for critical care management. Head CT scans were performed after surgery and at 24 h to assess hematoma clearance and detect any rebleeding. Subsequent CT scans were conducted based on the patient’s condition. Symptomatic treatment included osmotic diuretics, such as mannitol, glycerol fructose, furosemide, and albumin. In some cases, contralateral ventricular drainage was performed postoperatively to control ICP. Decompressive craniectomy involving the removal of a bone flap approximately 10 × 12 cm in size was considered for cases in which ICP remained elevated. Tracheostomy was performed in cases of severe impairment of consciousness, severe pulmonary infection, or poor sputum expectoration to maintain airway patency. Patient information, including sex, age, blood pressure at admission, history of hypertension, history of diabetes, coagulation function (international normalized ratio), intraventricular hemorrhage, cerebral herniation, GCS score, and time from onset to surgery, was recorded. Surgical data collected included duration of surgery, blood loss, preoperative and postoperative hematoma volume, hematoma evacuation rate, 24-h postoperative edema volume, ICP, rebleeding, complications (such as cerebral infarction, intracranial infection, pulmonary infection, gastrointestinal bleeding, epilepsy, and deep vein thrombosis), mortality rate, and length of hospital stay. Glasgow Outcome Scale (GOS) scores was recorded at 6 months. The 24-h postoperative edema volume was used as an indicator of iatrogenic injury during surgery , with hematoma and edema volumes measured using 3D Slicer. The hematoma evacuation rate was calculated using the following formula: (preoperative hematoma volume–postoperative hematoma volume)/preoperative hematoma volume × 100%. GOS scores were interpreted as follows: 1 for death, 2 for vegetative survival, 3 for severe disability, 4 for moderate disability, and 5 for independent living. A score of 4–5 indicated a good recovery .
Baseline Data Surgical and Prognostic Evaluation study analyzed 113 cases that met the inclusion criteria, with 65 cases in the endoscopic surgery group and 48 cases in the craniotomy group. Using propensity score matching at a 1:1 ratio, 34 cases were selected for each group. Post–propensity score matching analysis revealed no statistically significant differences in baseline characteristics between the endoscopic surgery and craniotomy groups. The mean age was 53.38 ± 11.36 years in the endoscopy group and 53.79 ± 11.65 years in the craniotomy group ( p = 0.883). Both groups had 67.65% male patients. In the endoscopy group, 15 cases (44.12%) had a GCS score of 4–8, compared to 11 cases (32.35%) in the craniotomy group ( p = 0.318). Hemorrhage in the basal ganglia region was present in most cases in both groups (88.24% vs. 91.18%, p = 0.690). The mean preoperative hematoma volume was 64.84 ± 11.02 mL in the endoscopy group and 66.57 ± 12.77 mL in the craniotomy group ( p = 0.554). In the endoscopy group, 9 cases (26.47%) exhibited unilateral cerebral herniation, compared to 11 cases (32.35%) in the craniotomy group ( p = 0.595). There were no statistically significant differences in admission blood pressure, coagulation function (international normalized ratio), creatinine, history of hypertension, history of diabetes, presence of intraventricular hemorrhage, or time from onset to surgery between the two groups (Table ).
Both endoscopic surgery and craniotomy effectively evacuate the hematomas, with evacuation rates of 93.27% and 89.34%, respectively, showing no statistical difference ( p = 0.141). However, blood loss during endoscopic surgery was significantly lower at 50 mL compared to 450 mL in the craniotomy group ( p < 0.001). The average surgical time for endoscopic surgery was also significantly shorter than for craniotomy (140 vs. 205 min, p < 0.001). Additionally, the average 24-h postoperative edema volume was significantly lower in the endoscopy group at 28.49 mL, compared to 61.85 mL in the craniotomy group ( p < 0.001). There was no significant difference in postoperative 24-h ICP between the two groups (11.58 ± 5.10 mm Hg vs. 11.79 ± 4.46 mm Hg, p = 0.858) (Table ). In both the endoscopic and craniotomy groups, four patients (11.76%) experienced postoperative rebleeding, all of which were managed with conservative treatment, avoiding the need for a second surgery. Regarding postoperative complications, the endoscopy group had 1 case (2.94%) of cerebral infarction, 4 cases (11.76%) of intracranial infection, 24 cases (70.59%) of pulmonary infection, 7 cases (20.59%) of gastrointestinal bleeding, 1 case (2.94%) of epilepsy, and 3 cases (8.82%) of deep vein thrombosis. In the craniotomy group, there were 4 cases (11.76%) of cerebral infarction, 3 cases (8.82%) of intracranial infection, 31 cases (91.18%) of pulmonary infection, 10 cases (29.41%) of gastrointestinal bleeding, 4 cases (11.76%) of epilepsy, and 7 cases (20.59%) of deep vein thrombosis. Although the rate of pulmonary infection was slightly lower in the endoscopy group, there were no statistically significant differences in overall complications between the two groups (Table ). The endoscopy group had two patients (5.88%) who died, whereas the craniotomy group had three patients (8.82%), with no significant difference ( p = 0.642). The length of hospital stay was significantly shorter for the endoscopy group, averaging 32.50 days compared to 48.00 days for the craniotomy group ( p = 0.002). At the 6-month follow-up, 11 cases (32.35%) in the endoscopy group achieved a favorable outcome (GOS score of 4–5), which was comparable to the craniotomy group with 12 cases (35.29%) ( p = 0.798) (Table ).
The debate over the surgical treatment of ICH remains unresolved, with no current consensus. Surgery theoretically offers several benefits, including hematoma evacuation, alleviation of mass effect, relief of neurological compression symptoms, improved perfusion, and reduction of secondary injury. These advantages have made surgery a common clinical practice [ – ]. However, large clinical studies such as Surgical Trial in Intracerebral Hemorrhage (STICH) and the STICH II have not demonstrated significant benefits of surgical treatment for patients with ICH . Notably, these studies excluded some critically ill cases. However, surgery could potentially save lives and improve prognoses in this subgroup. Currently, there is a scarcity of studies specifically analyzing surgical treatments, particularly endoscopic surgery, in critically ill patients with ICH with large hematomas. Most existing research consists of subgroup analyses rather than comprehensive studies. For patients with large hematomas, the mass effect and associated brain edema can elevate ICP, potentially leading to cerebral herniation and posing a life-threatening risk. The prevailing surgical approach is craniotomy for hematoma evacuation with or without decompressive craniectomy. Craniotomy effectively evacuates the hematoma, achieves hemostasis under direct vision, and reduces mortality. Decompressive craniectomy can further lower ICP following bone flap removal, significantly reduce midline shift, and provide a buffer for rebleeding and delayed edema . Research indicates that combining decompressive craniectomy with craniotomy yields better therapeutic outcomes than craniotomy alone . However, craniotomy involves excessive mechanical traction, electrocoagulation, and cauterization, which can cause huge brain injury. Additionally, the procedure is lengthy and associated with many postoperative complications. Craniectomy also alters the normal cranial structure, potentially leading to unpredictable side effects, such as subdural fluid accumulation and hydrocephalus, which may require secondary cranioplasty. These factors increase patient trauma and associated risks, rendering craniectomy a less preferred option [ – ]. In the context of adequate hematoma evacuation, edema is a crucial factor influencing ICP following surgical treatment. Edema primarily originates from two sources: the hematoma and its metabolic products and the impact of surgery on the brain. Posthemorrhagic edema mainly results from a cascade reaction, including the inflammatory response and oxidative stress, which leads to the development of edema that progressively worsens over time [ – ]. Therefore, early surgery can alleviate the edema caused by the hematoma itself and mitigate secondary damage, which is critical for surgical success. However, iatrogenic injuries, such as mechanical traction, electrocoagulation, and cauterization, can exacerbate edema. Postoperative edema typically peaks 24 h after surgery and becomes more pronounced with greater surgical trauma . In theory, early minimally invasive surgery can minimize brain edema, potentially eliminating the need for decompressive craniectomy. Minimally invasive procedures, including puncture drainage and endoscopic surgery, cause minimal damage, particularly endoscopic surgery, which is widely used in clinical treatment because of its excellent visualization . Endoscopic surgery causes minimal injury, reducing interference and damage to the brain tissue surrounding the hematoma, thereby reducing iatrogenic edema. This reduction in edema is crucial for managing ICP post operation. Endoscopic surgery provides a clear, multiangle operative field, allowing for comprehensive observation of pathological tissues and bleeding sites. This leads to more effective hematoma evacuation and hemostasis, resulting in a high hematoma evacuation rate and a low rebleeding rate. Multiple studies have confirmed that for small and medium-sized ICHs, endoscopic surgery reduces duration of surgery, decreases surgical complications, and improves prognosis, surpassing craniotomy [ , , ]. For large hematoma ICHs, early endoscopic surgery can clear most of the hematoma, minimize injury and edema, mitigate mass effect, and maintain lower ICP, potentially eliminating the need for decompressive craniectomy. Recent research supports the feasibility and safety of this approach. Ye et al. conducted a retrospective analysis of 112 patients with large hematoma ICHs, finding no significant differences in mortality rates and modified Rankin Scale scores between the craniotomy group and the endoscopy group. However, the endoscopy group experienced fewer postoperative complications compared to the craniotomy group . Leveraging our center’s neuroendoscopy expertise, we have applied endoscopic surgery in various types of ICH and traumatic brain injury . In ICH surgeries, we use a miniaturized endoscopic channel (outer diameter 1.1 cm, inner diameter 0.9 cm) to minimize brain injury. During localization, we carefully select the direction along the long axis of the hematoma to protect neural fiber bundles as much as possible. To minimize interference with the hematoma–brain tissue interface and reduce delayed edema, we operate strictly within the hematoma cavity during evacuation. For oozing of blood, hemostasis is achieved using compression with hemostatic gauze. In cases of vascular bleeding, the vessel is lifted before electrocoagulation and cauterization to minimize the impact on the brain. The key to successful surgery is ensuring sufficient hematoma evacuation to mitigate the mass effect of residual hematoma and the edema generated during its resolution. The endoscope’s excellent illumination enables clear observation of each bleeding point. While ensuring hemostasis, it is crucial to avoid overfilling the hematoma cavity with excessive hemostatic material. After hematoma clearance, brain pulsation and collapse should be carefully observed. If brain pulsation is good and the collapse depth is more than 0.5–1.0 cm below the bone window, the bone flap can be replaced. In our study, endoscopic surgery demonstrated comparable hematoma evacuation rates to craniotomy, while significantly reducing surgical times and blood loss. Analysis of the 24-h postoperative CT scans revealed a significantly lower volume of edema in the endoscopic surgery group compared to the craniotomy group, indicating that iatrogenic trauma from endoscopic surgery was notably less than that from craniotomy. After evacuating the majority of the hematoma and replacing the bone flap, ICP remained within normal ranges post endoscopic surgery, comparable to that observed in craniotomy, despite the increased cranial cavity volume resulting from decompressive craniectomy. Rebleeding rates were similar in both groups, with no need for second surgical treatments. The endoscopic surgery group exhibited a lower rate of pulmonary infection compared to the craniotomy group, likely attributed to reduced injury from endoscopic surgery, facilitating early postoperative ambulation. However, overall complications did not significantly differ between the two groups. Patients undergoing endoscopic surgery experienced faster recovery and shorter hospital stays than those in the craniotomy group. At the 6-month follow-up, there were no significant differences in the GOS scores between the two groups, indicating that endoscopic surgery did not provide obvious advantages in improving prognosis. This lack of distinction may be attributed to the severe primary neurological damage in patients with large hematoma ICH, indicating that even with minimally invasive surgery, there is no significant improvement in neurological function. However, endoscopic surgery has no significant impact on cosmetic appearance and eliminates the necessity for secondary cranioplasty, along with its associated trauma and risks. Based on our experience, several critical factors must be considered in endoscopic surgery without craniectomy: Time from onset to surgery: Early intervention can mitigate brain edema caused by the hematoma. Beyond 24 h, the edema becomes significant, and bone flap retention may lead to unmanageably high ICP postoperatively. However, research also indicates that ultra-early surgery (less than 6 h) may increase the risk of rebleeding, underscoring the importance of timing for surgery . Age: Brain atrophy worsens with advancing age, increasing the compensatory ability for high ICP. Location of the hematoma: The farther away from the brainstem, the lower the risk of cerebral herniation. ICP monitoring: Accurate and dynamic ICP monitoring facilitates timely detection of severe risks, such as rebleeding and severe edema. External ventricular drainage: This involves placing a ventricular drain on the same side during surgery or performing contralateral ventricular drainage postoperatively. It allows dynamic and precise control of ICP by draining cerebrospinal fluid and can be sustained for several days to manage ICP during the peak period of cerebral edema. Ventricular drainage carries minimal trauma risks, primarily bleeding during puncture and postoperative infection, with a low occurrence rate indicated by current research . Intensive care unit (ICU): ICU care is often necessary for timely observation of potential changes in the patient’s condition. Multimodal monitoring and respiratory assistance equipment in the ICU can promptly detect and manage changes. In cases of severe pulmonary infection or airway obstruction, early tracheostomy should be considered. Limitations of our study include its single-center and retrospective nature, as well as a relatively small number of cases. Endoscopic surgery demands a high level of skill, and the procedure’s success is closely linked to the surgeon’s proficiency, limiting the generalizability of our findings. It is crucial to consider comprehensively, as endoscopic surgery may not be suitable for all cases of large hematoma ICH treatment.
For large spontaneous supratentorial ICHs, endoscopic surgery appears safe and feasible, with efficacy comparable to that of craniotomy with decompressive craniectomy. However, these findings warrant further validation through multicenter prospective randomized controlled trials.
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Panoramic evaluation of external root resorption in mandibular molars during orthodontic treatment: a comparison between root-filled and vital teeth treated with fixed appliances or clear aligners | 29b3ea50-cb9d-496e-a11c-a45426627e41 | 11439240 | Dentistry[mh] | Orthodontic treatment (OT) carries the potential risk of a complication known as external apical root resorption (EARR). EARR is characterized by the irreversible loss or shortening of the hard tissue at the root apex. This phenomenon can be a detrimental unintended consequence of the forces applied during the process of tooth movement . Although studies report a wide range of prevalence, estimating that EARR affects 20–100% of orthodontic patients, the extent of resorption can vary considerably . While EARR can potentially affect any tooth undergoing orthodontic movement, maxillary and mandibular incisors are generally considered the most susceptible to resorption . A potential consequence of EARR is a compromised tooth structure, which can lead to an imbalance between root and crown length, and in severe cases, even tooth loss, ultimately impacting both the aesthetics and functionality of the OT outcome . OT triggers a complex interplay of factors that can influence susceptibility to EARR. These factors include a patient’s genetics, age, nutritional habits, malocclusion severity, the type of appliance used, the chosen treatment approach (extraction vs. non-extraction), the characteristics of applied force (magnitude, direction, and duration), and the overall treatment length . Despite extensive research into the prevalence and severity of EARR in orthodontic patients, the exact biological mechanisms underlying this process remain unclear . For decades, fixed appliance (FA) treatments have been the mainstay of OT. However, a paradigm shift is underway with the increasing popularity of clear aligner (CA) treatments . This patient-centered approach prioritizes aesthetics and comfort. Unlike FAs, CAs are virtually invisible and removable, improving aesthetics, comfort, and oral hygiene maintenance . This treatment approach offers an overall reduction in treatment and chair time, providing a more aesthetic and comfortable alternative to FA treatments . The type of FA used for OT is known to cause EARR . The impact of CA treatment on EARR remains a topic of ongoing research. Despite the theoretical advantages of CA treatment in minimizing EARR through the application of gentler forces, current research presents a mixed picture . Existing studies highlight the possibility of EARR with CAs, with reports of both severe EARR and greater than a 20% reduction in root-crown ratio . Additionally, varying results were obtained in studies comparing CA treatment with FA treatment. While some studies suggest a potential reduction compared to FAs due to the gentler forces applied , others report similar levels of EARR with both methods . The lack of consistent findings highlights the need for further investigation into this aspect of CA treatment. A recent comprehensive study reveals the prevalence of root canal treatment (RCT) worldwide, showing that more than half of the population studied has received at least one RCT . Therefore, orthodontists are likely to encounter root-filled teeth (RFT) in a substantial portion of their patient population, often requiring a coordinated approach involving both orthodontic and endodontic interventions . Existing literature suggests a correlation exists between EARR and a tooth’s prior RCT and pulpal status. Consequently, the magnitude of EARR may differ between RFT and vital pulp teeth (VPT) . Existing research regarding the comparative susceptibility of RFT and VPT during OT presents conflicting results, with some finding no difference, some suggesting more, and others suggesting less resorption in RFT . A recent meta-analysis suggests that RFT might experience a lower degree of EARR compared to VPT. However, the certainty of this finding remains unverified, and this difference might not be clinically significant . Consequently, the comparative susceptibility of RFT and VPT to EARR during OT requires further investigation with a rigorous, evidence-based approach . Few studies in the literature examined EARR in teeth after CA treatments. To date, no studies have directly compared EARR in RFT and contralateral VPT in the same patients after CA treatment. Additionally, there is a lack of research comparing EARR between RFT treated with FAs and those treated with CAs. Therefore, this study aims to investigate the following objectives: To evaluate and compare the amount of EARR associated with OT in RFT and their contralateral VPT during CA treatment in the same patient. This comparison will be conducted using both linear and root surface measurements. To investigate and compare the differences in EARR patterns between CAs and FAs in patients undergoing OT. This comparison will specifically focus on EARR in RFT or VPT. This study investigates two primary hypotheses: There is no statistically significant difference in the amount of EARR between RFT and their contralateral VPT during OT, regardless of whether CAs or FAs are used. The type of OT employed (CAs versus FAs) does not have a statistically significant impact on the extent of EARR in either RFT or VPT.
This retrospective clinical study employed a split-mouth design and received ethical approval from the Clinical Research Ethics Committee of Kutahya Health Sciences University, Kutahya, Türkiye (reference number: 2024/07–37). Informed consent was obtained from all the participants including both patients and the legal guardians of children involved in the present study. A power analysis was conducted to determine the minimum sample size necessary to detect a clinically relevant difference in EARR between groups. The analysis employed an alpha error probability of 0.05 and a power of 90%, targeting a minimum detectable difference of 0.95 mm with a standard deviation of ± 1.15 mm . Based on this analysis, a minimum of 21 patients per group was required. To enhance the study’s statistical power, a larger sample size was ultimately recruited. For this retrospective analysis, radiographs obtained from patients undergoing OT in our clinic from January 2022 to May 2024 were evaluated. The radiographs were subsequently divided into two groups based on treatment modality: CA treatment and FA treatment. Inclusion and exclusion criteria were then applied to select the final sample for assessment. Inclusion Criteria . High-Quality Radiographs: Panoramic radiographs (PRs) that are standardized and exhibit high image quality for accurate landmark identification . Complete Permanent Dentition: Patients with full permanent dentition and no missing teeth, excluding third molars . OT Modality: Patients who received OT with either FAs or CAs . Specific Malocclusion: Treatment who have skeletal Class I anomalies, moderate crowding, and did not involve tooth extractions . Adequate Endodontically Treated Teeth with a VPT counterpart: Patients who had at least one mandibular molar that underwent RCT at least a year prior. The treated tooth must exhibit a healthy periodontal ligament without any signs of periapical pathology. Additionally, VPT counterpart must be present on the contralateral side of the jaw. Acceptable root canal fillings criteria included complete obturation of all root canals, termination of root canal fillings within 0–2 mm of the radiographic apex, absence of voids in any region of the fillings. Criteria for an adequate coronal restoration included a permanent, intact coronal restoration with well-adapted margins, no evidence of recurrent caries, and a radiographically intact appearance . Absence of Parafunctional Habits: Patients who reported no history of bruxism or clenching . Treatment Group Consistency: Groups which utilized the same FA or CA system and aimed for the same amount of tooth movement (anchorage amount) . Exclusion Criteria . RCT During Orthodontics: Patients who underwent RCT during OT were excluded . Dental Anomalies: Patients with teeth exhibiting size or position anomalies, a non-vital tooth on the contralateral side, or unerupted teeth were excluded . Treatment History: Patients with a history of trauma, previous OT, missing treatment records, or teeth extracted for OT or before treatment were excluded . Molar Tipping: Radiographs were evaluated for molar tipping. Patients with tipping in their mandibular molars were excluded . Open Root Apex: Patients with open root apices were excluded . Systemic and Congenital Conditions: Patients with systemic diseases or congenital anomalies or craniofacial syndromes were excluded . Oral Health Issues: Patients with any signs of oral problems such as caries, periodontal disease, or existing root resorption on the examined teeth were excluded . Temporomandibular Joint Disorders and Supernumerary Teeth: Patients diagnosed with temporomandibular joint disorders or with supernumerary teeth were excluded . To ensure comparability between the two groups and minimize the influence of case complexity, the American Board of Orthodontics (ABO) discrepancy index was employed to assess case difficulty. Consequently, patients with high complexity scores were excluded from the study . Following application of the inclusion and exclusion criteria, a total of 37 patients (21 females, 16 males) who received FA treatment were recruited from a pool of 146 patients who completed their OT. The mean age of the FA group was 17.45 years (SD ± 2.67 years). Similarly, 29 patients (18 females, 11 males) who underwent CA treatment were included in the CA group from a pool of 108 patients who completed treatment. The mean age in the CA group was 18.33 years (SD ± 1.96 years). The CA treatment group comprised participants who underwent treatment with CAs (ClearCorrect ® , ClearCorrect LLC, Rock Round, TX, USA). This group was helped by the implementation of virtual treatment planning (ClearPilot™ - ClearCorrect’s Treatment Planning Tool, version 5; ClearCorrect ® ). The treatment sequence, encompassing procedures such as interproximal reduction, attachment placement, and the application of intermaxillary elastics, adhered to the established virtual plan. Patients in this group were instructed to change the CAs in 15-day intervals, with a recommended daily wear time of 22 h. In the FA treatment group, a conventional bracket (MBT prescription and a slot size of 0.022 inches; Razor SS brackets, International Orthodontic Service IOS™, California, USA) was used for treatment. Nickel-titanium archwires (0.014-, 0.016-, 0.018-, 0.016 × 0.022-, 0.017 × 0.025 and 0.019 × 0.025 inch) were used for the aligning and leveling phases, and stainless steel (0.019 × 0.025 inch) was used for the working phase. The FA treatment group exhibited a mean treatment duration of 1.96 years (SD ± 0.78 years), while the CA treatment group demonstrated a mean duration of 1.28 years (SD ± 0.51 years). The amount of EARR in both RFT and contralateral VPT was evaluated in mandibular molars in both groups. Measurements were obtained on taken digital PRs before OT and immediately after debonding. All radiographs were were taken in a standardized head position using a Castellini X-ray unit (Castellini X Radius Compact, Imola, Italy) to ensure consistent patient positioning. Crown and root length measurements were obtained using the measurement program of the Castellini X-ray unit following a methodology established in previous studies . The reference points and specific measurements employed for pre- and post-OT evaluations are detailed in Fig. . The crown and root length measurements involved several key steps. First, the cementoenamel junction (CEJ) was established as a straight line connecting the mesial and distal CEJ points. Subsequently, crown length was determined on both the initial and final radiographs for RFT and their contralateral VPT counterparts. This measurement comprised the longest distance from the occlusal edge to the CEJ. Root length measurements followed a similar approach for both RFT and VPT. The distance was measured from the CEJ to the root apices. Because mandibular molars have two roots, root length was calculated by measuring the distance from the CEJ to the midpoint of the line connecting the two root apices . Details pertaining to the calculation of EARR and its proportions, are presented in Fig. . The EARR was determined in millimeters by subtracting the post-OT root length from the pre-OT root length. Additionally, the ratio of pre-OT crown length to post-OT crown length was calculated. The final EARR value was obtained by multiplying these two values. To assess the relative amount of EARR in RFT compared to VPT, a proportional calculation based on the EARR values in VPT was employed . To complement the linear measurements employed in this study, digital root surface measurements were also obtained from the digital PRs (Fig. ). Following a digital calibration process, the root surface measurements were performed using the SketchAndCalc software program (Axiom Welldone, https://www.sketchandcalc.com/ ). The root surface was measured by meticulously outlining all internal and external root surfaces, starting at the CEJ. To accurately measure the entire root surface, points were meticulously plotted along the root surface at one-pixel intervals. An average of forty (± five) points per tooth was employed to ensure comprehensive representation of the entire root surface . Statistical analysis The normality of the current study’s data was assessed using the Kolmogorov-Smirnov test. Consequently, parametric tests were employed due to the normally distributed data. The Pearson correlation test was utilized to analyze the relationship between gender and the types of teeth measured in the groups. A random sample of 20 patients was selected to evaluate the reliability and reproducibility of measurement protocol. Forty PRs from these patients were measured twice, with a two-week interval between measurements. The intraclass correlation coefficient for the first and second measurements was 0.946 (0.916–0.977), indicating excellent agreement between the two measurements by the same researcher. Additionally, the interclass correlation coefficient was 0.936 (0.908–0.964), demonstrating excellent agreement between the two researchers. These high correlation values indicate excellent agreement between the measurements, and no statistically significant difference was observed between the measurements. Treatment-induced changes within each group were assessed using paired t-tests. Independent t-tests were then utilized to compare the mean age and treatment duration of individuals between the groups, as well as the difference in the EARR amount between RFT and VPT. In order to evaluate the effect of different OT methods on the amount of EARR, analysis of covariance (ANCOVA) was also performed to compare the two groups, taking initial tooth length measurements and initial root surface measurements as covariates ( p < .05). Statistical analyses were conducted using the SPSS software package (version 20.0 for Windows; SPSS Inc., Chicago, IL).
Analysis of demographic characteristics revealed no statistically significant differences between the FA treatment and CA treatment groups in terms of gender distribution and tooth type (Pearson’s chi-squared test) or chronological age (Student’s t-test) ( p > .05). However, as detailed in Table , the treatment duration was significantly longer in the FA group compared to the CA group ( p < .05). The ABO discrepancy index scores demonstrated similar baseline difficulty between the CA treatment group (mean; 11.93 ± 1.57) and the FA treatment group (mean; 13.41 ± 1.92) ( p > .05). Table presents a comparison of the mean EARR values and proportions between the FA and CA groups. Both the FA and CA groups exhibited statistically significant EARR in both RFT and their contralateral VPT ( p < .05). In addition, statistical analysis revealed a significant difference in terms of EARR between RFT and their contralateral VPT within both the FA and CA groups ( p < .05). Compared to the CA group, the FA group showed significantly greater EARR in both RFT and their contralateral VPT ( p < .05). When the initial tooth length values were taken as a covariate (initial tooth length [RFT] = 15.8 mm), the EARR in RFT and the ANCOVA and proportion evaluation showed that the differences between EARR of the FA treatment and CA treatment groups were statistically significant ( p < .05). Table presents a comparison of the mean root surface measurements and treatment-induced changes between the RFT and VPT groups. Statistically significant reductions in root surface measurements were observed across all teeth in both groups ( p < .05). When the pre-treatment root surface measurements were taken as a covariate (initial root surface measurements [RFT] = 103.41 mm²), the EARR evaluation with ANCOVA showed that the decrease in root surface measurements in RFT and VPT was significant between the two groups ( p < .05). Additionaly, in both treatment groups, a comparison of the decrease in root surface measurements between the two sides in the groups revealed significant differences between RFT and VPT ( p < .05).
OT frequently presents with EARR, a complication that can negatively impact both patient outcomes and treatment success. While CAs have gained significant popularity, their influence on EARR remains poorly understood. Additionally, current literature lacks sufficient scientific evidence comparing the effects of CAs on EARR in both RFT and VPT. Therefore, this study aimed to assess the changes in tooth lengths and root surface measurements of mandibular molars following OT with either FAs or CAs. The study evaluated both RFT and their contralateral VPT using digital PRs. Our findings revealed significantly greater EARR in VPT compared to RFT, as measured by both linear and root surface measurements. This contradicts our first hypothesis. Additionally, the treatment modality (FA treatment or CA treatment) significantly impacted the amount of EARR in both groups. Teeth treated with FAs exhibited greater EARR compared to those treated with CAs. Consequently, our second hypothesis was also rejected. CAs and FAs have different features, and both treatment methods are frequently used in OTs. Therefore, to ensure a more robust comparison, this study carefully evaluated inclusion criteria, including the ABO discrepancy index. This helped to guarantee that patients in both treatment groups had similar levels of treatment difficulty, required tooth movement, and anticipated treatment outcomes . In addition, Alqerban et al. demonstrated a link between the degree of EARR and the quality of RCT in RFT. Consequently, this study evaluated the quality of RCT, selecting only cases with appropriate RCT and coronal restoration according to previous studies . ClearCorrect aligners are a leading choice for CA treatment, known for their precision, comfort, and aesthetic properties. Decades of research and innovation in material science have culminated in these aligners, which incorporate the proprietary ClearQuartz™ tri-layer material, Performance Trimline design, and scientifically validated ClearControl™ clinical features. This combination aims to optimize treatment outcomes and achieve desired orthodontic results. The industry-leading triple-layer aligner material combines two layers of flexible polymer with an elastomeric core layer for greater orthodontic control, resulting in light and consistent force application . Furthermore, ClearCorrect aligners incorporate a scientifically validated high, flat trimline design. This design has been demonstrated to optimize force transmission, thereby facilitating more precise tooth movement and enhanced control over root positioning . Optimizing the force delivered to the teeth and delivering more controlled force application helps minimize EARR following OT. Previous research has explored the comparative effects of FAs and CAs on EARR. Li et al. observed significantly greater EARR in maxillary and mandibular incisors treated with FAs compared to CAs in a retrospective study of 70 patients. Similarly, Almagram et al. reported less EARR in the CA group compared to the FA group in their study of 40 patients focusing on maxillary incisors. Jyotirmay et al. further corroborated these findings with a larger sample size of 110 patients, demonstrating significantly less EARR in the CA group for both maxillary and mandibular incisors. Additionally, a meta-analysis suggests that while CAs may not entirely prevent EARR, the incidence and severity are likely lower compared to FAs . Our study’s results align with these previous findings, demonstrating a similar trend of reduced EARR with CA treatment. Orthodontic tooth movement, induced by mechanical stress, triggers an inflammatory response in the periodontal tissues. This response involves local changes, including altered blood flow and the release of various biological factors. The sterile inflammatory environment created by OT can lead to EARR . This process is influenced by various factors, including genetic predisposition , systemic conditions , patient demographics (age and gender) , and treatment parameters . The magnitude, duration, and continuity of orthodontic forces play a significant role . Studies have shown that heavier and continuous forces are associated with increased prevalence and severity of EARR . Conversely, intermittent forces allow for cementogenic repair, potentially mitigating resorption . CAs are hypothesized to deliver lighter, intermittent forces through a computer-aided design process, potentially contributing to less EARR compared to FAs . Additionally, the magnitude and direction of forces generated by aligners may vary from those generated by brackets and archwires. Moreover, force transmission in CA treatment is through the aligner and attachment, while in FA treatment, it’s through the bracket located on the tooth’s crown. These mechanical differences could potentially influence the rate of EARR . Furthermore, our study found that the duration of OTs with CAs was shorter than that of FAs. Our findings can be attributed to the combined effects of the aforementioned factors. Besides this, prior investigations have predominantly focused on incisors, whereas this study investigated EARR in molar teeth. Our findings demonstrate a similar pattern of reduced resorption with CAs compared to FAs, even in this posterior segment. This approach contributes to the existing body of literature by exploring the understudied relationship between OT and EARR in posterior teeth. While studies by Iglesias-Linares et al. and Toyokawa-Sperandio et al. reported no significant differences in EARR between FAs and CAs, these findings may be attributable to methodological variations. Notably, both studies solely evaluated incisors, whereas our investigation focused on molars. Additionally, Toyokawa-Sperandio et al. assessed EARR after only 6 months, a considerably shorter timeframe compared to our study’s treatment duration. These discrepancies in tooth type and treatment duration likely contributed to the observed differences in outcomes. Both physiological and pathological root resorption involve a regulated interplay between osteoclasts and osteoblasts, as well as odontoclasts and odontoblasts . Stimuli, such as localized increases in cytokines, activate T-cells, leading them to express RANKL. This, in turn, triggers the activation and differentiation of pre-odontoclasts. Fibroblasts and odontoblasts communicate through bioactive neuropeptides. Cytokines, prostaglandin E2, tumor necrosis factor-alpha, and hormones produced by the compromised periodontal ligament stimulate RANKL expression in fibroblasts. These factors exert their influence through vasoactive, chemotactic, and cellular effects. This cascade of events ultimately leads to the recruitment of active odontoclasts, initiating the process of root resorption . Despite a scarcity of scientific data on EARR in RFT, the debate on their response to orthodontic forces persists . Khan and Kumar, in a study involving 30 patients, suggested an increased risk of EARR in RFT . Conversely, Llamas-Carreras et al. found no significant difference in the degree of EARR between RFT and VPT . However, a recent meta-analysis yielded a contrarian conclusion, indicating significantly less EARR in RFT teeth compared to those with VPT . Yoshpe et al. even proposed the potential of endodontic procedures to manage or prevent EARR during OT. Yamamoto et al. reported that compromised and stressed pulp cells secrete inflammatory cytokines, receptor activator of NF-κB ligand (RANKL), and macrophage colony-stimulating factor (M-CSF), initiating odontoclastic activity. However, the absence of a pulp would negate the secretion of these factors, potentially explaining the increased EARR observed in VPT. Further investigation is warranted to elucidate the underlying causes of this disparity in EARR between RFT and VPT . A limitation of this study was the use of digital PRs to evaluate tooth length changes and root surface measurements. PRs have been employed in previous orthodontic EARR evaluations . While periapical radiographs and 3D imaging (cone-beam computed tomography, CBCT) offer greater accuracy, PRs are considered a less precise but more readily available option . Serial periapical radiographs and 3D imaging are not routinely used during OT. In addition, 3D imaging boasts higher accuracy and repeatability compared to 2D imaging in root resorption assessment. CBCT offers advantages in diagnosing and measuring root resorption but emits a higher radiation dose . Although PRs may overestimate or underestimate actual root loss due to potential distortion, they were chosen due to their routine use in orthodontic monitoring, allowing for retrospective analysis in this study . Here, we focused on comparing pre- and post-treatment radiographs to assess relative changes in root lengths and root surface measurements, rather than obtaining exact measurements. Stramotes et al. suggested that PRs taken at different times can provide sufficiently accurate linear measurements if the occlusal plane remains similar and tooth angulation doesn’t exceed 10 degrees. To minimize errors in this study, a radiology assistant used the same panoramic machine with a standardized head position ensured by the machine’s positioning light. Another limitation of this study is that the use of immediate post-treatment radiographs. Without a long-term evaluation, it is impossible to determine if the orthodontic effects of either treatment modality undergo spontaneous repair mechanisms over time . Future research designs should incorporate such a comprehensive assessment. Additionally, our investigation focused solely on mandibular molars. EARR patterns may differ in other tooth groups, highlighting the need for future studies to evaluate the full dentition. Moreover, the CA group received treatment solely with the Clear Correct system, whose aligners are made from a single plastic material. Material variations can influence the modulus of elasticity, potentially leading to disparate effects on the root apex from identical tooth movements. Therefore, different results may be obtained with treatments using different types of aligners. Additional studies, both retrospective and prospective, using larger sample sizes and advanced diagnostic imaging techniques such as CBCT are necessary to corroborate these findings. Several strengths differentiate our study from the existing literature. First, we employed a sufficiently large sample size, crucial for identifying both clinical and statistical significance. Furthermore, meticulous application of inclusion and exclusion criteria ensured standardized and comparable groups. Second, root surface measurements were examined along with EARR and proportions, and our linear measurements corroborated the root surface measurements, mitigating limitations associated with the absence of 3D imaging. Third, we assessed the impact of OT type and pulpal status on EARR within a single study. This approach is novel in the context of CA therapy, and we believe our findings offer a significant contribution to the field. Finally, whereas prior studies often focused on incisors, we opted for mandibular molars to promote standardization and address the scarcity of research dedicated solely to molars.
Within the limitations of this study, this study has several conclusions: All teeth exhibited varying degrees of EARR based on pre- and post-OT radiograph comparisons. While CAs may not eliminate EARR during OT, the prevalence and severity were lower compared to FAs. However, treatment selection remains a clinician’s judgment based on individual cases. A comparative analysis revealed that RFT, irrespective of OT modality (CA treatment or FA treatment), exhibited greater resistance to EARR than VPT counterparts. Consequently, the potential for EARR in RFT may be a less critical factor in OT planning.
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Update and European consensus on a patient-centered core outcome set for multiple myeloma in clinical practice and research | 54b7abdc-eb44-4e46-bff8-079759b5e29a | 11141662 | Patient-Centered Care[mh] | Supplementary Appendix
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Recognizing the Benefit of Telemedicine Before and After COVID-19: A Survey of Pediatric Surgery Providers | 34f077be-8079-4f2c-8024-58ec563263e0 | 8141788 | Pediatrics[mh] | An increase in access to reliable high-speed internet services combined with a growth in the number of communication platforms have made real-time, remote interaction possible for most Americans. , Within the healthcare domain, these technologies have been leveraged by providers to reach a growing number of potential patients through the use of telemedicine. Across the United States, the use of telemedicine has been increasing for more than a decade. In 2016, it was estimated by the department of Health and Human Services (HHS) that more than 60% of healthcare institutions made use of some form of telehealth, which includes telemedicine and other platforms that support patient-to-provider communication. Amongst pediatric surgeons, the use of telemedicine remained rare prior to the COVID-19 pandemic, despite evidence that supports video visits as a safe and effective method for evaluating patients in the pre- and post-operative phases of care. The low adoption rates by surgeons have been attributed to factors such as a lack of compensation, unfamiliarity, and security concerns. However, these limitations have not been fully explored in the scientific literature. At our institution, there was no consistent use of telemedicine in any surgical divisions prior to the COVID-19 pandemic. To curb the spread of the virus, restrictions were put in place in the early stages of the pandemic to limit physical contact between patients and providers in nonemergent settings. There was a rapid rollout of telemedicine across the institutional to replace in-person outpatient visits. The aim of this study was to assess the perspectives of surgical providers towards using telemedicine, defined for this study as either synchronous video encounters or synchronous audio only encounters, to evaluate and care for patients. Prior to the expanded use of telemedicine, a survey was distributed to surgeons with the goal of ascertaining their interest and perceived potential barriers for using telemedicine to care for pediatric surgical patients. Following the rapid expansion of telemedicine, a second survey was distributed to determine if the increased use of telemedicine impacted the perspectives of surgical providers at our institution.
An overview of telemedicine at our institution Pre-COVID-19 survey development Pre-COVID-19 survey distribution Post-COVID-19 survey development Post-COVID-19 survey distribution Telemedicine use within pediatric surgery Analysis The terms “telemedicine” and “telehealth” have been defined in my ways and may be used differently depending on the organization and the context. For all survey material and for this manuscript the term “telemedicine” refers to a synchronous encounter between a provider and a patient and can include video and audio or audio only (phone) appointments, unless otherwise specified. At our institution, the decision to support telemedicine use within a specific division was made at the organizational level. Once approval was granted for use within a division, it was at the discretion of the individual providers to decide how to implement telemedicine into their practice. Prior to COVID-19, telemedicine parity laws had not been enacted in our state, leaving decisions regarding reimbursement for telemedicine services to the individual insurers. All video appointments were carried out using software embedded within the electronic medical record, which can be accessed by computer or with a video capable phone. Before patients could complete a telemedicine appointment, they first provided authorization for remote access to the electronic medical record. During the video visit, patients and providers were connected remotely by the embedded application. The functionality of the software is limited to video and audio sharing, and there were no healthcare team members present with the family to assist with the appointment.
Before the COVID-19 pandemic, our group was in the process of assessing interest in telemedicine across the surgical divisions at our institution. As a part of that process, a survey was developed to determine the perspectives of surgical providers on the use of telemedicine within their practice. The aims of the survey were to determine the perceived barriers and potential benefits of using telemedicine to evaluate and care for pediatric surgery patients. Assessments of surgeonthe opportunity to write clarification or other comments. Modifications to the survey were made following feedback from members ofwith a waiver of written consent. The complete survey has been included as Appendix A.
On March 27, 2020, an invitation to complete the pre-COVID survey was distributed by email to all pediatric surgeons at our institution. The following divisions were included: General Pediatric Surgery, Cardiothoracic Surgery, Colorectal Surgery, Dentistry, Gynecology, Neurosurgery, Otolaryngology, Orthopedic Surgery, Plastic Surgery, and Urology. Clinicians who chose to participate completed REDCap surveys online via a secure network. To reduce the potential for bias or misreporting, responses were anonymous.
Following the implementation of telemedicine at our institution, we developed a survey for surgical providers to determine the impact of the increased use of telemedicine on their perceived barriers and interest in continuing the use of telemedicine beyond the restrictive period. The pre-COVID differed from the post-COVID survey due to the difference in the aims of the two surveys and to account for the difference in context regarding telemedicine use when the studies were distributed. The assessments of comfort using telemedicine and perceived cost used a 5-point Likert-type scale, and all other questions were presented at the categorical or ordinal level. Participants were given space at the end of the survey to write clarification or other comments. Modifications were made following feedback from the Department of Pediatric Surgery, resulting in the final list of questions. The survey was reviewed by the local IRB and approved for distribution with a waiver of written consent. The complete survey has been included as Appendix B.
On July 1, 2020, an invitation to complete the post-COVID survey was distributed via email to all attending surgeons and advanced practice providers across all pediatric surgical specialties at our institution. The choice of how to utilize telemedicine was made at the level of the divisions. It came to our attention after the pre-COVID survey had been distributed that the divisions of gynecology, neurosurgery, otolaryngology, and urology had extended the use of telemedicine to advanced practice providers (APP) while others had limited the use of telemedicine to surgeons. To be inclusive of all providers who were responsible for evaluating surgical patients the decision was made to send the post-COVID survey to APPs as well as surgeonsTo combat the spread of COVID-19 at our institution, a restriction on in-person contact was put in place on March 16, 2020. The restriction was eventually lifted on June 25, 2020. The medical record at our institution was queried to identify all outpatient clinic visits that were completed from January 1, 2020 to July 10, 2021. Telemedicine appointments were identified by using the following current procedural terminology (CPT) codes: 280097 (telehealth phone call new patient), 280098 (telehealth phone call follow up), 280095 (video visit new patient), and 280096 (video visit follow up).
Descriptive statistics were generated and percentages and frequencies were calculated for categorical variables. Comparisons between the pre-COVID and post-COVID periods and between the use of video or phone telemedicine were made using chi square or Fisher exact tests as appropriate. Significance was determined at a P value of <0.05.
Response rate and demographics The Impact of COVID-19 on telemedicine use within pediatric surgery The perceived barriers for implementing telemedicine in pediatric surgery The desire for ongoing use of telemedicine in pediatric surgery The pre-COVID survey was completed by 37 of 69 (54%) surgeons, and the post-COVID survey was completed by 36 of 83 (43%) providers . Pediatric general surgery was the most represented division in both the pre- (25%) and post-COVID (33%) phases. Most providers reported having been in practice between 2-10 years (pre-51.3%, post-44.4%). In the pre-COVID group, 21.6% of surgeons reported previous experience with telemedicine, compared to only 5.6% of providers in the Post-COVID group. Before the COVID-19 pandemic, at least half of the time, providers scheduled in-person post-operative appointments for patients undergoing ambulatory (56.7%) or inpatient (64.8%) procedures.
Prior to March 27, 2020, there was no use of telemedicine within the surgical divisions at our institution. During the restrictive period, surgical providers had reduced clinics and saw fewer patients each week as a result . For 44.7% of providers, at least 75% of visits during the restrictive period were performed using telemedicine. In response to the restrictions put in place from March 16, 2020 to June 25, 2020, to combat the response of COVID-19, the use of telemedicine to complete outpatient clinic appointments increased dramatically compared to the use of telemedicine in 2019 . Use by division varied widely during the restrictive period, with surgical dentistry completing zero appointments compared to more than 2000 for otolaryngology providers . Following the removal of the restriction on in-person encounters, there was a sudden decrease in the use of telemedicine across most divisions .
The expanded use of telemedicine coincided with a lessening of provider concerns regarding patient and physician satisfaction and was associated with a marginally significant increase in provider confidence in the ability to obtain an accurate diagnosis through video . Providers listed issues with compensation, staffing, and workflow as potential areas of concern in both the pre- and post-COVID periods. Of note, six of the eight respondents who choose “other” on the pre-COVID survey stated “the ability to do a physical exam.” Additional issues that were mentioned by respondents who selected “other” concerns included technology issues (five respondents) and confidentiality (two respondents).
Prior to COVID-19, 83.8% of surgeons were interested in adding telemedicine or expanding the role of telemedicine in their practice. Following its expanded use during COVID-19, 94.4% reported that they would prefer to continue offering telemedicine appointments . There were no differences in respondents’ choice of video or phone telemedicine appointments for completing appointments for initial evaluation, postoperative follow-up after ambulatory surgery, or postoperative follow up after an inpatient stay . However, when asked to rate their confidence level with completing various tasks, providers were more confident when using video to obtain information to make a common diagnosis, order the appropriate diagnostic study, prescribe the appropriate medication, and relay information to the patient in a way that is easy to understand
Despite concerns regarding workflow disruptions and compensation, pediatric surgical providers perceive the benefits of telemedicine to outweigh the potential barriers, leading to overwhelming support for its continued use in the evaluation and management of patients. Following a period of expanded use, support for telemedicine grew from 84% to 95%, and there was a marginally significant decrease in the proportion of providers concerned with the ability to obtain an accurate diagnosis. The COVID-19 pandemic has provided the impetus to evaluate the resiliency and effectiveness of the healthcare delivery model in the United States, including the current options for patients seeking evaluation from a surgeon. Previous studies have demonstrated the safety and effectiveness of using telemedicine to evaluate surgical patients and to reduce the disparity in access to specialists that exists across the United States. , Now more than ever, patients and providers are familiar with telemedicine and supportive of its use. Lessons learned during the COVID-19 pandemic can help guide future decisions regarding how best to incorporate telemedicine into a surgical practice. The restrictions on in-person contact during COVID-19 required surgeons to adjust their approach to patient care and provided an opportunity for many to gain first-hand experience using telemedicine in a surgical practice. Following the expanded use of telemedicine, there were only two areas that were identified by more than 40% of respondents as potential issues – compensation and workflow disruption. In a 2016 report on telemedicine from the Department of Health and Human Services (HHS), it was speculated that the regional discrepancy in telemedicine use was due in part to insurers’ uneven compensation policies. Inconsistent reimbursement policies may affect organizational support, potentially limiting opportunities for surgeons seeking to offer telemedicine to their patients. In the years following the HHS report, the financial landscape has changed in favor of telemedicine. With the 2018 Bipartisan Budget Act, telemedicine became a supported option for patients covered under Medicare Advantage. Despite movement in a positive direction, inconsistencies amongst private insurers were common prior to COVID-19. In recognition of the importance of telemedicine use during the COVID-19 pandemic, federal legislation was passed to temporarily expand the previously established compensation practices to include a wider group of patients covered through government-supported insurance. In addition, it was stipulated that video appointments were to be reimbursed at the same rate as in-person appointments. , The growth of telemedicine coincided with the adoption of policies that incentivized institutions to support telemedicine services, but many of these policies were temporary. The value of telemedicine is predicated in part upon it being a widely accessible technology, and continued use beyond the COVID-19 period will not be possible without organizational support. Ensuring availability beyond the pandemic will require the adoption of permanent legislation that includes both private and public insurers. To this end, research on the impact of telemedicine on patients and providers during the COVID-19 pandemic will be essential for maintaining the support needed to continue its use. The long-term success of telemedicine within pediatric surgery will require acceptance from both patients and providers. There is strong evidence that patients are highly satisfied with telemedicine, and in some instances even prefer it to the traditional in-person encounter. The patient perspective is important, but without surgeon buy-in the potential impact is limited. In our study, nearly 60% of respondents in both the pre- and post-COVID groups reported workflow disruptions as a potential issue with trying to use video appointments within their surgical practice. At our institution, the decision to expand the use of telemedicine was made to provide surgical providers with an opportunity to continue caring for their patients during a period of hospital-wide restriction on in-person contact. Following the removal of the restriction on in-person contact, telemedicine use dropped precipitously across most divisions. Possible explanations for the abrupt decline include patient or provider preference as well as decisions that were made at the level of the organization or individual division. It is not possible to delineate further with the currently available data, but future studies should be designed to evaluate the impact of telemedicine across all levels of the hospital system. There has been an explosion in the presence of technology within healthcare in recent decades, but most of these changes occurred slowly over time. For many surgical providers, the transition to telemedicine during the COVID-19 pandemic was abrupt, providing little time for adjustment. The dangers of not considering the long-term impact on providers when implementing new technology can be exemplified through studies examining the provider perspective on the electronic health record (EHR). Use of an EHR was strongly incentivized in 2009 with passage of the American Recovery and Reinvestment Act. Early support of the EHR was high amongst providers, but there were concerns about its impact on workflow, efficiency, and patient-provider communication. , Now, the EHR is viewed as a major contributor to provider burnout and use of the EHR is inversely related to physician satisfaction. , , Like the EHR, telemedicine is a tool with the capability of improving the patient experience. However, unlike the EHR, there is no federal policy in place to either mandate or incentivize its long-term use. It is imperative for pediatric surgeons and other provider. Our study included providers from ten different pediatric surgery divisions. Not surprisingly, there was a wide range of responses regarding the types of patient encounters that occur in each practice under normal working conditions. For surgical patients that require in-office procedures, such as those needing in-person treatment from an oral or orthopedic surgeon, the utility of telemedicine will be limited. Telemedicine is not a direct substitution for an in-person appointment with a surgeon. Given the currently available technology, telemedicine is also of limited value for ophthalmologists and other providers who require specialized equipment to complete most evaluations. For surgical patients who do not require an in-office procedure or the use of specialized equipment, video offers a high level of confidence to providers when completing most tasks. When compared to audio alone, video was viewed by providers as superior for developing a diagnosis and for communicating with the patient and their caregivers. The use of telemedicine is not without limitations, but remote evaluation by video is widely supported by pediatric surgeons and should be considered an option for evaluating and treating patients in both the pre- and post-operative phases of care. This study is not without limitations. Our goal with the post-COVID-19 survey was to obtain a sample of responses across all the pediatric surgical divisions at our institution. This included more than 60 providers, many of whom were not directly involved with the research. The survey used was anonymous and the sample size relatively small, leading to the possibility that the results of this study are not generalizable to all institutions or pediatric surgical providers. The use of an anonymous survey that was devoid of most identifiers, including respondent age and sex prohibited attempts to match responses between the two cohorts, making it difficult to evaluate unexpected findings in the pre-and post-COVID survey. For instance, a higher percentage of respondents reported previous experience with telemedicine in the pre-COVID group than in the post-COVID group. Without the ability to match responses, it is not possible to determine if the unexpected difference in reported previous experience was the result of recall bias or the product of a small sample size. Future studies should examine the outcomes of patients evaluated using telemedicine and determine whether there were errors in diagnosis or changes in operative plans after in-person evaluation.
Following the expanded use of telemedicine with pediatric surgery, there was a decrease in the concern for inaccurate diagnoses and a near uniform desire to continue its use. Going forward, it will be imperative for pediatric surgeons to take an active role in creating a process for implementing telemedicine that best fits their individual needs and the needs of their patients and their patients’ families.
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Beyond physical accessibility, bypassing health facilities offering caesarean section: insights from women in Dakar’s slums | f75c3c88-63e2-4dfa-8982-9b66f22fb857 | 11931907 | Surgical Procedures, Operative[mh] | The article highlights the particular challengocus on caesarean sections, whereas previous studies on bypassing of health facilities have mainly addressed delivery without specifying the type. We use modelled travel time rather than straight-line distances to determine the accessibility of Comprehensive Emergency Obstetric and Newborn Care (CEmONCs) and the additional travel time taken by bypassing women. Our model does not take traffic into account, which could be challenging in urban settings such as Dakar. The article focuses only on caesarean section, which limits the generalisability of the results to other forms of obstetric or medical care.
Physical accessibility to health facilities remains a major challenge in sub-Saharan Africa, where 70% of maternal deaths were recorded in 2020 and where the neonatal mortality rate is the highest in the world. Distance from health facilities is one of the main factors affecting maternal and neonatal mortality in low-income and middle-income countries. It leads to underutilisation of maternal health services leading to increased home deliveries, particularly in the absence of skilled health providers, and a higher risk of maternal deaths. In Kenya, for example, women are willing to give birth in a health facility if it is 2 km away. Beyond this distance, women have the same probability of giving birth at home or in a health facility. A similar finding has also been documented in Eastern Ghana. Additionally, findings from Ghana also indicate that women are willing to bypass the closest health facilities and spend more than 2 hours on the road to give birth in other health facilities, in order to benefit from a better quality of service. Bypassing health facilities is a recurring phenomenon and reflects the inefficiency of a health system. Several studies have addressed this issue and argued that the choice of place of delivery is often explained by the search for better quality care in higher level hospitals when the closest health facilities offer lower quality of care and underskilled staff. However, most of these studies did not specify the type of delivery (caesarean section vs normal delivery) and focused mainly on rural areas. Women in urban slums, despite the perceived advantages of urban life, may experience similar or even greater challenges. In addition, studies have used secondary data from specific research projects such as clinical trials, routine data, demographic and health surveys (EDS). The use of primary data, collected through surveys whose main aim is to provide evidence on the problem of accessibility to health facilities and the reasons why women bypass them, is limited. In order to improve access to emergency obstetric care in Senegal, c-section fees exemption was introduced in 2005 in five pilot regions. The policy was later extended to referral hospitals in other regions, excluding Dakar. In 2013, this policy was extended to the Dakar region, where the c-section rate is at 18.4%, compared with 11.4% at the national level. However, although Dakar has the highest concentration of high-level public health facilities (EPS) and is the wealthiest region in the country, little conclusive evidence has been produced to understand the access of poor women, mainly located in slums, to these EPS, especially to perform caesarean sections when medically necessary. This study aims to assess the physical accessibility of women living in the slums of Dakar to emergency caesarean section and to document the factors influencing their choice of health facility. Specifically, we aim to determine the percentage of women who bypass the nearest health facility, understand their reasons and formulate policy/programme recommendations to improve Comprehensive Emergency Obstetric and Newborn Care (CEmONC) services. This pioneering study in Dakar and in West Africa examines expected and observed flows between slums and CEmONC facilities while identifying direct and indirect factors contributing to bypass behaviour.
Study type and area Definition of slums Data Travel time modelling Statistical analyses The study is a spatial, quantitative, cross-sectional and retrospective analysis. Data were collected from health facilities performing caesarean sections and from women who underwent caesarean section between July and December 2022, which is 6 months before the start of the survey. The study area was limited to Dakar and seven of the region’s 10 health districts. The remaining three health districts were excluded due to their very low caesarean section rates. Each surveyed health facility was geo-referenced, with data on indicators enabling comparative and spatial analysis
We defined slums as irregular settlements and traditional neighbourhoods, based on the classification provided by Ndiaye and the Dakar’s 2025 Urban Development Strategy Document. Initially, we provided the supervisors who processed the delivery rooms and operating theatres registers of the surveyed health facilities with a list of identified slums to help them identify women living in eligible neighbourhoods. We then established the survey quotas for each health facility and coordinated appointments with randomly selected women who agreed to participate in the study ( ).
Two data sources were used in this study. The first was the database from the delivery rooms and operating theatres in six public health facilities, representing 32% of public health facilities performing caesarean sections in Dakar during our study period. The selection criteria included geographical location, type of health facility (hospital or health centre), number of caesarean sections performed and completeness of data on women having given birth by caesarean section between July and December 2022. This period was chosen because it enabled the selection of a representative sample of the target population while avoiding the excessive time required to analyse records spanning a longer period. A total of 763 women who had undergone a caesarean section in the six target health facilities and who lived in the slums of Dakar were identified. For each woman, we recorded her place of residence and the place where she gave birth by caesarean section. This information was used to estimate the road network distances and travel times covered by women to access emergency caesarean sections. Among these women, we conducted in-depth surveys with 108 participants, representing 14% of the total. This survey constituted our second data source and aimed to gather information on the sociodemographic characteristics of the women as well as on the reasons that influenced their choice of health facility for their caesarean section.
Motorised travel times were estimated using ArcGIS Online proximity tools from the slum location to the health facilities. For the nearest health facility, we used the ‘Find Nearest’ tool to estimate travel times between slums and the nearest health facility providing caesarean services. We then used the ‘Connect Origins to Destinations’ function to model travel times from slum locations to the health facilities where women underwent caesarean section. Environmental Systems Research Institute (ESRI) estimates the fastest travel times between locations using road network analysis in ArcGIS online with a high-quality cloud-based global road network. In locations like Senegal where ArcGIS lacks real-time traffic data, historical speed data, road conditions and average travel speeds are used to model travel times between locations. The driving times implement rules like one-ways, compliance with local restrictions and other road regulations. The maps were displayed using freely available OpenStreetMap basemap and administrative border outlines from Geoboundaries.
Bivariate and multivariate analyses were conducted. The bivariate analysis focused on the number of bypasses per health facility and explanatory reasons. The multivariate analysis used in this study was multiple correspondence factorial analysis (MCA), a tool for examining relationships between two or more qualitative variables without assigning them the roles of dependent and independent variables, as in regression analysis. The variables used to produce the MCA included the health facility where the caesarean section was performed, the woman’s travel time, the administrative location, the name of the slums, the associated health facility and the reasons behind the women’s choice.
Geographic accessibility Reason for bypassing caesarean care and show the expected flows from slum locations to the nearest health facility offering caesarean section services. The average travel time to the nearest health facility providing caesarean services was 6.3 min (SD: 4.6 min), with travel times ranging from 1.3 to 19.2 min. The majority of slums, 27 (60%), were within a 5 min by car of the nearest health facility offering caesarean services, while a few, 4 (8.9%), were more than 15 min away. Most women, 657 (88.4%), used a health facility within their district, while the remaining women travelled to another district for care. All the women closest to a health facility outside their district were from Pikine. Women travelled from Pikine to Rufisque (10), from Pikine to Dakar (6) and from Pikine to Guédiawaye (70). All the districts assessed had health facilities offering caesarean services. The average travel time for women whose nearest health facility was within their district was 6 min, compared with 11 min for those who had to travel outside their district to reach the nearest health facility. shows the actual movement of women from their slum to the health facilities where they underwent caesarean care. The average distance along the road network from the slums to the nearest health facility providing caesarean services was 3.8 km (SD: 4.4 km). The distance ranged from 0.5 to 24.4 km, with a median of 2.3 km (IQR: 2.4). Most slums, 31 (68.9%), were within 2 km of a health facility, compared with only 2 (4%) that were 10 km or more away. Regarding caesarean service utilisation patterns, 44.3% of women did not use a health facility within their district. Among women who bypassed their nearest health facility, more than half, 309 (51.1%), travelled outside their district for care. In contrast, 138 (87.3%) of those who did not bypass sought caesarean care within their district. Bypassing was more widespread in Dakar than in other districts. In Dakar, 222 (96%) of the women originating from Dakar bypassed a caesarean service within the city. There was also significant movement of women from other districts to Dakar, with 34 women from Guédiawaye, 174 from Pikine and 10 from Rufisque travelling there for caesarean services. Women from Rufisque primarily used a health facility within their district. The average travel time for women who travelled within their district to seek caesarean services was 10 min, compared with 16 min for those who had to travel outside their district to reach their health facility for care.
Medical referral is the main factor explaining where the caesarean section was performed ( ). It represents 64.7% of cases among women who did not bypass the nearest health facility and 43.2% among those who did. Women who bypassed their nearest health facility are more likely to seek higher quality care (13.5%) than those who did not (11.8%). They also rely more on their social or family networks to choose a caesarean section facility (14.9%) than those who did not bypass. A few women who bypassed their nearest health facility cited seeking more affordable treatment costs as their choice (1.4%). The MCA ( ) confirms the bivariate analysis and provides deeper insight into the factors associated with the observed flows. The factorial analysis highlights three major trends: The first trend focuses on slums located in the health district of Dakar South (eg, Fass and Rebeuss). These areas are characterised by a high density of health facilities performing caesarean sections. The geographical proximity of these health facilities is the primary factor explaining the women’s choice to use them. These women typically spend less than 15 min travel to undergo their caesarean section, often without leaving their municipality, and in some cases, even their neighbourhood. The second trend involves three health facilities which effectively attract women from nearby slums. Few women living near these facilities bypass them to seek care elsewhere. These facilities also serve as medical referral points for women coming from other municipalities. Women who had a caesarean section at these facilities typically spent between 15 and 30 min travelling. The third trend involves slums concentrated in the suburbs and other densely populated neighbourhoods of Dakar. This trend is characterised by flows where women bypass nearby health facilities to seek care at CS Gaspard Camara or health facilities in the Dakar South district. Women affected by these flows often spend more than 30 min, and sometimes more than an hour, travelling to reach the facility where they had their caesarean section. Medical referral is the primary factor explaining this trend. These areas are marked by a high population density, and the available health facilities performing caesarean sections often lack sufficient capacity. In emergency cases, these facilities refer parturients to higher level hospitals or those with available beds. Reasons for bypassing may also stem from family or social networks, or from women’s belief that health facilities in Dakar provide superior quality care for childbirth.
This paper examines the geographical accessibility of caesarean care in health facilities near slums in the Dakar region that offer caesarean sections. The first key finding reveals that the majority of slums are located within 6.3 min of motorised travel time (3.8 km along the road network) of a health facility providing caesarean sections. This indicates good geographical accessibility, as women can access these services within 30 min. This result aligns with findings from a study, which also demonstrated the relatively high level of access to caesarean section services in the slums of the Senegalese capital. This accessibility can be attributed to efforts by the Senegalese government since 2005 through the free caesarean section policy, which aims to overcome the physical and financial barriers that have often made this essential obstetric service inaccessible to disadvantaged populations. Additionally, this policy is supported by the National Health and Social Development Plan, which sets each decade the priority areas of health intervention for the State of Senegal. Despite the good geographical accessibility, the majority of women bypass health facilities offering caesarean sections that are closer to them in favour of those further away. Several factors contribute to this bypass, the primary reason being the medical referral. Contrary to what one might assume, there is no formal referral system for caesarean sections in the Dakar region. All health centres with a functional operating room and expertise in obstetrics and gynaecology are capable of performing this procedure. However, referrals only occur when the facility where the patient initially goes does not have available space or when its operating room is not functional. Therefore, it is usually the patients themselves who decide where to go for their caesarean section, sometimes with advice from nursing staff, based on criteria such as the reputation of the facility, the perceived quality of care, or geographical proximity. For example, the medical referrals observed in the slums served by EPS 3 Pikine (district of Pikine) can be attributed to the high demand for services at this facility, regardless of the women’s place of residence, while the number of available beds is limited. According to data from the Dakar medical region ( ), EPS Pikine recorded the highest number of vaginal deliveries (3334) and caesarean sections (2144) in Dakar in 2022. This totals 5478 deliveries, or 15 per day (including weekends), with six caesarean sections and nine vaginal deliveries. However, EPS 3 Pikine had only 35 beds for parturients in the obstetrics department and six gynaecologists at the time of our survey. Since women who undergo a caesarean section must stay in the facility for at least 3 days, and those who deliver vaginally can stay for 2 days, EPS Pikine is often forced to shorten the length of stay for new mothers. This leads to the referral of patients to other health facilities with available beds. This situation highlights the high demand for EPS 3 Pikine, which can lead health posts to refer caesarean section cases to other facilities and, conversely, women bypassing EPS Pikine in favour of less crowded facilities offering higher quality care. A similar pattern is observed in health centres located in densely populated areas, which each performed between 900 and 1200 caesarean sections in 2022, more than several hospitals. The fact that women bypass nearby health facilities in favour of more distant ones can be explained by the concept of spatial autocorrelation, as outlined by Filiponi and Manghera, who argue that two nearby locations are more likely to share similar characteristics than two distant ones. Furthermore, it is evident that the flow of women tends to head towards health facilities in the Dakar district, rather than the other way around. These facilities, therefore, attract women living in the slums, highlighting the central role of health services in more urbanised areas. The phenomenon of women bypassing the nearest health facilities for childbirth is not limited to Dakar and its slums; similar patterns have been observed in other parts of Africa and Asia. In Ghana, for instance, women often bypass nearby health facilities that provide poor quality of care, choosing instead to travel to hospitals much further away that offer better services. Their decision is often based on the availability of qualified personnel and essential medicines, and they are willing to spend more than those who choose local facilities. A study by Dotse-Gborgborts et al in the Eastern region of Ghana found that 56% of women were willing to spend more than 2 hours on the road to give birth in higher level hospitals, rather than using the nearest primary health facility. Similarly, in Tanzania, numerous studies have highlighted the tendency of women, particularly from rural areas, to bypass primary health facilities in favour of government hospitals or private religious clinics. The likelihood of bypassing increases for primiparous women, those with complications or those who perceive poor quality of care at the nearest facility. However, this risk decreases if the health facility has recently been renovated or has functional EmONC (Emergency Obstetric and Newborn Care) services. In rural Mozambique, women who bypassed health facilities tended to be older or from wealthier social backgrounds. In Kenya, the tendency of bypassing health facilities is more common among urban women, who avoid nearby facilities due to poor quality of care and instead opt for higher level hospitals further away. In contrast, women from rural or remote areas generally choose institutional delivery only if the nearest health facility is located within 2 km. Beyond quality of care and geographical proximity, financial factors can also influence the decision to bypass a health facility. A study by Gauthier and Wane in Chad examined the individual behaviour of women who bypass health facilities and the factors influencing their choice. The findings indicated that poorer women bypass high-quality health facilities in favour of those they can afford, while wealthier women bypass lower quality facilities in search of better care. The phenomenon of bypassing nearby health facilities for childbirth is widely documented in Asia and Africa. A study by Tappis et al in Afghanistan shows that although many women still give birth at home due to distance and transportation challenges, nearly 60% of women who had skilled assistance during their last birth bypassed the nearest primary care facility. They either delivered in a facility of the same level (10%) or travelled to more distant public or private primary hospitals (60%). In rural Nepal, more than half of the women surveyed by Shah bypassed the nearest maternity wards to give birth in higher level hospitals. In India, 37.7% of women in a study by Salazar et al bypassed the nearest health facility for delivery. As in Africa, women in Asia who bypass their local health facilities are often older, first-time mothers or belong to wealthier social categories. Common reasons for bypassing nearby facilities include poor quality of care at the nearest maternity hospitals, such as the absence of an operating room, lack of blood transfusions, insufficient medicines, unqualified personnel and the dilapidated condition of the facilities. In India, the quality of healthcare staff also affects significantly the likelihood of bypassing, with women more likely to bypass facilities that lack qualified personnel. A similar trend was observed in Japan by Aoki et al , who demonstrated that patients’ experiences with their primary care physicians correlate with the likelihood of bypassing them in favour of higher level healthcare facilities. Bypassing does not only affect women and childbirth but also other social categories and types of care. For example, a study in northern Uganda and China by Li et al highlights the bypass phenomenon in general healthcare, especially among older adults. In China, 40% of people aged 45 and above bypass primary care facilities to access higher level healthcare, often due to factors such as long travel times, higher levels of education, poor health, bad experiences at primary care facilities and the perception of better care in higher level health facilities. These findings raise questions about the geographical accessibility of health infrastructures, especially in emergency cases. Health facilities must be close to populations to prevent avoidable deaths due to delays in receiving quality care. While travel time estimates are commonly used to assess geographic access to various healthcare services, such as palliative care, emergency obstetric and neonatal care, mental health services, mammography centres and cervical cancer screening, the bypass phenomenon challenges the theory that proximity to care services ensures access. It also illustrates the difficulty in finding an appropriate methodology to study healthcare accessibility. The multiple iterations of the floating catchment area method underscore these challenges. While bypassing nearby facilities might seem intuitively risky for maternal and fetal health due to longer travel times, the situation is more nuanced. Most caesarean sections are planned in advance, with women informed beforehand if they require the procedure due to factors like fetal malposition, maternal comorbidities or other complications. Planned caesareans are typically performed at 37 or 38 weeks of gestation, reducing the risk of going into labour before the procedure. In emergency situations where labour has already started, women are generally cared for at the facility where they first present, minimising critical delays in care. Although travel time generally does not pose a significant risk in these planned scenarios, the phenomenon of bypassing facilities highlights perceived disparities in the quality of care for vulnerable populations, such as slum residents. Addressing this issue requires improving standards of care in peripheral health centres and ensuring consistent quality of care and patient reception across all facilities. Equipping these facilities with well-trained staff, supported by ongoing professional development, can help build confidence in the care they provide. Additionally, integrating patient feedback mechanisms would enable health administrators to identify areas for improvement in underutilised facilities, thereby fostering a patient-centred approach to enhancing healthcare. Travel times can be self-reported or modelled. Self-reported travel can be qualitative or quantitative. For instance, the DHS reports perceived distance as a challenge to healthcare accessibility. Also, women visiting a health facility could be asked about the time or distance they travelled. Comparatively, modelled travel times rely on using geographic topological data or censor-based data to estimate travel time and distance. Comparatively, self-reported or perceived travel time estimates are generally longer compared with modelled ones. This may explain why self-reported travel times ( ) are slightly higher than modelled travel times ( ) in this study. Furthermore, women in urban areas might find distance to care more challenging than rural women, although rural women travel longer to seek care. Therefore, this context should be considered when comparing our bypassing categories based on modelled travel times with self-reported travel times. Limitation Implication Our geographic accessibility estimation has some limitations. For medical referrals, the mapping analysis did not consider referral times/distance between home and the first health facility to which the woman went, and then referral from this facility to the health facility where the caesarean section was performed. This may underestimate the challenges of accessibility for referred women. Furthermore, although we estimated travel times, our model does not take into account the real-time traffic, which could be challenging in urban settings such as Dakar. In terms of the design of the study, exclusion of women living outside slums could be a limitation. In fact, the inclusion of women living in middle-income and affluent neighbourhoods would have added more value to the analyses, as bypassing health facilities offering caesarean sections may not be specific to slums, but may be a widespread public health problem in Senegal. The analyses did not consider women’s sociodemographic and socioeconomic characteristics, as many studies have done, to show the profiles of women who tend to bypass CEmONC facilities. Although slums are a homogeneous environment and women may share the same sociodemographic characteristics, a study that takes this factor into account, as well as other types of urban neighbourhood, could be relevant. Finally, the article focuses only on caesarean sections, which limits the generalisability of the results to other forms of obstetric or medical care.
The broader implications of bypassing health facilities on maternal-foetal well-being deserve further discussion. Bypassing can result in significant delays in accessing timely care, especially for women experiencing obstetric emergencies. Longer travel times are associated with increased risks of adverse outcomes, including maternal mortality, foetal distress, stillbirth and neonatal complications. These risks are compounded in slums with limited transportation infrastructure, where delays in reaching higher level facilities are often inevitable. In terms of recommendations, we can propose the following strategies for the government to address this issue: Strengthening the referral system: the government could improve the effectiveness and efficiency of the referral system by ensuring that lower level facilities are equipped and by enhancing communication and transportation between facilities. Improving quality of care in primary-level facilities: addressing perceived or actual gaps in the quality of care at local health facilities could reduce the need for women to bypass these facilities. Investments in staff training, equipment and the availability of essential medications could help build trust in primary-level care. Expanding emergency transport services: establishing or strengthening emergency medical transport systems, such as ambulances or community-based transport networks, could reduce delays for women needing to reach higher level facilities. Increasing awareness and education: community-level interventions to educate women and families about the importance of seeking care at the nearest facility in emergencies could help reduce unnecessary bypassing.
In Dakar, despite good access to health facilities performing caesarean sections, the phenomenon of women bypassing nearby facilities for reasons beyond their control deserves special attention. Bypassing these health facilities leads to longer travel times, higher costs for women and their families and inefficiencies in the healthcare system. The primary factors driving this bypass, medical referrals and seeking better quality care, reflect significant dysfunctions in the healthcare system. These issues can be attributed to poor quality of care, limited capacity of local facilities and lack of qualified healthcare personnel in facilities closer to the slums. This study makes several important contributions. First, it focuses solely on the bypassing of health facilities for caesarean sections, whereas previous studies focused mainly on general delivery without specifying the type of delivery. Second, it highlights the particular difficulties faced by women in disadvantaged urban neighbourhoods, as opposed to the more commonly studied rural populations. Finally, the study identifies the underlying reasons for bypassing through both women’s responses and multivariate analyses. This makes it one of the few studies to show that medical referrals are a significant factor behind bypass, suggesting that the issue is not solely under women’s control. To address the issue of bypassing in urban slums, it is crucial to improve the quality of care, increase the capacity of nearby health facilities and ensure the presence of qualified healthcare personnel to meet the needs of women in these underserved areas. Additionally, improving the medical referral system and investing in the infrastructure of local health centres can reduce the need for women to travel long distances, ensuring timely access to essential obstetric services.
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A computational tool to infer enzyme activity using post-translational modification profiling data | a0ad7066-7069-4702-a34d-77aec85a2b26 | 11751189 | Biochemistry[mh] | Protein enzymes are crucial in regulating cellular function, orchestrating activities through intricate signal transduction pathways . Dysregulation in enzyme activity has been implicated in numerous complex human diseases, such as cancer, diabetes, and neurodegeneration – . Given their central role in disease pathways, several groups of enzymes, including protein kinases, have emerged as the primary targets for cancer drugs – . However, responses to the drugs can vary significantly among individual patients, underscoring the necessity for accurate measurement of enzyme activities. Traditionally, enzyme activities have been primarily characterized through substrate events and assessed via small-scale in vitro experiments , . With advancements in high-throughput mass spectrometry (MS)-based proteomics, large-scale proteome profiling has become a powerful tool for measuring post-translational modification (PTM) events and understanding signal transduction pathways in diseases , . For instance, these studies identified 19,947 relationships of kinase-phosphorylated substrates in human, mouse, and rat , 3556 relationships of ligase-ubiquitinated substrates in human, mouse, and rat , and 503 human relationships of histone acetyltransferases (HAT)-acetylated substrate on a large scale. Despite these advancements, the analysis of PTM profiling data remains challenging due to the lack of effective tools for inferring the activities of enzymes, such as protein kinases, E3 ubiquitin ligases, and HATs. To address the challenges, several tools have been developed to derive activities for protein kinases using phosphoproteomics data. However, no tools are currently available for E3 ubiquitin ligases and HATs. These tools can be broadly categorized into three main types based on their underlying algorithms: enrichment-based methods, clustering algorithms, and machine-learning methods. Enrichment-based methods, including GSEA , KSEA , KEA , and VIPER , predict kinase activity by leveraging kinase-substrate interactions obtained from public databases (e.g., PhosphoSitePlus). While a major limitation of these methods is their reliance on prior knowledge of kinase-substrate relationships in databases, some extensions have been developed that use unbiased, fully data-driven approaches. These include the reverse-engineering of regulatory models from experimental proteomics data and even from expression profiles using ARACNe and VIPER , . However,. Clustering algorithms (e.g., CLUE) group phosphoproteomics data into clusters based on similar spatio-temporal kinetics of the phosphosites followed by an enrichment test. However, these methods often incorporate potential ‘noise’, such as low-abundance phosphopeptides and weak correlation between kinase-substrate relationships. Machine-learning methods (e.g., IKAP) attempt to globally fit a model of kinase activity and affinity to the phosphoproteomics data. However, these methods may face challenges in achieving a global optimum solution, and the derived activities from the model can vary with each computation. Structural equation modeling (SEM) has proven to be a powerful method and is extensively employed for analyzing biological networks in genomics . However, its application in proteomics, particularly for enzyme activity inference, remains largely unexplored. In contrast to existing tools for inferring protein enzyme activities, SEM offers several advantages. Firstly, it enables the inference of latent variables, such as protein enzyme activities, which cannot be directly measured. Secondly, SEM accounts for the interactions among enzymes, aligning closely with the inherent complexity of biological systems. Lastly, it incorporates measurement errors in all observed variables, such as an abundance of phosphopeptides in phosphoproteome experiments. This is especially true in MS-based PTM profiling data, where measurements inherently include errors and imperfections arising from biological and experimental variations. In this study, we aim to develop a computational tool of JUMPsem for inferring the activities of protein enzymes. The program infers enzyme activities based on enzyme-substrate relations and PTM profiling data. We then apply the JUMPsem tool to analyze kinase activities using phosphoproteome data, E3 ligase activities using the ubiquitinome, and HAT activities using the acetylome. To assess its performance, we compare JUMPsem with two widely used programs, IKAP and KSEA, for protein kinase activity. Another feature of the JUMPsem program is its ability to incorporate sequence motifs, significantly enhancing the identification of enzyme-substrate relationships. The JUMPsem program is made publicly available as an open-source R package, along with a user-friendly web-based R/Shiny application.
Design and implementation of the JUMPsem program Applications of JUMPsem to estimate kinase activity using mouse phosphoproteomic data Comparison of JUMPsem with other programs applied to human phosphoproteomic data Performance evaluation of JUMPsem using phosphoproteomic benchmark datasets Inference of activities of E3 ubiquitin ligases and histone acetyltransferases Modular and scalable R package with shiny web application We developed JUMPsem, a software tool specifically designed to infer protein enzyme activity using MS-based PTM profiling data (Fig. ). The JUMPsem program comprises three major components: (i) construction of enzyme-substrate relationships, (ii) inference of enzyme activity through a structural equation modeling algorithm, and (iii) output and comparison of enzyme activity. The program takes a quantitative table of modified peptides from PTM profiling studies as input, which can include analyses of phosphoproteome, ubiquitinome, or acetylome. Outputs from JUMPsem consist of a table containing inferred enzyme activities and a second table containing the affinities between protein enzymes and their substrates (Supplementary Fig. ). The quantitative table of PTM peptides can be generated by identifying peptides via database search, filtering PTM peptides at a user-specified false discovery rate (FDR), and quantifying PTM peptides from either labeled or label-free PTM profiling data. If the corresponding whole proteome data is available, JUMPsem provides an option for normalizing the measured expression levels of PTM peptides relative to the corresponding changes in protein abundance. This normalization allows the determination of changes in PTM state independent of variations at the protein level (Supplementary Fig. ). In the case of phosphoproteome analysis, the JUMPsem program initiates the process by extracting kinase-substrate relationship tables from public databases, such as the PhosphoSitePlus (PSP) database . Subsequently, the program constructs an adjacency matrix for those kinase-substrate relationships with detected phosphopeptides identified in the quantitative phosphoproteomics data. The adjacency matrix can be expanded to include kinase-substrate relationships derived from motif searches, which increases the depth of analyzed kinases (Supplementary Fig. ). The JUMPsem program uses the SEM algorithm to calculate enzyme activity, implemented through the lavaan R package . The core of its computation lies in the “sem” function, which fits the model by applying maximum likelihood estimation to determine the parameters. Both the adjacency matrix and PTM abundance table are used for the estimation. The outputs generated by the JUMPsem include the enzyme activity and the affinity coefficient (or connection strength) of each PTM site, representing the estimated effective connectivity with the corresponding enzyme.
We first applied the JUMPsem program to estimate protein kinase activity using the phosphoproteomic data derived from two mouse high-grade glioma (HGG) xenograft models driven by mutated receptor tyrosine kinase (RTK) oncogenes, platelet-derived growth factor receptor alpha (PDGFRA) and fusion genes of the neurotrophic tyrosine receptor kinase 1 (NTRK1), as well as from normal controls (CTRL) . Using 45,574 unique phosphopeptides quantified at the peptide FDR of <1%, the JUMPsem program detected a total of 324 substrates with phosphosites that can be found in the PSP database. The JUMPsem program estimated the activity of 67 protein kinases using 177 substrates. By comparing kinase-substrate relationships without motif-assisted prediction, the motif prediction strategy, on average, increases 14.7% (572/3896) kinase-substrate relationships (Fig. ). For example, SIK1 kinase has two substrates (CRTC3 and PDE4D) with three sites being found in the phsophositePlus database. With the motif-assisted function, we found two additional substrates (HMGA2 and NEFL). As expected, these two additional substrates showed a consistent trend, indicating that motif-assisted function selects biologically meaningful substrates instead of random assignment (Fig. ). Our analysis revealed significant alterations in the activity of 33 protein kinases between PDGFRA mice and CTRL are shared both from JUMPsem without motif assisted and IKAP, and 23 kinases between NTRK1 mice and CTRL are shared from these two tools (Fig. ). JUMPsem with motif assisted can alternatively infer 28 kinases compared with IKAP (Fig. ). These altered kinases were significantly enriched (FDR < 0.01) in cancer-related pathways (Supplementary Data ), including pathways associated with the checkpoint, and pathways in cancer and glioma. Compared to our previous analysis conducted using the IKAP program, we found a high degree of consistency in the majority of identified pathways between the IKAP and JUMPsem. Motif-assisted strategy allows for the uncovering of previously unknown kinase-substrate relationships, thereby enabling the estimation of the activity of additional kinases. By applying this strategy to the HGG phosphoproteomic data, we further revealed several kinases, such as MAPK7, STK4, DAPK1, CAMK4, MYLK, PAK2, PAK3, and ILK, which were not identified in the previous IKAP analysis. Many of these kinases were implicated in cancer-related pathways, including MAPK and ErbB pathways (Fig. ), exhibiting activity alterations without corresponding changes at the protein level (Fig. ). This motif-assisted prediction strategy enhances the scope of kinase activity analysis.
To assess the performance of JUMPsem, we compared the performance of JUMPsem with IKAP and KSEA, two widely used programs developed for kinase activity estimation, using a human phosphoproteomic dataset. The phosphoproteomic data were generated from the P31/Fuj and Kasumi-1 acute myeloid leukemia (AML) cell lines . P31/Fuj cells are deficient in PTEN (phosphatase and tensin homolog), whereas Kasumi-1 cells have wild-type PTEN, which is resistant to multiple drugs . The dataset was originally used to estimate the activity of protein kinases using the KSEA program. Utilizing a total of 4129 phosphorylated peptides quantified across the cell lines, we estimated the activity of protein kinases using JUMPsem, IKAP, and KSEA (Fig. ). When comparing the results from IKAP, JUMPsem revealed a cluster that shows a similar kinase activity pattern observed between P31/Fuj and Kasumi-1 cell lines. In addition, JUMPsem identified two unique clusters of kinase activity (Clusters 1 and 2). With a significance threshold set at a p -value < 0.05 and an absolute fold change greater than 1, we identified 75 up-regulated and 31 down-regulated kinases in activity between the P31/Fuj and Kasumi-1 cells (Fig. ; Supplementary Data ). For instance, PRKCA exhibited a notable 7.9-fold decrease in activity in Kasumi-1 compared to P31/Fuj cells. These kinases with differential activity were enriched in the pathways, such as glioma (e.g., MAP2K2), Toll-like receptor signaling (e.g., MAP2K2 and MAP2K4), acute myeloid (e.g., MAP2K2) (Fig. and Supplementary Data ).
To evaluate the performance of JUMPsem, we conducted a comprehensive analysis using benchmark data from two previously published studies , , which included 16 phosphoproteomic datasets covering a total of 31 conditions (Supplementary Data ). We assessed the performance by comparing the precision of JUMPsem and IKAP across various threshold cutoffs. Briefly, for each dataset and condition, we first processed the phosphoproteome, generated a kinase-substrate relationship table, and identified the target kinase (Supplementary Fig. ). The average kinase activity was then calculated across all replicates, and the rank of the target kinase was recorded among all inferred kinases for each dataset (Supplementary Fig. ). A true positive was defined as a target kinase whose rank was below the threshold cutoff. Precision was finally calculated for performance comparison, which was defined as the ratio of true positives to the total number of true and false positives. Overall, our analysis demonstrated that JUMPsem slightly outperformed IKAP in terms of precision (Supplementary Fig. ).
We next sought to infer the activities of E3 ubiquitin ligases from ubiquitinomic data using the JUMPsem program. The ubiquitinomic data were previously generated from cells under various conditions: unstressed, heat-stressed, or arsenite-stressed, in the presence or absence of 0.5 μM bortezomib, a proteasome inhibitor . The ubiquitinomic data were generated by the tandem mass tag (TMT) strategy from samples in response to a 60-min heat stress and following 2 h of recovery, quantifying 16,525 unique ubiquitin-modified peptides, corresponding to 4892 ubiquitinated proteins. The UbiBrowser database , which contains a total of 1599 ubiquitinated substrate proteins, corresponding to 408 human E3 enzymes, was used to construct the relationship table of E3 ubiquitin ligases and substrates. The JUMPsem program found 161 E3 ubiquitin ligases and 4892 unique substrate proteins in the database (Fig. ). Our findings revealed that 124 unique ubiquitin ligases exhibited significant alterations in their activity between the heat-shock conditions (HS) and the control condition (Ctrl), as determined by a t -test ( p -value < 0.05), with 80 showing differences between HS and Ctrl, 105 between HS and Ctrl with bortezomib treated (Supplementary Data ). For example, three ligases (i.e., TRIM28, TRIM13, and TRIP12) were among the ligases upregulated in both HS and Recovery stress conditions compared to the control with bortezomib treated (Fig. ). In contrast, two ligases, MKRN1 and TRIM25, displayed a decrease in activity in heat shock with proteasome inhibitor bortezomib (i.e., HS_Bortezomib and Recovery_Bortezomib) compared the control, respectively (Fig. ). The ligase activity showed consistency with substrate abundance. For example, TRIP12, especially after bortezomib treatment, a consistent increase in both substrate abundance and ligase activity in the HS and Recovery_Bortezomib groups compared to the control condition (Fig. ). To estimate the activity of histone acetyltransferases (HATs), TMT10 quantification techniques for global acetylation profiling, luminal and basal subtype breast cancer xenograft tissue samples were homogenized, lysed, and digested . A total of 4180 Kac peptides correspond to 3653 Kac sites, and 1804 Kac proteins were quantified. These acetylated proteins were mapped to 6 HATs in the compendium of protein lysine acetylation (CPLA) database, curated for lysine-acetylated substrates with their sites (Fig. ). The JUMPsem detected three HATs, including KAT2A, GTF3C4, and MGEA5, showed activities as inhibitors between luminal and basal xenograft samples.
JUMPsem is a modular and scalable package, consisting of distinct components designed to process specific types of PTM profiling data. This modular design allows for the future expansion of JUMPsem to incorporate other PTM data, such as methylation profiles. To streamline analysis and visualize results, we have developed a companion R/Shiny application (Fig. ) that integrates seamlessly with the JUMPsem R package. This web-based JUMPsem R/shiny application can be broadly categorized into three primary steps: loading the raw files, optionally configuring group parameters, and exploring and visualizing kinase analysis. For more comprehensive information and step-by-step guidance, the package vignettes and documentation are available online at the application website ( https://jumpsem.shinyapps.io/JUMPsem/ ).
Recent advancements in mass spectrometry-based proteomics technologies have enabled us to profile large-scale PTM data. The resulting PTM profiling data are instrumental in unraveling complex signal transduction processes and enzyme activities. Despite the wealth of PTM data generated, the interpretation remains challenging due to a dearth of tools specifically designed to handle and make sense of this complexity. Addressing this critical gap, our study introduces JUMPsem, an innovative computational tool that is capable of analyzing large-scale PTM datasets by facilitating the inference of enzyme activity. The JUMPsem program outperforms existing widely used tools, such as IKAP and KSEA, in terms of the number of enzymes it can analyze and its computational speed. JUMPsem is an effective tool for enzyme activity inference, available as an open-source R package and as an R shiny webtool. One significant advantage of the JUMPsem program over existing tools for predicting enzyme activity is its integrated motif search strategy. This strategy substantially broadens the potential kinase-substrate interactions that can be identified, going beyond the recorded entries in the PSP database. Despite the fact that proteomics technology is able to detect thousands of modified peptides in a single experiment, a mere fraction of the modified peptides is cataloged in the public database. For example, less than 10% of the quantified phosphopeptides in an MS-based experiment are represented in the PSP database. Consequently, a vast portion of the data remains underutilized for activity estimation. The JUMPsem program tackles this limitation by augmenting its motif search strategy, thereby extending the detectable range of kinase-substrate relationships beyond the confines of existing databases. This enhanced strategy permits JUMPsem to leverage a more significant segment of the phosphoproteomics data, thus facilitating a more comprehensive analysis of cellular signaling pathways. Another advantage of the JUMPsem program lies in its utilization of the SEM algorithm, which handles potential measurement errors present in all observed variables. This is particularly important in MS-based phosphoproteomics, where the measurement of modified peptides from large-scale PTM profiling data contains inherent uncertainties and is susceptible to various errors, including quantification measuring errors. In addition, a modified peptide could be incorrectly assigned to a spectrum and a modified site can be misassigned to an adjacent site due to missing informative ions in the spectrum. By accounting for these uncertainties, JUMPsem provides a more robust and reliable estimation of enzyme activities, reflecting the complex reality of biological data. One limitation of the current JUMPsem program is its design for inferring the activity of individual kinases without considering the interaction of different enzymes within complex signaling networks. These interactions could be incorporated into the SEM model. Most existing computational tools, including the current version of JUMPsem, are tailored to single kinases, which can introduce biases in activity estimation. This bias arises because changes in substrate abundance may be due to the influence of co-active enzyme partners. For instance, fluctuations in the substrate levels of a kinase might be the result of actions by other kinases. Furthermore, the PTM landscape can be influenced by a variety of factors, including kinases relocating within the cell, interactions with other proteins, or competition with de-activation enzymes (e.g., the dynamic between phosphatases and kinases). Such complexities could lead to incorrect estimation of the enzyme activity. In summary, we introduce the JUMPsem program, an innovative tool designed for inferring protein enzyme activity from PTM profiling data. The modular and scalable JUMPsem package, coupled with its user-friendly web application, makes it a valuable tool for inferring enzyme activity. We have successfully demonstrated the application of JUMPsem to three types of proteomic data—phosphoproteomic, ubiquitomic, and acetylomic—identifying and quantifying enzyme activities, as well as substrate affinities. The utility of JUMPsem in estimating enzyme activity and elucidating cellular signaling pathways marks it as an indispensable tool in the fiels Description of Additional Supplementary Files Supplementary Data 1-5 Reporting Summary
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Guidelines for the diagnosis and treatment of cutaneous squamous cell carcinoma: a GRADE approach for evidence evaluation and recommendations by the Italian Association of Medical Oncology | 23b35abb-3fab-45b9-b794-2467e9f847ec | 11067535 | Internal Medicine[mh] | Cutaneous squamous cell carcinoma (CSCC) accounts for ∼20%-25% of skin tumors. However, the precise incidence data for this neoplasm are not well-defined due to limited statistics and its frequent inclusion with mucosal forms. In Italy, the 2015 Italian Cancer Registry Association (AIRTUM) report estimated that ∼19 000 new cases of CSCC will be diagnosed in 2018, with higher incidence in males, especially after the age of 65 years, and a typical North–South gradient. In New South Wales, Australia, where one of the highest frequency of nonmelanoma skin cancers (NMSCs) has been recorded, the overall incidence rate of CSCC from 2016 to 2019 was 856 cases per 100 000 people. In the United States, estimates in 2006 reported 2.2 million people treated for NMSCs, with ∼600 000 identified as having squamous cell carcinoma (SCC). Another United States study estimated that between 4000 and 9000 patients died from CSCC in 2012. , Across Europe, in the past 20 years, there has been considerable variability in the incidence of CSCC, likely related more to differences in national case registration methods than to genuine phenotypic variation. In a 2019 national study in England, covering 2013-2015, the age-standardized rates for the first registered CSCC were 77.3 per 100 000 person-years for men and 34.1 for women. Within 36 months, 1.1% of women and 2.4% of men with this carcinoma developed metastases. Despite a favorable prognosis in over 90% of cases, some patients with primary CSCC, particularly those who are immunocompromised or face social challenges, may develop locally advanced or metastatic forms, presenting a growing clinical concern. Traditionally, chemotherapy and targeted therapy were the only available options for such cases, with limited response rates, of short duration (months) and with significant toxicities. However, recent approvals of immunotherapeutic agents, such as cemiplimab and pembrolizumab, have established immunotherapy as the standard of care for patients who are ineligible for curative surgery or radiotherapy, marking a significant advancement in the treatment landscape. , The recommendations presented here were developed using the Grading of Recommendations, Assessment, Development, and Evaluations (GRADE) approach by the Italian Association of Medical Oncologists (AIOM), aiming to provide guidance to health care practitioners dealing with patients diagnosed with CSCC. These guidelines encompass recommendations pertaining to diagnosis, treatment, and post-treatment monitoring, covering settings ranging from early-stage tumors to those that are locally advanced or metastatic. The prioritized aspects of CSCC management were identified by a panel of experts chosen by AIOM in collaboration with other national scientific societies. The application of these guidelines in routine clinical settings is anticipated to enhance the quality of patient care.
The AIOM Guidelines Panel for Cutaneous Squamous Cell Carcinoma includes clinicians with extensive expertise in dermato-oncology, hailing from all medical fields involved in the diagnosis and treatment of skin cancers (medical oncology, dermatology, surgery, pathology, and radiotherapy), in addition to members specialized in clinical research methodology. This multidisciplinary team annually updates the guidelines. Before the final publication on the AIOM website ( www.aiom.it ), the work is reviewed by external reviewers from the leading Italian dermato-oncological scientific societies (Italian Melanoma Intergroup; Italian Society of Medical, Surgical as Search strategy and quality of evidence evaluation Evidence to decision The following clinical questions all follow the PICO format, including Population (P), Intervention (I), Comparator (C), and Outcomes (O): - Question 1: Should sunscreen creams with solar protection factor 30-50 be recommended in subjects who are exposed to solar UV radiation (UVR) to reduce the incidence of CSCC? - Question 2: Should dermoscopy be recommended in subjects with suspicious skin lesions compared to visual inspection only for the detection of CSCC? - Question 3: Should chemoprevention be recommended in subjects at high risk of developing CSCC? - Question 4a: Should dermatological follow-up be carried out in immunosuppressed subjects? - Question 4b: Should clinical–instrumental follow-up be carried out in immunosuppressed subjects? - Question 5a: In patients with operable low-risk CSCC, should excision with margins ≥4 mm be recommended over <4 mm? - Question 5b: In patients with operable high-risk CSCC, should excision with margins ≥6 mm be recommended over <6 mm? - Question 6: In recurrent or high-risk CSCCs, should Mohs surgery be recommended over traditional excision? - Question 7aradiotherapy? - Question 7(b-c): In non-recurrent and operable CSCC, should surgical excision with clear margins be recommended over (b) cauterization or (c) cryotherapy? - Question 8: Should adjuvant radiotherapy be recommended after surgical excision of high-risk CSCC? - Question 9: Should sentinel lymph node biopsy be recommended in high-risk CSCC? - Question 10: Should prophylactic lymphadenectomy be recommended in high-risk CSCC? - Question 11: Should baseline radiological tumor assessment be recommended in subjects with high-risk CSCC? - Question 12: Should radiological tumor assessment be recommended in the follow-up of subjects with high-risk CSCC? - Question 13: Should concomitant chemoradiation be recommended over post-operative radiotherapy alone in patients with CSCC and histopathological high-risk factors? - Question 14: Should cemiplimab be recommended over chemotherapy for patients with recurrent and/or metastatic CSCC who are not eligible for curative treatment? - Question 15: Should concomitant chemoradiation be recommended over exclusive curative radiation therapy in patients with non-resectable CSCC? - Question 16: Should platinum-based chemotherapy be recommended over palliative care/best supportive care7The outcomes were identified by the panel members as either ‘critical’ or ‘important’, based on their degree of priority.
For each PICO, a systematic and cross-checked literature search was conducted on PubMed, Embase, and Cochrane Library [details about the search string and the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) flowchart are reported in the , available at https://doi.org/10.1016/j.esmoop.2024.103005 ]. Systematic literature reviews and randomized clinical studies were included. Where unavailable, non-randomized studies were retrieved. Narrative reviews and case reports were excluded. The quality of the evidence was assessed using the GRADE approach, which includes the evaluation of study limitations, imprecision, indirectness, inconsistency, and publication bias. Randomized controlled trials start from a high certainty, but any limitation found in one of these domains downgrades the certainty of the evidence. A judgment is then expressed among the following: high, moderate, low, and very low. A final summary of these judgments has been reported in the dedicated tables ( , available at https://doi.org/10.1016/j.esmoop.2024.103005 ).
The decision-making process is conducted and reported transparently in the evidence to decision (EtD) framework. The evidence is reported in relation to the priority of the problem while considering the certainty of the evidence, the balance of desirable and undesirable effects, patient values, resource use, equity, acceptability, and feasibility. Based on this, the panel can then formulate a vote between the intervention and the comparison: favorable, uncertain/favorable, uncertain/unfavorable, and unfavorable. The panel also votes on the strength of the recommendation: strong in favor, conditional in favor, conditional against, and strong against the intervention. The reporting of the recommendations was done according to the Appraisal of Guidelines for Research and Evaluation (AGREE) reporting checklist.
Primary prevention Secondary prevention Treatment of primary resectable CSCC Medical therapy, staging, and follow-up The prognosis for patients with CSCC is generally favorable, with a 5-year cure rate exceeding 90%. In a cohort of over 900 patients with CSCC followed for ∼10 years, 4.6% experienced recurrence, 3.7% had lymph node metastasis, and 2.1% succumbed to disease progression. In patients with >10 CSCC, the incidence of local recurrences and lymph node metastases was 37% and 26%, respectively, compared to 3% and 2% in those with a single CSCC. The risk of distant metastases is low, <5% after 5 years of follow-up in most patients. About 85% of metastases involve regional lymph nodes, while distant metastases are more frequent in the lungs, liver, brain, skin, and bones. There are no definitive recommendations regarding the use of instrumental staging procedures after the surgical removal of histologically confirmed SCC. NCCN guidelines suggest carrying out instrumental investigations for tumors with deep bone or soft tissue involvement or perineural invasion. Apart from the suggested use of magnetic resonance imaging in the presence of perineural involvement, there are no indications on the specific type of instrumental investigation to be employed. The early identification of regional nodal relapse could be of benefit in terms of surgical salvage. Therefore, according to EDF/EADO/EORTC guidelines, high-risk CSCC cases (diameter >2 cm, deep-infiltrating tumors, aggressive histology, perineural involvement, recurrent tumors, and those located on the lip or ear) should undergo nodal ultrasound every 3 months for the first 2 years, every 6 months for an additional 3 years, and then annually. Operated CSCC represents a highly heterogeneous category of disease. Within this, the definition of ‘high risk’ is used to identify a group of patients with a higher risk of locoregional or distant recurrence. This includes patients with head and neck cutaneous disease who have intra-parotid lymph nodes or cervical lymph nodes related to a primary cutaneous lesion with one or more of the following characteristics: presence of two or more lymph nodes, size >3 cm, or extracapsular extension of disease. High risk is also determined for the primary tumor when it has dimensions >5 cm (T3) or features of invasion into nearby tissues resulting in a T4 stage. For these patients, post-operative radiation therapy is suggested. However, for the definition of high risk based on T characteristics, there is no perfect concordance in the literature. EADO/EDF/EORTC guidelines define concepts only partially overlapping with ASTRO guidelines. CSCC is predominantly treated with surgery, but in cases of recurrent disease where surgical options are limited, radiation therapy may offer disease control. The presumed advantage of radiation therapy is often derived from results obtained in squamous carcinomas of the head and neck with mucosal origin. It should also be considered that in head and neck mucosal cancers, chemotherapy with platinum agents or treatment with anti-epidermal growth factor receptor (EGFR) antibodies has shown overall improvement in prognosis (both disease control and OS) compared to radiation alone. In cases of recurrent CSCC that is not amenable to curative surgical or radiation approaches, it often poses a dilemma for clinicians. Recurrence is a clinical challenge due to infections, bleeding, or pain. Additionally, patients often have comorbidities, toxicity from previous treatments, and age-related issues that may hinder the therapeutic path with chemotherapy. Due to the inherent fragility of patients with this type of disease, exacerbated by the complications created by the pathology itself, concurrent care pathways are often initiated from the beginning of treatment. In this regard, the main clinical question concerns the possibility of administering systemic oncological treatments alongside the already established best supportive care. The literature on this subject is relatively limited. An alternative to systemic chemotherapy, which can be challenging due to patient comorbidities or frailty, may be immunotherapeutic treatment. , Initial data on a relatively small sample of patients are particularly encouraging and may represent a shift in the therapeutic approach for patients in this stage of the disease. Recent years have witnessed a paradigm shift in the therapeutic landscape of CSCC with the advent of immunotherapy. Immunotherapy, particularly anti-programmed cell death protein 1 (PD-1) agents, has emerged as a groundbreaking approach in the treatment of advanced CSCC, capitalizing on the tumor’s high mutational burden and the presence of neoantigens. This transformation necessitates a multidisciplinary evaluation of each case to optimize treatment strategies in a population often burdened by severe comorbidities. The breakthrough status of immunotherapy is underscored by robust preclinical rationale, linking CSCC’s etiology to chronic UVR exposure and its subsequent high somatic mutation rate. Studies indicate a direct correlation between tumor mutational burden (TMB) and immunotherapy efficacy, with CSCC exhibiting the highest TMB among tumors. Age-related considerations, such as the increased likelihood of immunotherapy benefit in older patients, and the tumor’s elevated programmed death-ligand 1 expression further support the rationale for anti-PD-1 immunotherapy. , The CARSKIN trial and subsequent KEYNOTE-629 trial demonstrated the efficacy of pembrolizumab in unresectable CSCC, leading to Food and Drug Administration (FDA) approval. , Cemiplimab, approved by both FDA and European Medicines Agency (EMA), showcased significant clinical benefits in the EMPOWER-CSCC 1 trial, with an overall response rate (ORR) of 46.1% and a disease control rate (DCR) of 72.5%. The study highlighted the importance of considering cemiplimab as a first-line treatment, particularly in cases where curative surgery or radiotherapy is not feasible due to various factors. A retrospective study conducted in Italy further supported the real-world safety and activity of cemiplimab on 131 patients, with an ORR of 58% and a DCR of 71.7%. Clinical and biochemical factors associated with response were identified, emphasizing the need for personalized treatment approaches. While chemotherapy has historically yielded short-lived responses with considerable toxicities, immunotherapy, especially with cemiplimab, offers durable responses, improved quality of life, and a manageable safety profile. The EGFR inhibitor cetuximab, though less explored, demonstrated response rates in advanced CSCC. However, the overall efficacy and tolerability of immunotherapy make it a preferred choice in cases where curative surgery or radiotherapy is not suitable. , , In conclusion, the integration of immunotherapy has revolutionized the therapeutic landscape for advanced CSCC. Comprehensive evaluations considering patient characteristics and tumor factors are crucial for optimal treatment selection, ensuring the highest chances of long-term outcomes. The evolving evidence from prospective trials and real-world studies underscores the continued advancement and refinement of immunotherapeutic strategies for CSCC. Question 11 : Should baseline radiological tumor assessment be recommended in subjects with high-risk CSCC? Recommendation : In subjects with high-risk CSCC, a baseline instrumental staging may be considered for the detection of extracutaneous metastasesThe considered benefit outcomes encompassed the rate of extracutaneous recurrences, DFS, and OS, while complications associated with radiological procedures were evaluated as adverse outcomes. In the observational study conducted by Ruiz et al. in 2017, a cohort of patients with stage T2b or T3, consisting of 45 individuals who underwent staging procedures (for 48 CSCCs) at the initial diagnosis, was compared with 53 patients who did not undergo such procedures. Computed tomography was the most frequently used exam. Results from 65% of radiological procedures revealed abnormal findings, and in 33% of cases, the radiological procedure influenced the clinical management. There appeared to be no substantial uncertainty or variability in how the population assesses the analyzed outcomes. The balance of effects between desirable and undesirable outcomes favored the performance of staging procedures over not conducting staging. The recommended intervention seemed easily implementable, given its existing application for other types of neoplasms, even in non-specialized centers. Consequently, the distribution would likely be widespread from the outset, allowing facilities to incorporate this technique without excessively high costs or insurmountable technical–logistic difficulties. See (Question 1Question 12 : Should radiological follow-up tumor assessment be recommended in subjects with high-risk CSCC? Recommendation : Radiological follow-up tumor assessment may be considered as first option in subjects with high-riskincluded the rate of extracutaneous recurrences, DFS, and OS. The adverse outcomes involved complications related to radiological procedures. In the observational study by Ruiz et al., the percentage of lymph node metastases was higher in the group that did not undergo instrumental staging. Mortality in the two groups was statistically different, with 19 out of 45 patients deceased in the instrumental staging group (42.2%) and 32 out of 53 (60.4%) in the group without instrumental procedures (RR 0.70, range 0.47-1.05). The risk/benefit balance was assessed as probably favoring the intervention based on available data, particularly in identifying lymph node metastases. The panel deemed the issue a clinical priority, with no impact on equity given the widespread availability of clinical and instrumental investigations in every health care facility across Italy. Consequently, the feasibility of this intervention is substantially guaranteed, along with its potential acceptability by individuals and involved institutions. See (Question 123 with histopathological high-risk factors? Recommendation : Concurrent chemoradiation should not be considered as a first-line therapeutic option for patients with surgically resected high-risk CSCCwere DFS and OS, while the detrimental outcomes included incremental toxicities due to treatment and the worsening of quality of life. The radiosensitizing treatment with carboplatin was investigated in a randomized trial published in 2018 by Porceddu et al. The primary objective was the improvement of locoregional disease control. A total of 321 patients with head and neck region CSCCs were randomized. The study did not demonstrate an advantage in the primary endpoint [freedom from locoregional relapse (FFLRR)], as well as in secondary outcomes of DFS [hazard ratio (HR) 0.85 (95% CI 0.55-1.29)] and OS [HR 0.95 (95% CI 0.58-1.57)], and in quality of life. FFLRR at 2 and 5 years was 88% (95% CI 83% to 93%) and 83% (95% CI 77% to 90%) in the radiotherapy-only group, while it was 89% (95% CI 84% to 94%) and 87% (95% CI 81% to 93%; HR 0.84, 95% CI 0.46-1.55, P = 0.58) in the carboplatin + radiotherapy group, respectively. No increase in radiotherapy toxicities, such as mucositis, dysphagia, and acute or late dermatitis, was observed in the experimental arm. However, acute differences between the two arms appeared unfavorably in the chemotherapy-treated group for side-effects such as constipation, fatigue, and dysgeusia. Additionally, as expected, there were increased marrow toxicities related to the chemotherapy drug in the acute phase. Overall, the benefit-to-harm balance does not favor the addition of systemic radiosensitizing treatment in high-risk subjects after surgical intervention due to increased toxicities with no improvement in various outcome parameters. See (Question 134 Recommendation : Cemiplimab should be recommended as a first-line option over chemotherapyStrong in favor Overall quality of evidence : Low Motivation/comments on the benefit/risk balance: After carefully examining the issue and conducting a systematic literature review, the working group concluded that the recommendation proposed in the National Institute for Health and Care Excellence (NICE) guidelines addressed the question of interest, and its content was applicable to the Italian context. Furthermore, the NICE guideline was of excellent quality according to the AGREE II evaluation. For these reasons, the panel decided to adopt the NICE guideline. The panel decided to make a strong recommendation in favor of the intervention for the following reasons: - The lack of a therapeutic standard for locally advanced and metastatic CSCC had made it impossible to conduct randomized clinical trials. - In everyday clinical practice, there was currently no valid alternative therapy to the use of anti-PD-1 antibodies for the first-line treatment of locally advanced and metastatic CSCC. Note: Cemiplimab is the only anti-PD-1 agent approved by the EMA for the treatment of patients with CSCC who cannot have surgery or radiotherapy with a curative intent. Question 15 : Should concomitant chemoradiation be recommended over exclusive radical radiation therapy in patients with non-resectable CSCC? Recommendation : Concurrent chemoradiation may be considered as a first-line option for patients with unresectableOnly two studies evaluating the impact of concurrent chemoradiation in CSCC have been identified. , These retrospective studies had very limited sample sizes (12 and 11 patients treated in a curative setting), lacking a comparison arm, and including a mixed treatment approach with both platinum and cetuximab. Disease response rates ranged between 58% and 64%. , Acute side-effects reflected the well-known safety profiles of these drugs when used in combination with radiation therapy. Despite the absence of comparative data, considering the prognosis of these patients, the panel established that systemic treatment may be considered in this patient population. The desirable positive effects were deemed modest; the benefit/risk balance may favor treatment. The panel emphasized that the careful selection of patients for this treatment is crucial given the frailty conditions some patients with this disease may present, including age, comorbidities, or immunosuppression. See (Question 146 Platinum-based chemotherapy may be considered over palliative care/best supportive care in patients with recurrent and/or metastatic CSCC who are not eligible for curative treatmentstudy by Guthrie et al., published in 1990, assessed patients with mixed histology (BCC and SCC) in different settings (induction to surgery or radiotherapy and in palliative care), with a limited number of patients. In those not amenable to further treatment, a clinical response was observed in five out of eight patients treated with platinum-based chemotherapy. In the more recent study by Jarkowski et al., also retrospective and covering the period 2001-2011, a total of 25 patients with recurrent and/or metastatic disease were studied. The prevalent chemotherapy treatment included cisplatin or taxane, as well as the targeted anti-EGFR drug. The best responses were seen in combination therapy compared to monotherapy and in patients with locally advanced disease compared to metastatic disease. Patients who responded to systemic therapy had a significantly better prognosis. Note: It should be added that a study with cetuximab (anti-EGFR), not described in the current recommendations due to its lack of indication for use in Italy, achieved a response rate of 28% in a population not undergoing chemotherapy in a recurrent/metastatic setting. See (Question 157 : Should early integration of palliative care with oncological treatment be recommended in patients with advanced/metastatic CSCC over the ‘solo practice model’? Strength of recommendation : Strong in favor (if palliative care team available); weak in favor (if palliative care team unavailable) Overall quality of evidence : Very low Motivation/comments on the benefit/risk balance: The integrated care model for advanced/metastatic cancer patients, particularly the incorporation of early palliative care alongside active oncological treatment, has garnered significant attention in recent years. The integration of palliative care into the active treatment plans for advanced cancer patients has been a topic of interest since the early 2000s. Notably, the European Society for Medical Oncology (ESMO) initiated an accreditation program in 2003 to recognize oncology centers capable of ensuring the early integration of palliative care for symptomatic patients undergoing active oncological treatment. Over the past decade, various studies and expert opinions have consistently highlighted the benefits of this integrated approach on parameters related to the quality of life and symptom control. A meta-analysis of key studies, including Tattersall et al. (2014), Temel et al. (2010), Temel et al. (2017), Zimmermann et al. (2014), Maltoni et al. (2016), and Groeneveld et al. (2017), assessed the impact of early and simultaneous palliative care on quality-of-life parameters, showing a small effect in quality of life and symptom intensity. , , , , , Zimmermann et al. (2014) trials included blinded participants, while the blinding of outcome assessment was unclear in five out of six studies. Allocation concealment was considered at high risk in Temel et al. (2010) and Zimmermann et al. (2014). , Downgrading of evidence was applied due to potential biases and imprecision. The I 2 statistic was 67%, indicating moderate heterogeneity, and 92% for studies by Tattersall et al. (2014) and Temel et al. (2010), suggesting high heterogeneity. , The GRADE Handbook guided the decision to downgrade evidence for imprecision, as the 95% CI included 1, failing to exclude harm. The included studies used different scales for measuring outcomes, leading to a downgrade for indirectness. The GRADE assessment underscores the need for cautious interpretation, given the very low certainty of evidence. While there is a suggestion of small positive effects on quality of life and symptom intensity, the impact on OS remains inconclusive. The meta-analysis highlights the challenges and variations in study methodologies, emphasizing the need for further research with robust design to enhance the certainty of evidence in this critical area of early palliative care integration with oncology models. See (Question 17), available at https://doi.org/10.1016/j.esmoop.2024.103005 , for EtD results, quality of evidence, and implications for future results.
Risk factors associated with the development of CSCC include exposure to UVR, both natural and artificial, age, and fair skin phototype. The most significant environmental risk factor for the occurrence of CSCC is chronic cumulative exposure to UVR, which also explains the drastic increase in incidence with advancing age. Incidence is higher at lower latitudes, correlating with higher environmental light intensity. In 90% of cases, the tumor arises in anatomical areas chronically exposed to sunlight, such as the head/neck region and the dorsal surfaces of the hands and forearms, and is more common in individuals who work outdoors. Moreover, artificial sources of UVR, such as Psoralen and UVA (PUVA) therapy and indoor tanning devices, are implicated in the pathogenesis of CSCC, with a higher risk for individuals who are exposed at a younger age. , Sun protection is implemented through different effective means, with the use of sunscreen creams being one, but not the only, method. It also includes the use of clothing, hats, and protective eyewear, as well as avoiding direct sunlight. The significant role played by UVR in the development of skin cancer emphasized the importance of developing prevention strategies and adequate photoprotection and sun exposure. Measures to consider in this regard include raising awareness among individuals about the consequences of excessive sun exposure, protection from direct UVR exposure with appropriate clothing and hats, seeking shaded areas, and the regular and correct use of sunscreen creams. For roles requiring occupational exposure to sunlight, personal protective equipment (PPE) can be used as a secondary measure. However, it is crucial to emphasize that PPE, such as protective clothing, sunglasses, hats, and sunscreen, should not replace efforts to limit sunlight exposure. Notably, case-control or cohort epidemiological studies have analyzed the effects of sunscreen cream use on the development of skin neoplasms, with conflicting results. In the review conducted by Burnett and Wang in 2011, the analysis of literature data indicated that the application of sun protection creams led to a decrease in the incidence of CSCC without noteworthy reductions in vitamin D levels or adverse effects on overall health. Additionally, consistent and proper use of sunscreen creams has demonstrated effectiveness in diminishing the occurrence of actinic keratoses (AKs), a recognized indicator of prolonged sun-induced damage. An Australian study showed that the fraction of cutaneous cancers that could be prevented through proper application of sunscreen creams was 9.3% for CSCC and 14% for melanoma. Genetic factors, such as fair skin phototype, make the skin more sensitive to chronic UVR exposure and often enhance the effects of environmental factors in carcinogenesis (synergistic effect). An increased incidence of CSCC has also been reported in patients with genodermatoses, such as mucocutaneous albinism, xeroderma pigmentosum, and epidermodysplasia verruciformis. Finally, chronic long-term inflammatory processes, as present in some genetic diseases (e.g. epidermolysis bullosa), chronic wounds, burns, scars, and lower limb ulcers, can contribute to the development of CSCC. Another significant risk factor for the development of CSCC is immunosuppression, which can promote the development and progression of CSCC due to reduced immune surveillance against cancer cells or human papillomavirus (HPV) infection. Immunosuppression may be caused by solid organ or hematopoietic stem cell transplant, autoimmune condition requiring systemic immunosuppression, advanced solid organ malignancy, or a hematologic malignancy, such as lymphoma or leukemia, which are associated with an increased risk of CSCC. All immunosuppressive agents and biologic drugs have an impact on this risk, but to varying degrees. Iatrogenic immunosuppression is typically exemplified by organ transplant recipients, who have a 65-250 times higher risk of developing CSCC compared to the general population. , Other treatments, such as BRAF inhibitors, can promote the onset of eruptive CSCC through different mechanisms, for example, by enhancing the effectiveness of pre-existing mutations in chronically sun-exposed areas or by reducing defenses against HPV. Question 1 R to reduce the incidence of CSCC? Recommendation : In subjects who are exposed to solar UVR, sunscreen creams with solar protection factor 30-50 may be considered as a first option measure to reduce the incidence of CSCCIn the review and meta-analysis by Sánchez et al. in 2016, the only randomized study aimed at assessing the impact of sunscreens on the risk of developing both basal cell carcinomas (BCCs) and CSCC is the Nambour trial, named after the Australian region where it was conducted. , A total of 1621 individuals were randomly assigned to four different groups: daily application of sunscreen with a sun protection factor of 15 plus β-carotene supplementation; sunscreen plus placebo in tablet form; only β-carotene; or only placebo. A total of 1383 participants underwent full skin examination by a dermatologist in the follow-up period. The endpoint was the incidence of carcinomas after a 4.5-year follow-up. The results did not show any difference in the number of patients developing both BCC and CSCC across the various groups. However, although no difference was observed in the number of patients developing CSCC in the different groups, a significant reduction in the number of SCCs was noted in the group of patients applying sunscreen with a risk ratio (RR) of 0.61 [95% confidence interval (CI) 0.46-0.81]. The considered outcome was the number of patients developing new skin carcinomas, and the RR was 0.88 (95% CI 0.50-1.54). The risk of developing clinically and histologically confirmed CSCC was 3 out of 100 in both groups (with and without sunscreen). Regarding the risk of developing AKs, the RR was 0.95 (95% CI 0.75-1.20). However, concerning the number of CSCC, an RR of 0.61 (95% CI 0.46-0.81) was obtained, with an absolute value of 184 per 100 in the group without sunscreen and 100 per 100 in the group with sunscreen. This question represents an issue, and the available evidence is of low quality. It has been established that there is no significant uncertainty or variability regarding the assessment of the primary outcome, and the overall balance did not favor the intervention or comparator. There was no impact on equity, and the intervention was deemed acceptable by the parties involved, with potential for improvement. The overall recommendation was in favor of the intervention. The difficulties in conducting such studies and obtaining reliable results are associated with various factors, including the time required for a thorough assessment of the potential onset of these neoplasms, the presence of potential confounding factors, and the challenges in measuring solar radiation intensity and defining the use of sun protection creams. These aspects need to be considered when designing appropriate prospective studies that allow for adequate follow-up periods to assess the development of neoplasms such as BCC, which may potentially require a long induction period. See (Question 1Secondary prevention aims to detect disease at early stage. The clinical and dermoscopic diagnosis of CSCC presents greater challenges compared to that of BCC. These difficulties primarily stem from the diverse stages in which CSCC clinically manifests. While fully developed nodular CSCC usually does not represent a diagnostic challenge, early forms may resemble BCC or even inflammatory diseases, for which histopathological confirmation is generally not carried out in daily routine. Depending on the stage of tumor progression, CSCC may present as plaque or nodule exhibiting a dermoscopic vascular pattern of initially coiled vessels with yellow scales and hemorrhages and later a polymorphic pattern, with irregular linear vessels, corkscrew vessels, and glomerular vessels. Additionally, in hyperkeratotic varieties, the presence of whitish keratin material and, in ulcerated forms, the presence of ulceration and blood spots conceal and modify the dermoscopic characteristics of the lesion, consequently complicating the diagnosis. The pigmented variant of AK shares many features with lentigo maligna, both clinically and dermoscopically: in these cases, carrying out a biopsy, even incisional, is mandatory to confirm the diagnosis. , , Currently, there are no controlled studies in the literature specifically validating, for CSCC, procedures that improve diagnostic accuracy compared to clinical examination alone, such as dermoscopy or other non-invasive diagnostic methods like confocal microscopy. Individuals at high risk of developing CSCC encompass diverse groups, including those with lowered immunity, a history of NMSC, rare genetic disorders (e.g. xeroderma pigmentosum), and exposure to specific factors such as trauma, arsenic, albinism, or psoralen and ultraviolet A treatment. Noteworthy subsets at elevated risk include individuals with precursor lesions, previous NMSCs, and those with compromised immunity due to organ transplants or conditions like human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS). Additionally, specific genetic conditions like albinism and recessive dystrophic epidermolysis bullosa contribute to an increased susceptibility to CSCC. Understanding and addressing these varied risk factors are crucial for developing targeted preventive strategies and interventions. Chemoprevention is the use of dietary or pharmacologic agents to inhibit or reverse cancer development and is a promising approach for individuals at high risk of NMSCs. Oral retinoids, such as isotretinoin and acitretin, have been shown to effectively reduce the number of new NMSCs in high-risk patients. , , Other potential chemopreventive agents include difluoromethylornithine, T4 endonuclease V, and polyphenolic antioxidants. Question 2 Recommendation : In subjects with suspicious skin lesions, the use of dermoscopy should be recommended as the first option compared to visual inspection only for the detection of CSCC. Strength of recommendation : Conditional in favorIn the current literature, aside from anecdotal cases, there are no studies validating dermoscopy or other non-invasive diagnostic methods, such as confocal microscopy, as procedures that enhance diagnostic accuracy compared to clinical examination alone, specifically for the diagnosis of CSCC. However, studies on the effectiveness of dermoscopy for diagnosing melanoma and other skin conditions suggest the significant role of this technique in the differential diagnosis of skin lesions. It has been established that this question posed an issue. There is no significant uncertainty or variability regarding the assessment of the primary outcome, the overall balance favors the intervention, there is no impact on equity, and the intervention is deemed acceptable by the parties involved. The overall recommendation was in favor of the intervention. Question 3 : Should chemoprevention be recommended in subjects at high risk of developing CSCC? Recommendation : Chemoprevention treatment may be considered as a primary option compared to no treatment in individuals at high risk of developing CSCC. Strength of recommendation : Conditional in favor Overall quality of evidence : Low Motivation/comments on the benefit/risk balance : Few studies have been conducted in individuals with higher risk of developing CSCC to assess whether a chemoprevention strategy could reduce this risk. These studies have also evaluated different drugs, specifically one study on nicotinamide ; three on retinoids: acitretin versus placebo, oral retinol versus oral isotretinoin versus placebo, acitretin versus placebo ; two on antioxidants: one study on oral selenium versus placebo, one on β-carotene versus placebo ; and finally, one more recent study on nonsteroidal anti-inflammatory drugs, specifically celecoxib. The benefit endpoint considered is the incidence of new lesions. Regarding nicotinamide, the standardized mean incidence of new lesions in the 386 patients treated in the study was comparable to that in untreated subjects (ranging from 0.2 lower to 0.2 higher). In studies with retinoids, the overall standardized mean difference was 0.63 lower (ranging from 1.16 lower to 0.09 lower), , , , while with antioxidants, it was 0.14 times higher (ranging from 0.03 higher to 0.25 higher). , Finally, regarding celecoxib, in a single study involving 240 patients, the incidence was 0.41 times lower (ranging from 0.66 lower to 0.16 lower). Overall, in the eight randomized studies, considering a total of 626 patients, the standardized mean incidence in subjects who underwent chemoprevention was 0.23 times lower than in untreated patients (with a range from 0.44 lower to 0.02 lower). Overall, the evaluation of randomized studies considering a chemoprevention strategy versus no treatment has shown a reduced but evident benefit (the CI of differences in standardized mean incidence does not intersect the value 0). Differences between the various analyzed drugs have been highlighted; for nicotinamide and antioxidants, there is no significant impact (CI around 0), whereas for celecoxib and retinoids, the benefit is confirmed (CI <0), especially for retinoids, which have an evaluation in three studies, while only one study is available for celecoxib. Regarding the outcome of harm represented by adverse events associated with drug intake, the toxicity profile was described in Bavinck et al. (1995) for acitretin and analyzed exclusively in Chen et al. (2015) for nicotinamide, showing an increase of 0.33 in the odds ratio (OR) in 386 patients for hepatotoxicity (ranging from 0.01 to 8.19) and similarly 0.33 for nephrotoxicity (low-quality evidence). , The evidence for favorable effects has been confirmed but judged as low. The assessment regarding the balance between positive and negative effects was considered to probably favor the intervention. It was also evaluated that there is probably no impact on equity for the implementation of the intervention. See (Question 4a : Should dermatological visits be carried out in immunosuppressed subjects? Recommendation 1 : Dermatological visits versus no dermatological visits may be considered as a primary option in immunocompromised individuals. Strength of recommendation 1 : Conditional in favor—expert opinion Recommendation 2 : Dermatological follow-up visits versus no dermatological follow-up visits may be considered as a primary option in immunocompromised solid organ transplant recipients (SOTR) Strength of recommendation 2 : Strongs, i.e. immunocompromised subjects and SOTRs, are on the rise. It is not possible to providedermatological checks are probably very limited, while the benefits could be moderate or substantial (although there are no literature data on this), derived from the early diagnosis of potentially aggressive lesions. Taking all this into consideration and considering the risk of the population to be sufficiently high to anticipate the need for early diagnosis of disease recurrence or the appearance of a new lesion, the possibility of dermatological follow-up is moderately favored, especially in the subpopulation of immunocompromised patients undergoing solid organ transplantation. Dermatological follow-up in immunocompromised patients does not pose a major obstacle to the feasibility and equity of this approach and should be acceptable to the main stakeholders. There is no uncertainty or variability in how individuals may assess this approach. See (Question 4ab : Should clinical–instrumental follow-up be carried out in immunosuppressed subjects with a diagnosis of CSCC? Recommendation : Clinical–instrumental follow-up versus no clinical–instrumental follow-up may be considered as a primary option in immunocompromised individuals with a diagnosismagnitude of the problem is significant, as the reference population is on the rise. It is not possible to express an assessment of the desired/undesired effects with a more intensive follow-up since comparative studies are lacking. The potential risks from a strategy of clinical and instrumental checks are probably moderate, arising mainly from false-positive suspicious lesions requiring invasive diagnostic procedures (further radiological investigations that may expose to radiations; histological confirmation procedures that may cause complications, such as biopsies on visceral lesions). Considering all this and deemsuch a follow-up path is moderately favored. The follow-up will be personalized based on the type of transplant, the degree of immunosuppression, the drug used to reduce the risk of rejection, and the characteristics of CSCC (extension, recurrence, type of treatment received). Clinical–radiological follow-up in this population ofsince the number of patients to monitor is well limited compared to all patients with CSCC. There is probably not much uncertainty or variability in how individuals may assess this approach and, in its acceptability, especially given the increased risk of cutaneous recurrence or new cutaneous or extracutaneous neoplasms that this population may present due to sustained immunosuppression. See (Question 4btreatment of CSCC is based on complete surgical excision. Surgical removal allows histological examination and confirmation of the clinical diagnosis as well as evaluation of surgical margins, either intraoperative or post-operative, with very high rates of effectiveness and healing rates of 95%. There may be conditions in which alternative techniques to surgery are used in daily clinical practice. In patients where CSCC arises on multiple AKs or in areas with multiple in situ tumors, different destructive modalities (cryotherapy, curettage and electrocoagulation, photodynamic therapy with aminolevulinic acid or methyl-aminolevulinic acid) or topical agents (imiquimod 5% or 3.75%; diclofenac gel 3%, ingenol mebutate 500 μg/g or 150 μg/g) are also employed to clear the field of cancerization, although these therapeutic procedures do not allow for histological margin analysis. There are no studies comparing the therapeutic efficacy of these options to traditional surgery in invasive carcinomas. However, a multicentric placebo-controlled randomized study compared the rate of complete clinical responses for non-invasive SCCs/Bowen’s disease in a group of 225 lesions, with randomization into four arms (photodynamic therapy with aminolevulinic acid, cryotherapy, topical 5-fluorouracil, and placebo photodynamic therapy). Photodynamic therapy achieved the highest response rate (93%), followed by cryotherapy (86%) and 5-fluorouracil (83%). Another randomized study compared photodynamic therapy and topical therapy with 5-fluorouracil, finding a higher rate of complete clinical responses for photodynamic therapy (88% versus 67%) with a lower recurrence rate (6.8% versus 27.3%) after 12 months of follow-up. A retrospective study on 263 non-invasive lesions/Bowen’s disease compared photodynamic therapy, cryotherapy, and surgical excision in terms of recurrence rates after 8 years of follow-up. The recurrence rate after photodynamic therapy (18%) was significantly higher than that with surgery (0.4%) and cryotherapy (5%). However, the lesions treated with photodynamic therapy were larger and more infiltrated than those treated with cryotherapy. In cases where there is clinical uncertainty about the invasiveness of the lesion or doubt between in situ tumor and invasive CSCC, surgical excision or a biopsy followed by histological examination confirms the non-invasive nature of the lesions. Question 5a Recommendation : In subjects with operable low-risk CSCC, surgical excision with margins ≥4 mm should be considered as a first-line option compared to excision with margins <4 mmThe guidelines unanimously emphasize the importance of radical surgical excision with clear margins. However, there are no available clinical studies specifying the minimum appropriate dimensions for clear margins, and thus, there is no consistent guidance in this regard. The American National Comprehensive Cancer Network (NCCN) guidelines based their recommendations on the findings of a prospective American study by Brodland and Zitelli in 1992. The results highlighted for well-defined low-risk CSC, with a diameter of <2 cm, that excision with a margin of 4 mm from the clinical margins of the lesion resulted in complete excision of the neoplasm in over 95% of cases. For larger low-risk lesions exceeding 2 cm, the recommended margin to ensure histologically complete removal of the neoplasm is 6 mm. European guidelines from the European Dermatology Forum (EDF), European Association of Dermato-Oncology (EADO), and European Organization for Research and Treatment of Cancer (EORTC) recommended a standardized minimum margin of 5 mm for low-risk carcinomas, i.e. tumors with a vertical thickness <6 mm and no risk factors. Carrying out excisions with larger clearquestion represented an issue, with no significant uncertainty or variability regarding the assessment of the primary outcome, a balance favoring the intervention, no impact on equity, and the intervention being deemed acceptable by all parties. The overall recommendation is in favor of the intervention. Question 5b Recommendation : In subjects with operable high-risk CSCC, surgical excision with margins ≥6 mm may be considered as a first-line option compared to excision with margins <6clinical studies available to determine the minimum appropriate dimensions for free margins, and therefore, there are no consistent indications in this regard. The margins free from disease after surgical excision must be assessed based on tumor size and aggressiveness according to clinical–pathological parameters. The American NCCN guidelines based their recommendations on the results of a prospective American study by Brodland and Zitelli in 1992. For lesions >2 cm, the recommended margins to ensure complete histological removal of the neoplasm are 6 mm. For high-risk locations (scalp, ears, eyelids, nose, lips) or other high-risk characteristics (histological grading ≥2, invasion of subcutaneous tissue), lesions with diameters <1 cm, 1-1.9 cm, or >2 cm should require free margins of 4 mm, 6 mm, and 9 mm, respectively. The guidelines of the German Dermatology Society indicate that for SCCs >2 cm in diameter, or lesions with a thickness >6 mm, or with other high-risk prognostic features (poor cellular differentiation, recurrent tumors, perineural invasion, deep extension into the subcutaneous layer, and/or localization on the ear or lip), a free margin of at least 6 mm is necessary to achieve a 95% complete response at 5 years. The European EDF/EADO/EORTC guidelines recommend, for tumors with a thickness <6 mm but with high-risk features (histologically undifferentiated, perineural invasion, recurrent tumors) and for tumors with a histological vertical thickness >6 mm, a free margin of 6-10 mm. Carrying out excisions with larger free margins could inevitably lead to a higher incidence of treatment-related complications, such as surgical outcomes, scarring, pain, and bleeding. It has been established that the issue represents a problem, and there is no significant uncertainty or variability regarding the assessment of the main outcome. The balance favors the intervention, and there is no impact on equity. The intervention is certainly acceptable to all parties. The overall recommendation is in favor of the intervention. Question 6 : In recurrent or high-risk CSCCs, should Mohs surgery be recommended over traditional excision? Recommendation : In subjects with high-risk or recurrent CSCC, the Mohs technique may be considered over simple excision. Strength of recommendation : Conditional in favor Overall quality of evidence : Very low Motivation/comments on the benefit/risk balance : From the available literature, we considered five retrospective monocentric studies. The 2008 study by Brantsch et al. included 615 patients with CSCC treated with traditional surgery over a period of 10 years, with a median follow-up of 43 months (range 1-163 months). The 2002 study by Cherpelis et al. included 200 cases of CSCC treated with Mohs micrographic surgery (MMS) from 1988 to 1998, with a follow-up ranging from 6 months to 10 years. The study by Pugliano-Mauro and Goldman (2010) included 260 high-risk CSCC patients treated with MMS, with a mean follow-up of 3.9 years, involving neoplastic lesions in the H zone of the face, tumors >2 cm, or rapidly growing tumors with perineural involvement, and lesions occurring in immunosuppressed patients. Of these lesions, 231 (89%) were primary, and 29 (11%) were recurrences, with 20% of the patients being immunosuppressed. The study by Vuyk and Lohuis (2001) reported the experience of a single surgeon on 56 patients with CSCC treated with MMS over an 8-year period, with a mean follow-up of 33 months (range 1-99 months), of which 3 (5%) were recurrent lesions. The study by Silapunt et al. (2005) included 144 CSCCs in 117 patients with lesions located on the ear treated with Mohs surgery, with a mean telephone follow-up of 34.6 months (range 7-67 months). Of these cases, only 122 were subjected to follow-up, and the remaining ones were not reachable. Our objective was to compare MMS and standard excision in the treatment of CSCC in a high-risk population or with recurrent CSCC. We relied on the analysis of multiple benefit outcomes (essential: percentage of local recurrence, number of re-interventions, percentage of metastasis) and harm outcomes (essential: scarring outcomes; important: infections, bleeding). The benefit outcomes had limited importance, with a local recurrence rate of 2.7% in the population of 1045 individuals collected from four observational studies, , , , while the rate of distant metastasis was 12.4% in a population of 460 patients collected from three observational studies. , , None of the studies reported the benefit outcomes of re-excision and the undesirable ones (scar results, infections, and bleeding). Regarding the population’s perception of the importance of outcomes, there was no significant uncertainty or variability, and this aspect is not analyzed in the studies under consideration. Moreover, it was not assessable whether the balance between desirable and undesirable results favors one technique over the other due to the lack of studies on this topic. The Mohs technique is likely more expensive than traditional surgery because, despite the absence of pharmacoeconomic studies comparing the two techniques, the Mohs technique involves a greater number of professionals and more hours of surgical activity. No studies analyzed the required resources or considered whether the cost-effectiveness balance favors one technique over the other. The equity of the Mohs technique probably appeared reduced because the high costs and the need for specialized personnel would prevent a widespread and uniform distribution nationwide. The Mohs technique, compared to traditional surgery, could be considered depending on the figures involved in the process under consideration. Therefore, it might be considered economically unsustainable despite the potential superiority of the MMS technique, yet to be demonstrated. The Mohs technique was probably not very implementable for the reasons mentioned. The high-risk patient or those with recurrent SCC could undergo treatment with true MMS if carried out in specialized and competent centers. See (Question 6aRecommendation : In patienover radiotherapyre are no randomized studies comparing surgery with radiotherapy; literature consists solely of case series of patients treated with either method. The considered benefit outcomes were the percentage of recurrences and relapse-free survival, while the harmful outcomes included surgical complications and the incidence of radiodermatitis. Four observational studies involving a total of 395 cases of CSCC were analyzed. , , , The rate of recurrences after surgery was 3.5%, while that of surgical complications was 8.7% (41/469 patients). , , , The balance of effects in terms of benefit/harm was indicated as probably in favor of surgery. The question was evaluated as definitely representing a clinical problem, with anticipated positive effects being moderate, and unknown negative effects. There is probably significant uncertainty or variability regarding the assessment of outcomes. Concerning equity, it was assessed as probably having no impact, and the intervention was deemed acceptable to stakeholders. Note: In an observational radiotherapy study by Barysch et al., involving a total of 180 high-risk CSCC patients, the percentage of relapse-free survival at 10 years was 80.6% (35 recurrences/180). The percentage of recurrences after a median follow-up of 4.9 years was evaluated in two observational studies and was 8.1%. Data on the side-effects of radiotherapy were not available in the same studies. , See (Question 77(b-c) cauterization or cryotherapy? Recommendation : In subjects with non-recurrent and operable CSCC, surgical excision with clear margins may be considered as a first-line option compared to cauterization or cryotherapy. Strength of recommendation : Conditional in favor (for cauterization); expert opinion (for cryotherapy) Overall quality of evidence : Very low (for cauterization); no included studies (for cryotherapy) Motivation/comments on the benefit/risk balance : Surgical excision is the treatment of choice as it allows histological confirmation and assessment of resection margins. Surgery is rarely contraindicated, even in elderly patients or in cases of tumors that are challenging to treat due to extensive size and anatomical locations with potential functional and cosmetic consequences, provided that these patients are managed appropriately by experienced personnel. There are no randomized studies comparing surgery with diathermocoagulation/cauterization or cryotherapy. There is only one retrospective observational study published in 2002 by Werlinger et al., comparing surgical excision to curettage and diathermocoagulation in a cohort of 268 patients with cutaneous BCC or SCC, of which 110 underwent surgical excision and 158 underwent curettage and diathermocoagulation. These were small-sized tumors (median diameter 7 mm), and only 76 were SCCs. The considered benefit outcomes were the rate of recurrences and relapse-free survival, while the harmful outcomes included complications and the outcomes of different techniques. The study results did not show significant differences in recurrences between the two methods, although the study is burdened by a very high risk of bias due to its retrospective nature, lack of stratification, and a high number of patients lost to follow-up (8 in the surgery group and 32 in the curettage and diathermocoagulation group). However, when analyzing only the group of patients with CSCC, the recurrence rate for patients with available follow-up was 0/20 (0.0%) for those treated with surgery and 2/56 (3.6%) for those treated with other methods. For this study, the risks of inconsistency and imprecision were evaluated as not serious, while the risks of imprecision were considered serious due to the low number of events. The retrospective observational study by Nordin and Stenquist in 2002 reported data on 100 cases of NMSC located on the auricle, mostly BCCs, with only 13 invasive and 6 in situ CSCCs, showing only 1 recurrence in 76 cases followed over time. In a prospective study of 100 cases of superficial and non-facial NMSCs, including 11 in situ and 6 invasive SCCs treated with the same method (curettage + cryosurgery), there were no recurrences evident at a 1-year follow-up. The quality of the evidence is low; however, the panel deemed this issue very relevant and considered that there are no uncertainties regarding its evaluation, and no additional costs or equity problems are expected. Note: In patients where CSCC arises on multiple AKs or areas with multiple in situ tumors, various destructive modalities (cryotherapy, curettage and electrocoagulation, photodynamic therapycan be used, as reported in the EDF guidelines (Werner, 2015) on AK and EDF/EADO/EORTC on CSCC. See (Question 7b8 : Should adjuvant radiotherapy be recommended after surgical excision of high-risk CSCC compared to no adjuvant treatment? Recommendation : In subjects with resected high-risk CSCC, adjuvant radiotherapy may be considered compared to no treatment. Strength of recommendation : Conditional in favor Overall quality of evidence : Moderate Motivation/comments on the benefit/risk balance : After a systematic literature review, , , , the working group concluded that the recommendation proposed in the American Society for Radiation Oncology (ASTRO) guidelines addressed was applicable to the Italian context. Moreover, the ASTRO guidelines were of excellent quality according to the AGREE II assessment. For these reasons, the panel has decided to adopt the ASTRO guidelines. Specifically, the adoption is referred to the following risk factors: Key Question 2, points 1 (clinically or radiologically evident perineural invasion), 3 (CSCC operated after previous resection with clear margins), 4 [T3 and T4 tumors according to American Joint Committee on Cancer (AJCC) eighth edition staging], and 5 (desmoplastic or infiltrative cutaneous SCC in the context of chronic immunosuppression). Question 9 : Should sentinel lymph node biopsy be recommended in high-risk CSCC? Recommendation : In subjects with high-risk CSCC, sentinel lymph node biopsy should not be considered as a primary option compared to only follow-up. Strength of recommendation : Conditional against Overall quality of evidence : Very low Motivation/comments on the benefit/risk balance : In a retrospective monocentric observational design study by Maruyama et al., including 169 treated patients (with neoplasms more advanced than in situ tumors and without baseline metastasis) followed up for at least 6 months (average follow-up of 31.4 months), 49 patients underwent sentinel lymph node biopsy during the same period. The considered benefit outcomes were the recurrence rates, disease-free survival (DFS), and overall survival (OS); the harmful outcomes were the surgical complications of the sentinel lymph node biopsy. DFS had a risk of 11% with only follow-up, while with the sentinel lymph node biopsy, it was 6% (range 2%-21%), resulting in an RR of 0.55 (range 0.1-1.85). Consequently, there were 5 fewer disease recurrences per 100 patients undergoing sentinel lymph node biopsy, with a 95% CI ranging from 9 fewer to 10 more patients. Regarding OS, no reported differences were found between the population undergoing sole follow-up and patients undergoing sentinel lymph node biopsy. Concerning harmful outcomes, three cases of surgical complications were reported: one case of bacterial lymphangitis in a 77-year-old man, one case of lymphorrhea in a 49-year-old man, and one case of post-operative bleeding, all successfully treated. There was no likely uncertainty or variability regarding how the population evaluated these outcomes. Undesirable effects were considered small compared to irrelevant desirable effects. Therefore, the balance of effects between desirable and undesirable outcomes probably favors sole follow-up over sentinel lymph node biopsy. The procedure would be easily implementable, considering it is already in use for other types of neoplasms and is carried out in non-specialized centers. Consequently, the distribution would be widespread from the beginning, allowing facilities to implement9Question 10 : Should prophylactic lymphadenectomy be recommended in high-risk CSCC? Recommendation : Prophylactic lymphadenectomy should not be considered for high-risk CSCC compared to sole follow-up. Strength of recommendation : Conditional against—expert opinion Overall quality of evidence : No included studies Motivation/comments on the benefit/risk balance : Based on the literature, no studies addressing this issue were identified. The panel, therefore, proceeded to formulate a recommendation based on its clinical experience. It was concluded that carrying out prophylactic lymphadenectomy in the high-risk population affected by CSCC is not recommendable. Prophylactic lymphadenectomy is not recommended also due to potential side-effects, such as lymphedema, surgical site infection, and regional paresthesia. See (Question 10), available at https://doi.org/10.1016/j.esmoop.2024.103005 , for quality of evidence and implications for future results.
The management of patients with CSCC poses a clinical challenge, particularly when in its advanced stages, necessitating comprehensive and multidisciplinary care. Historically, a standard of care for advanced CSCC was elusive, leaving a substantial proportion of patients untreated due to concerns about low clinical efficacy and high risks of severe toxicities. The advent of immunotherapy, specifically anti-PD-1 agents, has revolutionized the landscape of CSCC management. Cemiplimab, as the first PD-1 inhibitor to gain regulatory approval for advanced CSCC, demonstrated remarkable efficacy with rapid and durable responses in >40% of patients, presenting a compelling case for its use in this challenging clinical scenario. Ongoing trials explore the potential of PD-1 inhibitors in adjuvant (NCT03969004, NCT03833167) and neoadjuvant (NCT04632433, NCT04808999, NCT04315701, NCT04428671) settings, aiming to establish them as the new standard of care. The encouraging results of these studies suggest a paradigm shift in the approach to high-risk and advanced CSCC, with anti-PD-1 agents poised to play a central role in reshaping treatment strategies. Neoadjuvant cemiplimab has been investigated in a phase II confirmatory study. , This multicenter, non-randomized trial focused on assessing cemiplimab as neoadjuvant therapy in patients with resectable stage II, III, or IV (M0) CSCC. Cemiplimab was administered for up to four doses before curative-intent surgery. The primary endpoint was a pathological complete response. A total of 79 patients received neoadjuvant cemiplimab. The outcomes were striking, with a pathological complete response observed in 51% of patients on independent review. A pathological major response was noted in 13%, and an objective response on imaging was seen in 68% of patients. Neoadjuvant therapy with cemiplimab holds significant promise for patients with resectable CSCC, demonstrating a high rate of pathological complete response. This breakthrough represents a crucial advancement in the management of CSCC, offering a potential curative approach for a disease that lacked a clear standard of care in its advanced stages. The study marked a critical milestone toward reshaping treatment paradigms in CSCC.
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Assessing the need for a doctor of philosophy (Ph.D.) degree in Endodontics: perspective and implication for advancing dental education and research in Saudi Arabia | e0b6651e-fd75-4fda-9e42-6c453d67f8bf | 11654090 | Dentistry[mh] | Endodontics is a branch of dentistry that deals with the etiology, diagnosis, prevention, and treatment of diseases of the pulp and periapical tissues compatible with good health . Endodontics as a specialty has undergone significant advancements in recent years . This considerable advancement includes the introduction of magnification devices, engine-driven file systems, materials for regeneration, and Cone Beam Computed Tomography (CBCT). This progress contributes to a shift in the understanding and satisfaction of endodontic specialties . General practitioners are the first line of dental care who provide endodontic treatment for patients who are in need . However, cross-sectional studies show that the quality of root canal treatment performed by general practitioners is substandard or inadequate . This can be caused by either the complexity of the cases or the graduation of inexperienced practitioners . The introduction of modern technologies in the field, such as nickel-titanium rotary files, bioceramic sealers, and magnification devices made root canal treatment procedures simpler and more predictable . As the field develops, the need for highly qualified professionals who can contribute to clinical practice and academic research becomes crucial. The current dental education system in Saudi Arabia primarily focuses on undergraduate Bachelor’s degrees, Master’s degree programs, and specialized training residency programs (Saudi Board in Endodontics) , which provide a solid foundation in clinical practices. In the meantime, there is a gap in integrating advanced research training and academic development at the doctoral level within the endodontic specialty. This has led to many debates about the possible benefits of introducing a Doctor of Philosophy (Ph.D) degree specifically designed for Endodontics. In Saudi Arabia, the landscape of dental education is rapidly exchangeable, reflecting global trends and the increased complexity of dental care. This necessitates reevaluating the educational pathways available to aspiring endodontists, particularly the potential introduction of a Ph.D. degree within Endodontics programs. The demand for doctoral programs is evident in other health-related specialties. A study by Badreldin et al. in 2022 showed that 80% of respondents felt the establishment of a Doctor of Pharmacy/Master of Public Health Dual Degree in Saudi Arabia would increase their job opportunities . The integration of a Ph.D. program in Saudia Arabia could address several pressing needs within the field. First, it would foster a culture of research and innovation, essential for the advancement of endodontic practices. As new techniques, materials, and technologies emerge, having a cadre of well-trained researchers is crucial to evaluate and implement these advancements effectively . A Ph.D. program would equip graduates with the skills necessary to conduct stringent scientific research, critically analyze existing literature, and contribute original findings that could enhance patient outcomes, and elevate the standards of care in Endodontics. Moreover. a Ph.D. program would emphasize the importance of research literacy, enabling future endodontists in Saudi Arabia to become not only skilled clinicians but also reasoning leaders in the field. This is particularly important in a country where the dental health needs are progressively developing and there is a growing emphasis on preventive care and the management of complex cases. Furthermore, the international dental landscape is progressively affected by evidence-based practice, requiring practitioners to stay side by side with current research and incorporate it into their clinical decision-making. Shetty et al., 2021, did a university-based survey of career choices in higher education among dental students in the United Arab Emirates (UAE) . They concluded that dental undergraduate students in the UAE prefer to pursue specialization in clinical branches like Orthodontics and Endodontics. Moreover, the survey respondents felt that there was a need to offer more dental post-graduate and dental ph.D. programs by UAE universities. Another study was conducted in the College of Dentistry, Ajman University to assess the perception and understanding of the Endodontic specialty among dental students and newly graduated dentists. It also evaluated their interest in pursuing Endodontics as a future career. Results indicated that Ajman University dental students and interns showed a satisfactory perception of the Endodontic specialty. Also, perception did not significantly differ based on gender, level of study, or the experience of the supervising instructor . Contrary to these results, Newton et al. 2000 assessed whether differences existed between male and female dental clinicians in the types of positions they occupy in the United Kingdom . Their findings indicated that most females are employed in general dental practice. Another study made by Chan et al. 2006 concluded that 95.8% of the 386 general dentists who participated in the 26th Asia Pacific Dental Congress declared that they would take continuing professional dental education programs and courses within the next 5 years . In 2016, Puryer, Kostova, and Kouznetsova conducted a cross-sectional survey to explore the attitudes toward postgraduate specialization of final-year students at one UK dental school and to identify possible influencing factors. The results indicated that 6.9% of undergraduates had an intention to specialize in Endodontics as their first choice of specialty and 17.2% as their second choice of specialty . Age and ethnicity did not affect the intention to specialize. Gender influences were more apparent among females who did not wish to specialize . Al-Dlaigan et al., 2011 studied the career development of males who graduated from King Saud University (KSU) in terms of the pursuit of postgraduate dental education, higher degree, or Board certification after qualification using a questionnaire survey. They concluded that most of the male dentists who graduated from the College of Dentistry, KSU, were motivated and eager to continue their postgraduate education to get higher academic degrees from nationally or internationally well-recognized universities to improve their career and self-esteem . In another study, Al-Dlaigan et al., 2012 investigated the number of female bachelor of dental surgery graduates who earned a postgraduate education degree from KSU and a degree of qualification using a questionnaire survey. They concluded that 54% of the female dentists had pursued postgraduate education. Out of these 5% had Saudi board, 5% had obtained degrees in Endodontics, 59% had a master’s degree, 7% had a doctorate, and 2% had fellowship certificates . lrashdan et al.2018, explored the professional career plans among final-year dental students from different backgrounds at a single Middle Eastern institution using a self-administered questionnaire. Results concluded that several differences in career plans were found between dental students from variable backgrounds studying a single institution . Many of these disparities could reflect variations in socioeconomic backgrounds. A cross-sectional study across seven dental colleges in Saudi Arabia by Fita et al. 2020, assessed dental students’ opinions about their future career challenges in the dental profession and factors associated with the perception of career challenges. It was concluded that establishing a private clinic and getting a government job were the most common career challenges . Senior students, female students, and students with low academic scores had an increased likelihood of facing employment and academic-related difficulties . The importance and value of establishing a research-based program in the field of Endodontics need further assessment of its feasibility and desirability by potential candidates and key stakeholders. Such a program will provide dental schools with highly educated faculty members who can provide better clinical training for graduate and undergraduate students and write and develop Endodonitc curricula that meet national and international demands. This study aims to evaluate the necessity and feasibility of integrating a Doctor of Philosophy (Ph.D) degree into the endodontic programs at King Abdulaziz University Faculty of Dentistry (KAUFD), in Saudi Arabia. In addition, we aim to gather insights from key stakeholders including practitioners and students to understand their perspectives on the value and necessity of a Ph.D. in enhancing the field of Endodontics leading to improved patient care and health outcomes.
The research was conducted in accordance with the Declaration of Helsinki. The research was approved by the Ethics Research Committee at the Faculty of Dentistry, King Abdulaziz University (Propocal No: 84-09-24). We conducted an online survey to evaluate the need for and design considerations of Ph.D. programs in Endodontics in Saudi Arabia. The survey targeted potential Ph.D. candidates and key decision-makers in the field of Endodontics and was distributed through social media and emails to members of the Saudi Endodontic Society and the Saudi Endodontic Journal. We used a structured questionnaire (Appendix A), which was developed based on a literature review and expert consultation. The questionnaire was designed in the form of closed questions, as a ‘‘tick box’’ that included 14 multiple choice questions. The validity of the questions was evaluated by several Endodontic faculty members to establish the reliability and validity of the questions. The Delphi method was used during the process of developing the questionnaire . The final version of the questionnaire. The sample size was estimated based on the WHO recommendations. The required sample size should be equal to or greater than 125 participants to achieve a 95% confidence interval and a 5% significance level (p-value), estimated using OpenEpi VER 3.0 . The questionnaire included sections on demographics, professional background, interest in Ph.D. programs, program preferences, perceived need for Ph.D. programs in various sectors (assessed using a 5-point Likert scale), and university selection criteria. We developed the survey using Google Forms and distributed the survey link via email, social media, and professional networks. Statistical analysis The data were analyzed using IBM SPSS Statistics (Version 23; Armonk, NY: IBM Corp.). Descriptive statistics, including frequencies and percentages, were calculated for demographic variables and categorical responses. Continuous variables were reported as means and standard deviations. For inferential statistics, the chi-square test of independence was used to examine the relationships between categorical variables and the Mann-Whitney U tests for Likert scale. A significance level of 0.05 was used for all statistical tests. P-values less than 0.05 were considered indicative of statistically significant differences.
We received 141 responses for the survey. According to the regulations of the Ministry of Education in Saudi Arabia, one must have a master’s degree in Endodontics from an accredited program. The demographics of the participants, as shown in (Table ), indicate that the majority of respondents are in the age range of 31–40 years (44.0%), with an almost equal gender distribution (52.5% male, 47.5% female). A significant part holds a Master’s degree (29.1%) or a Bachelor’s degree (24.8%), and the largest group among the current level of dental education had board certification (45.4%). According to the Saudi Commission for Health Specialists (SCFHS) classification, participants were mostly Consultants (44.0%), and the most common place of work was Government hospitals (51.8%). Table shows opinions regarding the development of the Ph.D. program. Participants’ purposes for pursuing a Ph.D. were equally distributed for promotion reasons and research interests. The top 3 factors shaping participants’ decision regarding a Ph.D. program are duration (69.5%), SCFHS recognition (48.9%), and the possibility of being part-time students (45.4%). In terms of transferable skills that should be acquired during a Ph.D., a significant majority (80.1%) emphasized the importance of learning new research technology, followed by gaining publication experience (76.6%). Lastly, the qualities that make a university’s Ph.D. program appealing to participants include good research facilities (86.5%), availability of research funding (74.5%), and a diverse staff of Ph.D. holders (73.8%). Table presents the demographic and professional characteristics of probable prospective Ph.D. candidates in Endodontics. A higher percentage of probable prospective students were in the younger age groups of 20–30 years and 31–40 years (41.4% each) ( P = 0.001). Gender distribution was balanced with equal representation of males (50.0%) and females (50.0%), with no significant difference found. In terms of the current level of dental education, a significant number had board certification (35.7%) and Endodontic residents/Master students (28.6%) ( P = 0.006). Regarding SCFHS classification, many probable prospective students were classified as general dentists (37.1%) and consultants (32.9%) ( P = 0.013). In terms of place of work, the majority were employed at government hospitals (57.1%) and government universities (32.9%), with no significant difference found. Additionally, a considerable proportion of probable prospective students had less than 5 years of expertise (45.7%) ( p = 0.013), and the highest degree held by many was a Bachelor’s degree (35.7%) ( P = 0.007). The key considerations for probable prospective Ph.D. students in Endodontics are shown in Fig. . When considering registering for a Ph.D. program, 62.9% of students highlighted program duration, 52.9% noted SCFHS recognition, 50.0% valued the ability to be part-time, and 42.9% considered the cost important. Only 7.1% considered the acceptance of non-Saudis. When choosing a particular university, the majority prioritized good research facilities (87.1%), diversity of Ph.D. staff members (84.3%), and research funding availability (80.0%). For transferable skills needed during a Ph.D., 84.3% of probable prospective students emphasized the importance of gaining publication experience, and 80.0% prioritized learning new research technology. Enhancing clinical training was important for 52.9%, and 50.0% valued grant writing. Regarding the purpose of pursuing a Ph.D., 65.7% expressed interest in research, 50.0% aimed to pursue promotion in SCFHS reclassification, and 48.6% cited academic job requirements. These insights are presented in Fig. . Table details opinions on the need for a Ph.D. degree in Endodontics. Candidates who see a need for more Ph.D. degree holders in academic institutions report the highest agreement, with a mean score of 4.67 ± 0.58. This is followed by the general need for a Ph.D. degree program in Endodontics in Saudi Arabia, with a mean score of 4.46 ± 0.83. The need for more Ph.D. holders in the private sector has a mean score of 3.81 ± 1.21, and governmental hospitals show the least agreement with a mean score of 3.74 ± 1.18. All results are statistically significant with p-values < 0.0001.
The current landscape of dental education in Saudi Arabia offers several postgraduate programs in Endodontics, primarily through the Saudi Commission for Health Specialties (SCFHS) and university-affiliated residency programs. However, there remains a significant gap in research-based doctoral programs, specifically a Ph.D. in Endodontics. This gap raises crucial questions about the future direction of Endodontics education in Saudi Arabia and the role of research in advancing the specialty. The development of a Ph.D. program in Endodontics is debatable based on the need for such a program. Moreover, the career choice of dental students is often affected by several factors, depending on their perspective of higher education and their goals in life. According to the General Authority for the Statistics Demography survey of 2016, the total population of Saudi Arabia is 31,742,308 and it has 410 licensed endodontists, 62% of whom are non-Saudi . This study reflects the demand for highly trained Endodontists in Saudi Arabia. The Ministry of Education in Saudi Arabia outlines the national qualification framework for all academic programs in the country, including graduate and postgraduate dental programs . This framework was made to meet nationally and internationally qualified dental graduates. Dental graduates are committed to lifelong dental education to meet the requirements of Saudi Licence registration . The development of higher education programs is mandatory to provide dental schools in Saudi Arabia with highly trained Endodontists capable of teaching the specialty and able to design graduate and undergraduate Endodontic programs and courses . The demand for highly trained Endodontic educators is reflected in the difficulty in treating molar teeth in undergraduate students. Studies show that undergraduate students have confidence issues when performing root canal treatment . In Saudi Arabia, dental students reported a high level of stress during their clinical training . The Endodontic procedure, particularly, was one of the most stressful clinical events in dental training . Tanalp et al. suggest that students are less confident in treating complex cases because they tend to refer these cases to postgraduate residents . These findings were also found internationally . Highly qualified Endodontic faculty members with a higher degree of education are essential for training graduate and undergraduate dental students and in establishing Endodontic curricula that meet international standards such as Commission on Dental Accreditation (CODA), the European Society of Endodontology (ESE), and the Australian and New Zealand Academy of Endodontists . A total of 141 participants responded to this questionnaire regarding the need for a Doctor of Philosophy degree in Endodontics in Saudi Arabia. The majority of responses were particularly from younger age groups (20–30 and 31–40 years) with almost equal gender distribution between males (52.5%) and females (47.5%). This is in line with broader trends seen in the Middle East and globally, where specialized education and research training are becoming essential for career advancement . The responses show that the majority of the respondents were board-certified, classified as consultants by SCFHS, and worked in governmental hospitals. In particular, participants highlighted the importance of Ph.D. holders in academic institutions, suggesting a clear demand for such qualifications to fill teaching and research positions. The demand is not limited to academia, as respondents also recognized the need for more Ph.D.-trained professionals in governmental hospitals and the private sector, albeit to a lesser degree. Two different studies were done at King Saud University to assess the career characteristics and postgraduate education of male and female dentists who graduated from the same university. Among female dentists, 54% completed their postgraduate education, from which 5% obtained their degree in Endodontics . Among male dentists, 77% completed their postgraduate education, from which 7.7% obtained their degree in Endodontics . In a study done by puryer et al., 2016, it was observed that 37.95 dental students in the UK preferred to specialize, with 24.1% choosing restorative dentistry and 20.7% choosing orthodontics. In this study, 79% of the responses were from females . A study by Halawani et al., 2016, on dental specialty career preferences and their influencing factors among final-year dental students in Saudi Arabia showed that the most preferred specialties were restorative and aesthetic dentistry (17.7%), followed by Endodontics (14.1%) and then Prosthodontics (11.7%). The most important factors in choosing a specialty were family influence, preference for private practice, and specific interest in the patient population . In our study, 41% of the age group 20–30 years and 41% of the age group 31–40 years consider themselves as a probable candidate for a Ph.D. program in Endodontics, which reflects the importance of developing such a program. However, it is important to understand the perception of dental students toward the Endodontic specialty. This is a key factor in developing postgraduate programs. Several studies have been done on the career perspective of dental students. A study was done by Alrashdan et al., 2018 on the career perspectives of senior dental students. The data obtained from the results about the choice of career revealed that the majority of the respondents wanted to join specialization courses. These results are observed in a study done by Rashid et al., 2013 . On the other hand, in another study performed in Jordan, it was observed that most of the respondents preferred private practice . In a study by AlMaslamani et al., 2023, at Ajman University, dental undergraduate students were asked about their perception of the Endodontic specialty. Among the respondents, 47.3% expressed their interest in pursuing an Endodontic specialty, with 55.95% due to career interest, 20.6% due to high patient flow and monetary gain, and 20.1% due to county/city need . A study performed in the Middle Eastern region by Halawany et al., 2014 shows that Endodotnics was one of the preferred specialty choices. The study concluded that the career motivation of dental students in Saudi Arabia is associated with socioeconomic aspects, and the future of dentistry is associated with postgraduate education . In a study done by El-Housseiny et al. in 2014 on factors Motivating Career Choices for Dental Students, it was found that grades, family influence, and economic factors with females are more affected by family influences . In Saudi Arabia, motivations for pursuing a Ph.D. are closely tied to career advancement, particularly regarding SCFHS classification and academic job opportunities. Half of the respondents indicated that they seek Ph.D. qualifications to meet academic job requirements, while others expressed interest in research and grant writing as key drivers . This aligns with findings from international studies, where dental professionals increasingly recognize the value of research literacy and publication experience in enhancing their careers. A study in the UAE, for example, found that 28% of dental students were inclined toward pursuing a specialization in Endodontics, and a majority felt that Ph.D. programs opened up opportunities for research and teaching. Most of the respondents (64%) felt that medical universities should open Ph.D. programs as it opens multiple opportunities in teaching and research . A study in the United States of America by Herzog et al., 2018 on Ph.D. program enrollment shows that many Ph.D students can secure the National Institute of Health grants along with dental school funding streams . The study analyzes the enrollment, graduation, and placement outcomes of PhD students from 1994 to 2016, using data from the American Dental Association (ADA) and surveys of 22 oral sciences PhD programs. More females (54.7%) than males (44.5%) were enrolled in PhD programs . In a study by Andriole and Jeffe, 2016, it was shown that 52.5 MD-Ph.D. graduates between (2000–2005) held full-time academic faculty positions by 2013 . Endodontic research has been showing tremendous growth in the dental community in Saudi Arabia. A study by Alrubaig et al., 2023, on Endodontic research performance in Saudi Arabia in the period from 2010 to 2022 shows a growth tendency from 1.29% in 2012 to 7.6% in 2022 from a global perspective. King Saud University was the most prolific institution, and most publications were published in the Journal of Endodontics . In our study, the key consideration that affects a candidate’s choice of joining a Ph.D. program were mainly the availability of good research facilities, staff diversity, and research funding. This further justifies the need for a structured Ph.D. program that can cultivate research skills and provide an academic framework for future endodontists to contribute to scientific advancements. In Saudi Arabia, the interest in joining postgraduate education programs in dentistry is usually governed by job promotion in the governmental sector. In our study, around 50% of the responses showed that the key factors in pursuing a Ph.D. program are the promotion in SCFHS classification and academic job requirements. Other 50% of the respondents showed interest in learning grant writing. In a study by Khami et al., 2008, five motive dimensions affected career choices for Iranian dental students and they were altruism and intellectual challenges, characteristics of the profession, social status and security, other person’s recommendation, and failure to be admitted to other study programs . Similarly, a study was done in Saudi Arabia on the associated influences and motivating factors in choosing a dental specialization. These factors were found to be job security and financial stability, encouragement from others, the nature of dentistry as a profession, family encouragement, interactions with dentists, and work-life balance flexibility . This study identifies several key factors that influence prospective students’ decision to pursue a Ph.D. in Endodontics. The program’s duration, recognition by SCFHS, and the ability to study part-time were critical considerations for participants. These practical concerns suggest that any Ph.D. program in Endodontics must offer flexible options to accommodate professionals already engaged in clinical practice. Furthermore, the availability of good research facilities, a diverse staff of Ph.D. holders, and research funding are essential to attracting high-caliber candidates. Steps should be taken to motivate dentists to pursue Ph.D. degrees. The emphasis on research infrastructure is particularly important in the context of the rapid technological advancements in Endodontics, such as new diagnostic tools, treatment techniques, and materials. Ph.D. programs are expected to equip candidates with the skills to critically evaluate these advancements and contribute to their development. As noted by previous studies, Endodontics is increasingly influenced by evidence-based practices, and Ph.D. holders will play a pivotal role in driving innovation and maintaining the highest standards of care. Candidates who are interested in pursuing a Ph.D. degree in Endodontics should focus on specific skills such as critiquing articles, writing scientific papers, and curricular development.
This study demonstrates a significant need for a Ph.D. program in Endodontics within Saudi Arabia. The high value placed on factors such as program duration, SCFHS recognition, and part-time options underscores the need for a program design that accommodates the needs of practicing professionals. Furthermore, the emphasis on research infrastructure, including readily available research funding, advanced facilities, and a diverse faculty, reflects the importance of establishing a research-focused program to address the rapidly evolving landscape of. Endodontics. Future researches are needed such as longitudinal studies on the career trajectories of Ph.D. holders in endodontics.
The representative sample size of the participants of the population of dental professionals in Saudi Arabia is small. Response bias of participants such as overestimating or underestimating their interest in a ph.D. due to social desirability or perceived expectations. The study focused on a limited number of institutions and this will affect the generalization of the results. Further studies should be done to compare the skills gained different doctoral programs in both the dental and the medical fields. Moreover, further studies could compare the research productivity in schools with postgraduate programs in comparison to schools that do not have such |
Factors associated with recent and regular non-use of dental services by students from a university in southeastern Brazil: a cross-sectional study | 36198f73-2b7c-493b-a9fe-b0f859bfa772 | 9753854 | Dental[mh] | The use of health services is at the core of the functioning of health systems . However, recent studies point to the existence of great inequities in the use of dental services by the general population [ – ]. Many factors can act facilitating or restricting the use of health services by individuals . According to the theoretical model proposed by Andersen and widely used in literature , the use of these services results from the interaction of individual factors, characteristics of the health system, and the social context and past experience of using the services. In addition to the pattern of use of dental health services, the literature has pointed to the importance of assessing the regularity of the habit of visiting the dentist, identifying those individuals who consult this professional only in cases of pain or curative treatment and those who seek them out for preventive treatment . Different population groups have been evaluated regarding the use of dental services. However, most epidemiological studies follow the parameters recommended by the World Health Organization (WHO) and the age index does not include young people aged between 18 and 24 years old . The university environment brings together many individuals in this age group, who have relatively similar characteristics but, at the same time, have diverse experiences and lives . These young people are characterized as individuals undergoing constant behavioral and lifestyle changes, which may interfere with both general health standards and oral health . A study conducted in southern Brazil with the university population revealed inequalities related to socioeconomic factors in the regular use of dental services and a lower use among university students with worse oral health conditions. In addition, students reported using the service more to solve oral health problems and not on a regular basis to prevent aggravations . However, there is still little studies about the patterns of dental service use among the young university population in the literature. Considering the specificities of this population, more studies are needed that aim to understand the factors associated with non-use of dental services, identifying the portions of the population with greater difficulty in access and thus, assisting in the development of public health policies aimed at specific populations in an equitable manner . Thus, this study aims to assess the factors associated with recent and regular non-use of dental services by young university students, using the Andersen model as a reference .
Study design and participants Data collection Variables Data analysis This was a cross-sectional study carried out through a census of university students admitted in 2021 to the Federal University of Juiz de Fora (UFJF). The public university is based in the city of Juiz de Fora (MG), and also has an advanced campus in Governador Valadares (MG). In 2021, 2480 students entered the university in the first semester and 1501 students in the second semester, for a total of 3981 students entered in presencial courses. The study included students entering undergraduate courses at UFJF in 2021 aged between 18 and 24 years. Students who did not respond to the survey questionnaire sent by e-mail after three attempts at contact were considered as sample losses. The parameters used to calculate sample size for finite populations were: a 45% prevalence in the regular non-use for this type of population , a 95% confidence interval and sampling error of 5%, resulting in a total of 347 individuals. The study was approved by the Human Research Ethics Committee of the UFJF, under protocol number 4.617.665.
Data collection was carried out during the COVID-19 pandemic, between May and November 2021, a period in which emergency remote teaching had been adopted by UFJF. Thus, a survey was created on the Google Forms platform was made available via email to all students admitted in 2021; their access was conditioned to signing the Informed Consent Form. The survey included objective questions about socio-demographic and socioeconomic characteristics, information related to the student’s major, their admission to the University and questions regarding the use of dental services and oral health status. A pilot study was carried out to test the instrument prior to data collection and allowed estimating a response time of around four minutes. Based on the pilot study, some changes were made to the survey used in order to improve understanding and interpretation of the questions.
Two outcomes that characterize the use of dental services by the studied population were investigated. The first outcome was the recent non-use of dental services, assessed through the question “Have you accessed dental care in the last 2 years?”, with “yes” and “no” as possible answers, and non-recent use assigned to the answer “no”. The time parameter for recent use of dental services adopted in this study was 2 years, and not 12 months, as has been advocated in most studies. The choice of this interval stems from the pandemic situation caused by COVID-19, which limited patients access to dental care worldwide for a long period of time. The World Health Organization (WHO) and the Pan American Health Organization (PAHO) conducted a survey in 128 countries to establish the degree by which attention to Noncommunicable Diseases (NCDs) was disrupted by the impact of the COVID-19 pandemic. The effect of the COVID-19 pandemic on the number of clinical dental consultations has also been examined, and a significant decrease observed [ – ]. A study in Brazil compared the mean number of clinical dental consultations in the period March-July in the years 2015–2019 with the same period in 2020, and a decrease of 65.6% in dental consultations was found . The second outcome investigated was the regular non-use of dental services, measured through the question: “Which of the statements below describes your access to dental care?”, with the following answer options: “I never go to the dentist”; “I go to the dentist when I have a problem or when I know I need to have something treated”; “I go to the dentist occasionally, whether or not I have some kind of problem”; and “I go to the dentist regularly.” The first two answer choices were assigned to regular non-use. The independent variables were grouped into three categories, according to the theoretical model for determining the use of health services proposed by Andersen (Fig. ). This model addresses the complexity of health services use in a comprehensive way, categorizing the determinants of service use into predisposing factors (that make the individual more or less susceptible to using health services), enabling factors (related to the possibility of accessing the service) and factors related to the individual’s need. Predisposing factors, in turn, are divided into demographics, social structure and health beliefs. In this study, predisposing factors included: gender (cisgender woman; cisgender man; transgender, agender or nonbinary), skin color (black or brown; white), marital status (single or married/Common-law marriage), Father’s level of education and mother’s level of education (did not study/did not finish elementary school; elementary school/did not finish high school; high school degree/did not finish undergrad degree; university degree/graduate degree), living situation before starting University (alone/with friends/with partner or With family), type of high school (public or private), admission through affirmative actions (yes or no), area of study (Biological Sciences/Health Sciences or Exact and Earth Sciences/Engineering/Agricultural Sciences/Human, Sciences/Applied Social Sciences/Linguistics, Letters and Arts), reason for last dental appointment (symptomatic or prevention), use of dental services in childhood (no or yes). The enabling factors were: monthly family income (up to 1.5 minimum wage; from 1.5 to 3 minimum wages; from 3 to 6 minimum wages; over 6 minimum wages), Current job status (working or not working), type of service accessed in last appointment (public or private), status at last dental appointment (very bad/bad/regular or very good/good), oral health guidelines (no or yes). And the factors related to the needs of the individuals were: self-perceived oral health (bad/regular; or excellent/good/very good), satisfaction with the appearance of teeth and mouth (very unhappy/unhappy/neither happy nor unhappy; or very happy/happy), toothaches over the last 2 years (yes or no), perceived need for dental care (yes or no).
Data analysis was conducted using the Statistical Package for the Social Sciences (SPSS) software, version 20.0 for Windows. Initially, descriptive analyses were performed using absolute and relative frequencies. The association of dependent and independent variables was investigated through bivariate analysis and robust Poisson regression with estimation of crude and adjusted prevalence ratios, using confidence intervals of 95%. The associated independent variables with a value of p ≤ 0.05 entered the multiple model, while the variables with a value of p < 0.05 remained in the final model.
Of the 3,981 entering undergraduates, 581 responded to the survey questionnaire, which corresponded to a response rate of 14.6%. Of these, 477 met the inclusion criteria of the study. Therefore, the final sample consisted of 477 university students aged between 18 and 24 years old. The majority were cisgender women (66.9%), aged between 18 and 19 years (74.0%) and white (64.5%). Most students attended public high schools (51.8%), had a monthly family income between 1.5 and 3 minimum wages (26.2%), lived with their parents and/or other family members (80.5%), and did not work (81.8%). In addition, most reported that their father or mother had a high school degree (31.0% and 29.1% respectively). Regarding their area of study, most participants were enrolled in courses in the health sciences (41.7%). Of the participants, 87.8% self-rated their oral health as excellent, very good or good, and 57.4% were happy with the appearance of their mouth and teeth. Despite this, 43.4% reported having had a toothache in the last two years and 59.3% reported needing dental treatment. Most participants used private dental health services (86.3%). Table presents the demographic, socioeconomic and oral health characteristics of the young university students grouped according to Andersen’s theoretical model 6 and distributed by the investigated outcomes. The prevalence of recent non-use was 19.5% (95% CI 16.0–23.3%) and of regular non-use was 53.5% (95% CI 48.9–58.0%). In the crude analysis, the following variables belonging to the predisposing factors showed significant differences when associated with recent non-use of dental services: gender, father’s level of education, type of high school institution, reason for last dental appointment and use of dental services in childhood. Among the variables listed as enabling factors, the following stand out: monthly family income, type of service used and status at last dental appointment. And of those classified as need factors: self-perception of oral health, satisfaction with the appearance of teeth and mouth, and perceived need for dental treatment. After the adjusted analysis, the following variables remained associated with recent non-use: type of service used and perceived need for dental treatment. In the crude analysis phase, regular non-use of dental services was associated with the following variables of predisposing factors: father’s level of education, type of high school institution, area of study, fear of dental treatment, reason for last dental appointment and use of dental services in childhood. Among the variables related to enabling factors, this outcome was associated with the following: monthly family income, type of service used and status at last dental appointment; and the following were related totoothaches over the last 2 yearsgular non-use: father’s level of education, area of study, reason for last dental appointment, use of dental services in childhood, self-perception of oral health, and toothaches over the last 2 years. Crude and adjusted prevalence ratios for recent and regular non-use of dental services are presented in Tables and , respectively.
The prevalence of recent non-use of dental services among university students in the present study was of 19.5%, a finding lower than what has been observed in population-based epidemiological surveys. In the last epidemiological survey on dental health, SB Brasil 2010, the prevalence of non-use of dental health services in the last 2 years among young people aged 15 to 19 years old in the Southeast region of the country was of 70.2% ; in the 2019 National Health Survey, the non-use of dental services in the year prior to the interview by adults aged over 18 years, was at 46.8% . However, it should be noted that these surveys, despite including a part of the young population, do not cover the specific age group selected in this study. In addition, no studies were found in the literature on the recent use of dental services by university students, which makes this comparison difficult. In the adjusted analysis, the type of service used and self-perceived need for dental treatment were associated with recent non-use of dental services. Young people who used private services and who did not perceive a need for treatment were more likely to not use recent dental services. The importance of income as a determinant of access to dental services is already widely known in the literature, and is possibly aggravated in the young population, which has historically been disregarded by the public sector in defining priorities for oral health care . The lack of perception of the need for treatment has also been indicated in the literature as one of the main reasons for not seeking dental care . Corroborating this non-perception is the fact that some oral diseases are asymptomatic at the beginning of their course, being identified by the individual only later . Moreover, the perception of an individual feeling sick comes, besides the physical sensations of pain and discomfort, also from the social and psychological consequences that the evolution of oral diseases can cause. In both situations, the perception of the need for treatment and, consequently, the use of dental services may occur, many times, late and for curative purposes. Despite the low prevalence of recent non-use of oral health services found in the present study, when the non-use of dental services on a regular basis by the population studied was assessed, a significant increase in this prevalence was observed, reaching 53.5%. This finding corroborates the limited national literature on the subject. A population-based study carried out by Carreiro et al. , in Minas Gerais, found a prevalence of 64.2% for regular non-use of dental services among individuals over 18 years of age. Echeverria et al. assessed the regular use of dental health services by university students over 18 years of age in Pelotas (RS), finding a prevalence of 55%of non-use of these services, even closer to that found in this research. It is important to highlight that the sample of this study is composed of college students and, despite of the university inclusion policies adopted in recent years, our study does not reflect the Brazilian population profile in this age group, because it does not include populations with greater social vulnerability, which have the greatest oral health problems and have less access to health services . Moreover, it is noteworthy that regular use was obtained through self-report, which may generate an information bias. The self-reported regular use of dental services by undergraduate students was associated with regular use of services during childhood, negative self-perception of oral health, toothache in the last 2 years, reason for the last dental visit, level of paternal education and area of knowledge of the course. The habit of visiting the dentist in childhood showed association with a higher prevalence of not using it regularly. On the one hand, it is believed that if children develop patterns of regular dental care, they are likely to maintain this habit in adulthood . However, access to dental care in youth may be hindered for various reasons, ranging from lack of maturity to seek health care, characteristic of age, to financial reasons related to the ability to pay for services. Brazil is going through a complex scenario both from the economic and political point of view, with direct repercussions on the health sector. There is evidence of a decrease in the number of individuals who can afford private services and, consequently, an increase in the number of patients who depend on public services; however, the financing of oral health policies has not followed this increase . It should also be considered the fact that public dental services in Brazil have historically been offered in a priority manner to children of school age , and this privilege occurred to the detriment of care to other population segments . To date, there is a gap in oral health policies and programs in relation to the young population. In this sense, it is possible that an individual who has had access to dental services in childhood, will have more difficulty in access to these services in youth, highlighting the need for actions aimed at this population. Regarding the factors of need, the adjusted analysis revealed that young college students with negative self-perception of oral health and toothache in the last 2 years were less likely to not use dental services regularly. Self-perception of oral health measures the value placed on oral health and determines the likelihood of seeking care with the goal of achieving optimal oral health status . Thus, both negative self-perception of oral health and recent experience of pain may result in the individual’s perceived need for treatment and, consequently, influence care-seeking. Students who used services for symptomatic or curative reasons at their last visit were less likely to not use dental services regularly. This result seems to show that even those students who claim to use services even in the absence of problems are still more motivated by curative reasons. Echeverria et al. also found that university students in southern Brazil used the service more to solve oral health problems and not on a regular basis to prevent aggravations. Other studies have demonstrated limited knowledge of oral hygiene practices and seeking dental care only in case of severe pain or discomfort by university students from various fields . The latter two associations were contrary to what was expected by the evidence already existing in the literature [ , , ]. Young people whose parents had lower levels of education were less likely to not use regularly. Moreover, students from health or biological sciences courses presented higher probability of not using regularly when compared to undergraduates from other fields of knowledge. As already mentioned, an important limitation of the present study concerns the collection of self-reported information on the regularity of dental visits. Considering that this is a socially desirable behavior, it is likely that there was an overestimation of the report of regular dental visits for preventive reasons, causing an information bias. Further studies on the regular use of dental services by this population with the use of objective indicators are suggested in order to minimize information bias. Other limitations include the study design (since being a cross-sectional study cause and effect relationships cannot be determined) and losses in the sample, which may have been enhanced by the use of the online questionnaire and email contact. In addition, it should be considered that the motivation to participate in the study can influence the response patterns. For example, individuals who attach greater importance to oral health may feel more motivated to participate in the study; on the other hand, individuals with greater need for treatment may also be more interested in participating in the study. Thus, some parameters may have been over- or under-estimated. Despite the limitations, this study contributes by broadening the focus on a population still poorly investigated in the literature and lacking oral health public policies that understand their specificities. Furthermore, we highlight the verification of two different patterns of use of dental services by young university students. This evidence points to the need to develop programs and actions aimed at young university students, who have been neglected within the scope of oral health policies, in order to break the cycle of using dental services for curative treatments or for emergency care. The data described in this article can be freely and openly accessed at figshare: 10.6084/m9.figshare.20980357.v2 .
It was concluded that young university students use dental services motivated by curative treatment needs and not with a preventive purpose, as would be the ideal. Thus, public policies for the prevention and promotion of oral health in higher education institutions must be planned and implemented, as well as expanding access to public dental services to the young adult population, in order to guarantee improvements in the quality of life of this population.
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Functional profiling of the rhizospheric Exiguobacterium sp. for dimethoate degradation, PGPR activity, biofilm development, and ecotoxicological risk | ef4bffa0-62e9-4be8-a5cb-a9c70f525d58 | 11599895 | Microbiology[mh] | A surge in the human population to more than 9 billion by the year 2050 parallelly demands an urgent rise in crop production by 60% . This urgent demand has led to the widespread adoption of intensive agricultural practices, which often rely heavily on agrochemicals such as fertilizers and pesticides. While these practices have elevated crop yields, they have also severely impacted soil health, leading to the depletion of soil fertility, loss of essential nutrients, and diminished water-holding capacity . Furthermore, the overuse of these chemicals contributes to environmental issues such as eutrophication and the decline of biodiversity. Among the pesticides commonly employed in agriculture, dimethoate, an organophosphate, is used in abundance regardless of the environmental threat it bears. It poses significant risks to non-target organisms, including humans and aquatic life, causing genotoxicity and developmental issues . Despite regulations aimed at limiting the use of such xenobiotics, dimethoate remains prevalent due to economic pressures and a lack of awareness regarding its environmental consequences , . Its persistence in groundwater and surface water, despite a half-life of 10–14 days , raises serious concerns about its long-term ecological impact. Sustainable agriculture emphasizes the improvement and maintenance of soil health, the production of high-quality crops, and the enhancement of socio-economic balances . A promising approach involves leveraging the potential of soil microorganisms, often referred to as “natural soil engineers” . These microbes play crucial roles in nutrient cycling, soil enrichment, and the bioremediation of contaminants. A number of studies have been conducted for the bio-degradation of dimethoate using microbial agents like Kocuria turfanensis isolated from soil , Bacillus licheniformis and Pseudomonas aeruginosa discerned from fish intestines and water respectively , Pseudomonas kilonensis etc. The rhizosphere is an important zone of the soil where a lot of biochemical exchange takes place between plants and soil microbiota. For this reason, in this zone, the microbial colonization rate is quite higher than the rest of the bulk soil . A typical group of bacteria is often found associating with roots and rhizosphere soil that promotes growth of the plants and is known as plant growth promoting bacteria (PGPB). Beside this, it also plays an exclusive role in nutrient cycling, soil enrichment, breakdown of complex toxic compounds like pesticides, and combating environmental stress like high salinity, high pH, drought, or other extreme conditions. PGPBs can also produce various metabolites like siderophores, antibiotics, enzymes, Volatile Organic Compounds (VOC) that show antagonistic effects on phytopathogens . An efficient PGPB to be applied in agriculture should be able to colonize plant roots and phyllosphere and also develop protective barriers like biofilm or microcolony formation . Biofilm is an important protection strategy in microorganisms in various ways, like mechanical stability, nutrient accumulation, gene exchange, survival in stress conditions, and tolerance to toxic agents. Microorganisms release extracellular polymeric substances (EPS), facilitating attachment to biotic or abiotic surfaces, and gradually form an extracellular matrix (ECM) by embedding the bacterial cells . Bacillus , Rhizobium , Rhodopseudomonas and Agrobacterium from the roots of Larrea divaricata , Bacilli isolated from native maize landraces as seed-endophytic , Bacillus vallismortis from the tea rhizosphere are few evidences of biofilm forming PGPB, providing protection to the bacteria as well as promoting distinctive plant growth. Biofilm often works as a stress response strategy for bacteria exposed to pesticides. However, obtaining an eligible PGPB candidate with biofilm forming ability is quite challenging, as the survival of targeted PGPBs often becomes questionable in in-vitro, when exposed to agrochemicals or fluctuations in environmental conditions. Biofilm-forming bacteria indigenous to organophosphate contaminated sites metabolically utilize and gain resistance to the pesticides and develop efficient mechanisms for their biodegradation. Rhizospheric soil collected at contaminated sites provides a superior source of microorganisms having the potential to degrade pollutants . This study aims to isolate and characterize bacteria from rhizospheric soil contaminated with dimethoate, focusing on their pesticide-degrading capabilities and plant growth-promoting traits. The novelty of this research lies in its comprehensive approach to identifying bacterial isolates that not only effectively degrade the organophosphate dimethoate but also possess strong biofilm formation abilities. This dual functionality is crucial for their resilience in field conditions, where they must endure environmental stressors while promoting plant growth. Moreover, the study will analyze crude metabolite extracts to identify and elucidate the metabolic pathway, evaluate the safety and viability of degraded by-products, as well as the suitability of selected microbial candidates. Addressing a significant research gap, this work seeks to identify bacteria that possess a unique combination of pesticide degradation, biofilm formation, and plant growth promotion. The findings of this work could lead to the development of eco-friendly strategies that mitigate the impacts of agrochemical pollution while improving soil health and crop productivity. Study area and soil sample collectionIsolation and screening of potential dimethoate degrading bacteriaDetection of dimethoate biodegradationIdentification of bacterial isolateScanning electron microscopy (SEM) analysisCharacterizations of the isolatesEcotoxicity studies Phytotoxicity analysisMicrobial toxicity study The microbial toxicity of dimethoate and its biodegraded products was assessed using the Agar well diffusion method . In this study, lawn cultures were prepared using model organisms Bacillus subtilis and Escherichia coli , selected for their well-characterized genetics, role in soil health, resistance mechanisms and are representative of enteric bacteria that can be impacted by environmental pollutants, making them an ideal candidate for assessing the effects of agricultural chemicals like pesticides , (Sturme et al. 2021; Abd El-Ghany et al. 2023). Wells were created in the agar plates, into which crude dimethoate and the metabolic extracts were introduced for testing against the target microorganisms. The plates were incubated at 37 °C for 24 h to facilitate microbial growth and interaction with the test compounds. Following incubation, the presence of any clear zones of inhibition around the wells, indicating the antimicrobial efficacy of the compounds tested was observed and noted. The experiment was conducted in triplicates to ensure the reproducibility and reliability of the results, providing a comprehensive insight of the microbial toxicity of both dimethoate and its degradation products. A sugarcane cultivation field in Sevur, Vellore district, Tamil Nadu, India (12.9649° N, 79.1831° E), which had a history of organophosphate pesticide dimethoate exposure for over 10 years was selected for soil sampling. Collection of rhizospheric soil was done from uprooted sugarcane plants of 15 random areas in the field. By tapping on roots, the adhered soils were collected in a sterile plastic bag, which was then taken to the laboratory. The soil samples were mixed thoroughly, and lumps were broken down into powder. The soil was tested for its physiological and chemical properties. Fresh soil samples were used for the experiments conducted. The soil samples brought into the laboratory were artificially enriched with technical grade dimethoate (30% emulsifiable concentrate (EC), Rallis India Ltd., Mumbai, India.), according to the procedure described by Naphade et al. with few modifications . Under aseptic conditions, in a 150 ml conical flask, 1 g of soil sample was added to 50 ml sterilized nutrient broth, and a 50 ppm concentration of dimethoate was added. The flask was kept in a rotatory shaker (180 rpm) for 5 days at room temperature. At day 5, 1 ml of stock (spiked soil sample) was taken, serial dilutions were performed (10 −1 to 10 −10 ) and spread plated on nutrient agar (pH 7, Hi-Media) [Peptone 5.000 g/L, Sodium chloride 5.000 g/L, HM peptone B# 1.500 g/L, Yeast extract 1.500 g/L, Agar 15.000 g/L]; incubated at room temperature (28–30 °C) for 24 h. Subsequent subcultures of individual colonies were performed to obtain and ensure pure cultures. The pure cultures obtained after isolation from rhizospheric soil samples, were further screened for potential dimethoate degrading strains. Onto selective mineral salt media (MSM, pH 7.0, Hi-Media) [NaCl 0.50 g/L, KH 2 PO 4 3.00 g/L, MgSO 4 0.12 g/L, CaCl 2 .2H 2 O 0.013 g/L, yeast 3.00 g/L, Na 2 HPO 4 6.00 g/L] supplemented with 150 ppm of dimethoate, the isolates were streaked. The plates were incubated for 24 h at 28–30 °C. The strains showing maximum viability were selected for further studies. The taxonomic characteristics and the colony features of the selected isolates were observed keenly. To detect the basic morphology and properties of the strain, biochemical tests and Gram staining were performed. UV-spectrophotometer analysis of dimethoate biodegradationExtraction of crude metabolites by liquid-liquid extraction and FTIR analysisThe crude chloroform extract containing the secondary metabolites produced by the bacterial isolate was analysed using GCMS . GC–MS (VIT-SIF Lab, Division of Chemistry for NMR and GC-MS Analysis). The Clarus 680, Perkin Elmer GCMS instrument was specified with an Elite-5MS column with 30.0 m length, 250 μm film thickness and 0.25 mm internal diameter. To separate the components, helium was employed as carrier gas at a constant flow rate of 1 mL/min. The chromatographic run was executed at 260 °C injector temperature. 1 µL of the extracted residue was injected into the instrument for 2 min with the initial oven temperature at 60 °C, followed by 300 °C at the rate of 10 °C/min, and 300 °C was possessed for 6 min. Total GC running time was 64 min. The obtained spectra of the metabolite components were compared with the database of known components’ spectra correlating the GC-MS NIST (2008) library using Tuerbomass Version 5.4.2 software. To propose a prospective biodegradation pathway for dimethoate, GC-MS data were analyzed to identify degradation products. The PathPred pathway prediction server from GenomeNet was used to predict metabolic pathways based on these compounds, facilitating the identification of intermediate metabolites and associated enzymes. Additionally, the KEGG (Kyoto Encyclopedia of Genes and Genomes) database was used to visualize the curated metabolic networks related to dimethoate degradation. The resulting pathway was adapted and redrawn from the findings of Sharma et al. , . The biodegradation of dimethoate in Mineral Salt Medium (MSM) was assessed using UV-spectrophotometric analysis. Cultures grown overnight in MSM media spiked with dimethoate, were centrifuged at 4000 g for 5 min at 4 °C. The pellet formed was washed with sterile 0.9% NaCl (saline) solution and resuspended in saline to achieve an optical density (OD) of 0.7. A 2% (v/v) aliquot of this seed culture was then inoculated into Erlenmeyer flasks containing 150 ml MSM media, supplemented with 150 ppm of dimethoate and adjusted to an initial pH of 7. The experimental setup was monitored for 5 days to track the degradation process of dimethoate by the isolate L.O. Uninoculated media with the identical parameters served as the control. At an interval of 24 h, 2 mL of broth sample was collected to measure cell density at OD 600. Following 5 days of incubation, the sample was centrifuged at 7000 g for 10 min to obtain the cell-free supernatant (CFS), which was then analysed by UV-spectrophotometry to quantify dimethoate degradation at 214 nm. The experiment was performed in triplicate to ensure accuracy and reproducibility. The % degradation was calculated by the formula: [12pt]{minimal} $$\% {}={}{} {}00,$$ where A i is the initial absorbance and A f denotes the final absorbance. The crude metabolite extract of isolated strain L.O was subjected to Fourier transform infrared spectroscopy analysis (FTIR), to detect any alterations in chemical bonds or functional group modifications occurring, due to the metabolic activity of the isolate in the course of dimethoate degradation . Liquid–liquid extraction was performed to obtain crude extract from the culture medium . The isolate was cultured in a conical flask containing MSM broth supplemented with dimethoate on shaker for 5 days at 180 rpm, 28–30 °C. Uninoculated dimethoate supplemented media was used as a control (untreated). After 5 days, the broths were centrifuged at 6000 g for 10 min to obtain cell free supernatant (CFS). In a separating funnel, metabolites were extracted by mixing equal volumes (1:1) of supernatant with chloroform (solvent). The supernatant was thoroughly shaken with the solvent and allowed to stand for about 30 min to facilitate the complete separation of the chloroform layer from the aqueous phase. This crude chloroform extract was collected, dried to remove residual solvent and used for FTIR analysis. The FTIR analysis was conducted using the Nicolet iS50 instrument from Thermo Scientific, USA. The analysis covered a spectral range from 4000 cm −1 to 500 cm −1 with a resolution of 4 cm −1 . Peaks in the FTIR spectrum are developed based on the percentage transmission of IR light at specific frequencies. Comparative changes of chemical bonds and functional groups associated with the peaks were observed for detecting bio-degradation of the dimethoate (untreated) and the treated sample. The data were identified using the frequency tables provided by Nandiyanto et al. . o identify the potential pesticide degrading bacterial isolate L.O, 16 S rRNA sequencing was performed by the Sanger sequencing method using the ABI 3130xl platform. From cellular DNA, the 16 S rDNA gene was amplified by PCR using 16 S rDNA-specific primers (Forward: GGATGAGCCCGCGGCCTA, Reverse: CGGTGTGTACAAGGCCCGG) . The polymerase chain reaction amplification was achieved in a final volume of 50 µl. The amplification reaction containing 144 ng of extracted DNA is used for amplification along with 10 pM of each primer, dNTPs 2.5 mM, 10×Taq DNA polymerase assay buffer and Taq DNA polymerase enzyme. It was run on an ABI3130 genetic analyzer. The amplification reaction was set for 30 cycles. PCR conditions were set to initial denaturation at 94 °C for 3 min, 32 cycles consisting of denaturation at 94 °C for 1 min, annealing at 50 °C for 1 min, extension at 72 °C for 1 min, and final elongation at 72 °C for 7 min. Using NCBI Blast , the sequence similarity was analysed and compared with the reference strains from Genbank and the Ribosomal Database. Fastree software was used to construct the phylogenetic tree incorporating the neighbor-joining method . The impact of dimethoate on the cell surface morphology of the isolated strain LO was investigated using scanning electron microscopy (SEM) . Cell culture grown overnight in MSM media, both with and without dimethoate treatment at log phase, was used for sample preparation. For SEM sample preparation, the bacterial biomass was fixed in a glutaraldehyde solution (4:1 ratio) to preserve cellular structures, followed by washing with phosphate-buffered saline (PBS) at a 1X concentration to remove excess fixative. A gradual dehydration process was further conducted using a series of ethanol solutions (20%, 40%, 60%, 80%, and 100%), ensuring the maintenance of cell morphology. The samples were subsequently set for SEM observation, operated at a voltage of 10 kV, and imaged at an 8KX magnification. Cultures grown without dimethoate treatment served as the control group, which allowed a comparative analysis of any morphological changes induced by the pesticide treatment. The SEM images were analysed to assess alterations in cell surface structures of the isolate, providing insights into the effects of dimethoate on the isolated strain. Analysis of PGPR activityPhosphate solubilizationDetection of biofilm formation by the tube methodThe isolate was tested for plant growth promoting activity like phosphate solubilization activity and production of HCN, IAA, ammonia and siderophore as per the protocols described by Sharma et al. . All the tests were performed in triplicate. Phosphorus solubilizing activity of isolates was determined qualitatively according to the method stated by Nautiyal . To perform the experiment, 2.5 µl of the isolate L. O (O.D. 600) was spread plated onto Pikovskaya’s agar medium [(g/L) Yeast extract 0.500, Dextrose 10.000, Calcium phosphate 5.000, Ammonium sulphate 0.500, Potassium chloride 0.200, Magnesium sulphate 0.100, Manganese sulphate 0.0001, Ferrous sulphate 0.0001, Agar 15.000] containing calcium triphosphate (0.5%) as the inorganic form of phosphate. The plates were incubated for 5 days at 28 °C. A transparent halo zone around the grown colonies indicates the phosphate solubilizing activity of the bacterial isolates. Production of HCNProduction of indole acetic acid (IAA)Production of ammoniaProduction of siderophoretargeted pesticide-tolerant bacterial isolate L.O was streaked onto nutrient agar plates supplemented with 4.4 g/L glycine. Filter paper discs, pre-treated with 0.5% picric acid were dissolved in 2% sodium carbonate, and placed inside the lid of each Petri dish. The dishes were then sealed and incubated for 5 days at 28 °C. A gradual colour change in the filter paper from deep yellow to orange and then to brown would indicate the production of hydrogen cyanide (HCN) by the target bacteria . The isolate L.O was inoculated in tryptophan (1–2%) supplemented nutrient broth. The flasks were kept on rotary shaker and incubated for 24 h at 28 °C. Cultures were centrifuged at 10,000 rpm for 15 min. The supernatant was taken and mixed with Salkowski’s reagent, and incubated at room temperature for 25 min. Appearance of the pink colour indicates production of Indole acetic acid (IAA) . The bacterial isolate L.O was cultured in peptone water for 4 days at 30 °C. 1 ml of Nessler’s reagent was added to the tube. Development of a dull yellow color indicates small amounts of ammonia production, and a deep yellow to brownish color indicates maximum ammonia production . Siderophore production of the isolate was detected by the chrome azurol S (CAS) assay . Bacterial isolate grown on nutrient agar was spot inoculated on CAS agar [Agar 15 g/l, Chrome Azurol S 0.02 g/l (1% solution and used as 10 ml/l), nutrient broth 5 g/l, KCl 0.1 g/l, MgSO 4 ·7 H 2 O 0.2 g/l, CaCl 2 ·2 H 2 O 0.01 g/l, FeCl 2 ·6 H 2 O 0.001 g/l] and incubated for 7 days at 28 ± 0.2 °C in a dark room. An orange zone around the isolate indicates positive siderophore production. The isolate L.O was tested for biofilm forming ability by the tube method . The strain was inoculated in a set up containing MSM with a 150 ppm dimethoate supplement. The culture was incubated for 5 days at room temperature. After incubation, the tube was poured out, washed with phosphate buffer saline (pH 7.3) and air dried. The tube was then stained with 0.1% crystal violet. Excess and residual stain was removed with deionized water and then dried inside out. Tubes containing only media and no inoculation were used as control. The control tube was used as a reference to assess the differences in biofilm formation of the sample tube. Visible purple film lining the walls and bottoms of the tubes indicates a positive indication of biofilm formation. The experiment was performed in triplicate. The biodegraded metabolites were assessed for their toxicity compared to the parent compound dimethoate. Phytotoxicity tests were performed as per the method described by Sahoo et al. . In this experiment, the phytotoxicity of metabolites obtained from dimethoate biodegradation in comparison to the parent compound was performed against mustard seeds ( Brassica juncea ) due to its widespread cultivation in India and rapid germination rate. The seeds were initially washed three times with distilled water to remove surface contaminants, followed by surface sterilization using 70% ethyl alcohol, and then rinsed again with distilled water. Experimental setups involved preparing cotton beds within Petri dishes, overlaid with Whatman No. 1 filter papers, which were subsequently moistened with sterile distilled water to create optimal germination conditions for the seeds. Minimal Salts Medium (MSM) solutions containing specific concentrations of dimethoate and crude extracts of biodegraded metabolites were added to individual Petri plates, while control plates consisted of MSM and tap water only. Ten surface-sterilized seeds were placed in each Petri dish, maintaining adequate spacing to prevent overlapping of roots. The Petri dishes were covered with aluminum foil to maintain humidity and shield the seeds from light, and incubated in the dark for 24 and 48 h. Following incubation, germination rates and root lengths were measured. Each treatment was replicated in triplicates to ensure statistical reliability, allowing for a comprehensive analysis of the effects of the biodegraded metabolites and dimethoate on seed germination and root development. Isolation and screening of isolatesDetection of dimethoate biodegradationIdentification of secondary metabolites and analysis of probable dimethoate biodegradation pathwayIdentification of bacterial isolateScanning electron microscopy (SEM) analysisCharacterizations of the isolatesEcotoxicity studies Phytotoxicity analysisMicrobial toxicity study The toxicity of dimethoate and its degraded products was investigated in model microbes like Bacillus subtilis and E. coli by agar well diffusion method. Figure a–f shows the experimental results for microbial toxicity study. No zone of inhibition was observed for control (only MSM) and metabolite extract treated (degraded products) plates for all the microorganisms tested. However, clear zones were observed in the plates with crude dimethoate for both the tested bacteria. This result validates that the biodegradation products formed by the metabolic activity of L.O, in treatment of dimethoate, are less toxic than pure dimethoate towards the targeted microorganisms. The initial screening generated morphologically distinct, mixed colonies on nutrient agar (NA) plates from spiked soil sample. The soil used had a pH of 7. In the control plate, no growth was seen. A potent dimethoate degrading strain L.O was selected based on the growth of the strain on MSM agar plates supplemented with 150 ppm dimethoate as the sole carbon source after incubation at 28–30 °C for 24 h. Subsequent subcultures were performed to obtain pure cultures. The isolated bacteria under investigation demonstrated several distinguishing characteristics relevant to its identification and ecological contribution. Colonies are opaque, glossy, and orange in colour, indicating robust cell structure and pigment production capability. The organism is classified as Gram-positive, stipulating a thick peptidoglycan layer that may confer sustainability against environmental stresses. The negative result of spore stain, implies a possibility of alternative survival strategies. A positive Voges-Proskauer test reflects the acetoin production ability, indicating fermentative metabolism, while negative results for citrate utilization and indole production suggest restricted metabolic versatility. Additionally, the negative oxidase test and positive catalase test highlight specific metabolic pathways that facilitate the detoxification of hydrogen peroxide. The positive reaction to starch hydrolysis demonstrates its ability to utilize complex carbohydrates, highlighting its adaptability in diverse ecological niches. Details of the morphological and biochemical characters have been listed in Table . The relation between the growth of isolate and the biodegradation of dimethoate pesticide has been demonstrated in Fig. deducing the UV-spectroscopic readings. The analysis was carried out for five days. From Fig. , it is seen that, cell culture density (O.D 600 ) is at its maximum after one day, and after three days bacterial concentration was decreasing marking the deterioration stage. Bio-degradation of dimethoate was achieved maximum after five days. The control showed no turbidity. The percentage of dimethoate degradation was enumerated as per the formula mentioned in Section " ". The strain L.O. was found to be capable of degrading 95.87% of dimethoate in five days. The structural changes in dimethoate upon degradation by the isolate L.O were determined by FTIR-analysis. A lucid difference in spectrum was noticed between the untreated (control) and isolate-treated sample, indicating degradation of dimethoate, due to the activity of isolate by conformational changes or bond alterations. The spectrum has been shown in Fig. . In untreated dimethoate (control), peaks were noticed at 3370 cm − 1 , 2920 cm − 1 , 2850 cm − 1 , 1705 cm − 1 , 1190 cm − 1 , 1050 cm − 1 , 995 cm − 1 , 845 cm − 1 , 685 cm − 1 and 570 cm − 1 . Whereas, for the treated sample, peaks were obtained at 3224 cm − 1 , 2949 cm − 1 , 2925 cm − 1 , 2855 cm − 1 , 1655 cm − 1 , 1454 cm − 1 , 1259 cm − 1 , 1076 cm − 1 , 1019 cm − 1 , 796 cm − 1 , 703 cm − 1 and 634 cm − 1 . Peak pertaining to aliphatic POC phosphate (1050 cm − 1) present in control, disappeared in the treated sample. There was a distinct reduction and peak shift in a range of 3000–3500 cm − 1 , 2850 –2815 cm − 1 , 710 –685 cm − 1 and 705–570 cm − 1 . At 2935 –2915 cm −1 range, splitting of peaks was also observed in treated extract compared to untreated sample. In the untreated sample, the presence of peaks at 3370 cm −1 and 1705 cm −1 corresponds to hydroxyl and carbonyl functional groups, respectively, which are typical in organophosphate compounds. The disappearance of the peak at 1050 cm −1 , corresponding to aliphatic POC phosphate, in the treated sample is indicative of a successful degradation process, possibly reflecting the breakdown of the phosphonate group. Additionally, the shifts and reductions in peaks observed in the spectral regions around 3000–3500 cm −1 (aliphatic 2° amine stretch), 2850 –2815 cm −1 (methoxy CH stretch), and 710–570 cm −1 (disulfides) further reflect that the isolate not only degraded the organophosphate compound (dimethoate) but also modified its chemical environment, influencing its toxicity and environmental persistence. The splitting of peaks in the 2935–2915 cm −1 (methylene CH stretch region) in the treated sample demonstrates changes in the molecular conformation, which may originate from interactions between the isolate and the dimethoate molecule during degradation. Overall, these spectral differences mark the effectiveness of isolate L.O. in degrading dimethoate, highlighting its potential application in bioremediation strategies targeting organophosphate pollutants. GC-MS analysis of by-products formed upon dimethoate bio-degradation imparts valuable insights into the chemical transformations by the metabolic activity of the isolate L.O (Fig. ). The initial detection of dimethoate (m/z 125) in the untreated sample establishes its baseline presence. Following treatment, the identification of intermediate compounds such as methyl diethanol amine (m/z 42) and aspartyl glycine ethyl ester (m/z 87) justifies the event of hydrolytic and substitution reactions. The hydrolysis of dimethoate possibly proceeds with the formation of methyl diethanol amine by cleaving the ester bond, releasing a methyl group, and incorporating ethanolamine. The formation of aspartyl glycine ethyl ester indicates enzymatic transformations utilizing these intermediate compounds in microbial metabolic activity, possibly denoting a pathway for assimilation of amino acids. The presence of phosphorothioic O, O, S-acid (m/z 151) in the treated sample signifies complete degradation of the dimethoate structure, directing towards a possible successful breakdown of the phosphorothioate moiety. This transformation provides an insight that the isolate L.O. utilizes oxidative and hydrolytic mechanisms to nullify dimethoate toxicity, resulting in less harmful products. The proposed bio-conversion pathways (Fig. A, B) demonstrate two probable distinct routes of degradation. Figure A shows the conversion of dimethoate to omethoate via nucleophilic substitution, while Fig. B depicts a carboxylation route, indicating further degradation into non-toxic end products. These chemical pathways uphold the versatility of metabolic mechanisms of isolate L.O., emphasizing its potentiality in dimethoate detoxification strategies. The molecular identification of the selected bacterial strain L.O was performed using 16 S rRNA sequence analysis. The reference sequences (FASTA) were retrieved using the Basic Local Alignment Search Tool (BLAST) against the NCBI database, and the phylogenetic tree was generated incorporating the neighbour joining method. A phylogenetic tree was generated based on the collection of the 16 S rRNA sequences of the related species acquired from NCBI along with the query. The tree was constructed using MEGA 7 software and the constructed tree was visualized in iTOL v6 . The similarity of the identified species is accounted by the closest match with a short genetic distance with the closest neighbor. The strain was identified as Exiguobacterium profundum with 100% similarity, and was submitted to NCBI against accession number, OR965293 (Fig. ). Comparative scanning electron microscopy (SEM) images of dimethoate-treated and untreated cultures of isolate L.O. (Fig. ) at 8 kX magnification exhibits that the strain conserves the rod-shaped morphology with no prominent structural changes in cell surface post-dimethoate exposure. This structural integrity of the isolate L.O. signifies exclusive adaptation to the toxic impacts of dimethoate, suggesting effective cellular mechanisms like sophisticated cell wall structures or the presence of efflux pumps for detoxification or tolerance of the organophosphate pesticide. Such features are important for survival in contaminated environments and may enhance the efficiency of the isolate in bioremediation targeting organophosphate pollutants. Analysis of PGPR activityDetection of biofilm formation by the tube methodThe isolate L.O. was tested for plant growth-promoting traits, including phosphate solubilization, hydrogen cyanide (HCN) production, indole-3-acetic acid (IAA) synthesis, ammonia production, and siderophore production. The results were found to be positive for HCN production, IAA synthesis, and ammonia production, while no siderophore formation was observed (Table ). The production of HCN and IAA, a key auxin, plays a crucial role in root development and overall growth of plants, suggesting that isolate L.O. may positively influence plant health through hormonal regulation. Positive indication of ammonia production further denotes increased nitrogen availability, supporting better nutrient uptake and growth of plants. The lack of siderophore production may constrain the ability of the isolate to sequester iron in iron-deficient soils; however, the positive traits exhibited suggest that isolate L.O. can still make a significant contribution to plant enhancement through its other mechanistic approaches. Overall, these findings highlight the promising potential of the isolated L.O. as a beneficial microbial inoculant in agricultural developments targeting improved crop quality. The results of the PGPR activities shown by the isolates are demonstrated in Fig. . The strain L.O were checked for biofilm forming ability using tube method, based on the deposition of crystal violet on biofilm formed in the inner surface of tubes. The results indicated that strain L.O. adhered to the surfaces and generated biofilm. The tube appeared purple due to the retained crystal violet stain on the sides and bottom of the tubes, indicating biomass accumulation and effective adhesion properties of the strain. In contrast, the control showed no such purple coloration. The visual differences have been demonstrated in Fig. . The observation of the experiment establishes the potential of strain L.O. as a useful microorganism in agricultural settings, where biofilm formation may enhance its survival in field conditions. toxicity of secondary metabolites resulting from the bacterial biodegradation of dimethoate was evaluated in mustard seeds through phytotoxicity to assess their suitability for environmental application. The results have been represented in Figs. and , illustrating that average seed germination took almost 24 h for all the treatments. It was observed that MSM and dimethoate treated seeds showed less germination in 24 h as compared to control (Tap water). In contrast, a nominal difference in germination patterns between the control and metabolite-treated seeds was seen. Maximum root elongation was observed in seeds treated with the metabolite extract, suggesting a possibility of positive influence of the biodegraded products on the seeds, while dimethoate-treated seeds exhibited the least root growth. This finding proposes that the metabolites derived from the biodegradation of dimethoate are comparatively safer than the toxicity associated with the parent compound, making them more suitable for environmental applications. solation and characterization of strain L.O from dimethoate-contaminated rhizospheric soil portray its promising potential as a bioremediation agent. Cultured in minimal salt media supplemented with organophosphate dimethoate as the sole carbon source, this strain exhibited remarkable tolerance, effectively degrading 95.87% of the pesticide within five days. This finding is quite significant in context to the persistence of dimethoate in the environment and its associated risks to soil health and non-target organisms , . Exiguobacterium sp. have been reported to degrade organophosphate pesticides like acephate , chlorpyrifos , profenofos and malathion , but there is no report found on dimethoate degradation for literature review so far. Strain L.O was identified as Exiguobacterium profundum through 16 S rRNA sequencing, which are often considered extremophiles that thrive in diverse and often harsh environments. Earlier literature studies have documented the presence of Exiguobacterium species in various substrates, including mine soil , plastic dumps , saline sediments , arsenic rich soil , petroleum contaminated soil , as well as Tibetan glaciers . This adaptability suggests that strain L.O possesses resilient metabolic pathways that enable it to sustain under adverse conditions, enhancing its applicability in context to bioremediation. Significant alterations and shifts in peaks were observed in spectral data, indicating a possible biodegradation occurred due to activity of the isolate on dimethoate. A similar work has been reported by Silambarasan et al., where alterations in FTIR spectrum were observed between treated and untreated chlorpyrifos samples . Analysing the GCMS data, a biodegradation pathway was elucidated , . From the data, it was illustrated that the biodegradation of dimethoate supposedly could have followed two routes, the dimethoate carboxylic acid pathway or the omethoate pathway, ending in phosphorothionic O, O, S acid and mineralization respectively which are probable safe end products that might possibly be released post bioremediation. High degradation efficiency of the isolate emphasizes its potential in mitigating the ecological impact of dimethoate contamination. Rapid degradation is crucial in bioremediation, as it reduces the duration of toxic exposure and minimizes the risk of harmful effects on soil, soil microbiome, water bodies, and plant life. Additionally, the morphological stability of strain L.O, evidenced by scanning electron microscopy (SEM), endorses its ability to maintain structural integrity in the presence of dimethoate. This contrasts with findings in other prior studies where pesticide exposure led to significant cell alterations, indicating that strain L.O exhibits effective protective mechanisms against pesticide-induced stress . Beyond its degradation capabilities, Exiguobacterium profundum exhibited various beneficial plant growth promoting (PGPR) properties such as hydrogen cyanide (HCN), indole-3-acetic acid (IAA), and ammonia, establishing L.O as a dual-function agent. These properties contribute to enhanced root development and nutrient uptake, simultaneously improving crop health in contaminated soils , . The ability to promote plant growth while degrading pollutants represents a promising approach for sustainable agricultural practices, improving soil qualities in affected regions. The capability of the strain to form biofilms under stress conditions further enhances its potential as an effective bioremediation candidate. Biofilms can provide protection against environmental stresses to the bacteria and increase the degradation efficiency of the contaminant . Hypothesizing, if dimethoate serves as an inducer to biofilm formation, this could further enhance the functionality of the strain in contaminated environments. However, survival of the potential bioremediation candidate is not the only concern. Sometimes, the by-products of biodegradation are more toxic than the parent compound . Assessing the toxicity of the intermediate biodegradation product is an absolute necessity to prevent adverse effects of the treatment on the environment and ecosystem. Hence, ecotoxicity studies of the biodegradation by-products were conducted on mustard seeds and model bacteria like B. subtilis and E. coli , which were found to be less toxic than dimethoate, therefore can be considered safe. From the phytotoxicity test, an inhibitory effect was observed in seed germination and root elongation in dimethoate and MSM treated seeds. Pesticide toxicity and the strong ionic effect of MSM could be the possible reasons. This reverse effect was seen in metabolite treated mustard seeds and, thus, can be considered less toxic to the plants. In microbial toxicity studies, no zone of inhibition was observed in agar well diffusion assay with crude metabolic extract, which validates the safety of the strain Exiguobacterium sp. L.O and its dimethoate degradation by-products for the environment. Strain L.O emerges as a promising candidate for the bioremediation of dimethoate-contaminated soils, offering remarkable advantages in degradation efficiency, morphological stability, and plant growth promotion. To maximize the potential of strain L.O, it is important to address the identified research gaps, including detailed mechanistic studies that elucidate the specific enzymatic reactions involved in the degradation process. Additionally, evaluating the long-term effects of the secondary metabolites on overall ecosystem health is necessary. This approach will help ensure that the bioremediation process does not inadvertently introduce new ecological risks, making the strain safer for application in agricultural systems and restoring contaminated ecosystems. This research presents a novel application of rhizospheric soil bacteria Exiguobacterium sp . (L.O), integrating the bioremediation of the organophosphate pesticide dimethoate with agricultural enhancement. This study contributes significant insights into the potential of the strain Exiguobacterium profundum as a bioremediation agent as well as a plant growth promoter, establishing the way for sustainable practices in managing pesticide pollution and enhancing agricultural productivity. This dual approach is crucial for managing the challenges posed by pesticide pollution in agricultural practices. Additionally, the capability of the strain to form biofilm enhances its resilience and survivability in contaminated environments, providing a strategic advantage under actual field conditions. To our knowledge, this study is the first to demonstrate the efficacy of this strain in the dimethoate degradation, as supported by an extensive review of the literature. However, the study also highlights limitations that require further investigation, which include the need for long-term assessments of the effectiveness of the strain across diverse environmental conditions and its interactions and impact on the broader soil microbiome. While ecotoxicity studies on intermediate metabolites have been conducted for seed germination, future research should prioritize the evaluation of their effects on fully grown plants, assessing root and shoot growth, chlorophyll content, and flowering to obtain an insight into prolonged impacts on plant health and yield. This research provides a significant understanding of the potential of Exiguobacterium profundum as a bioremediation agent and plant growth promoter, facilitating sustainable practices for managing pesticide pollution and enhancing agricultural productivity. |
Attracting Users to Online Health Communities: Analysis of LungCancer.net’s Facebook Advertisement Campaign Data | c9f98d1e-847a-48a1-9ab9-b568a47a749f | 6861997 | Health Communication[mh] | Online Community Growth LungCancer.net In this study, we reported the feasibility and cost-effectiveness of Facebook advertising to promote online community growth in the context of the LungCancer.net community. LungCancer.net provides patients and caregivers a platform to learn, educate, and connect with peers and health care professionals. The content published by LungCancer.net is written by patients, caregivers, and health professionals and supplemented by editorial content. In August 2017, LungCancer.net catered to 1575 users and sought to expand their community base through a series of social media advertisements. With 69% of US adults on Facebook and 74% of users on the site daily , Facebook seemed to be an ideal platform to promote community growth. The goal of this study was to assess the engagement results of Facebook advertisements designed to increase the number of opt ins to the LungCancer.net online community (ie, the number of users that provided their email to join the community).
Currently, 72% of adults seek health information on the Web, and 16% search for peers with similar health concerns . Online communities can effectively extend health education and facilitate social support and have been linked to improved self-management and enhanced health outcomes . The number of online communities has grown substantially over the past decade, with countless websites increasing traffic from patients and caregivers through user-engaged communities . Patients are motivated to join these communities to access support, advice, and accountability in reaching health goals . Online community growth is crucial to meeting these user needs, as it builds communities’ pooled knowledge and increases access to quality informational and social support . Larger online networks have the power of network effects—where more users increase the usefulness of the community . For those seeking others with shared experiences, larger communities offer a greater number of individuals with the potential for cognitive empathy, particularly from people outside ones’ close network where sharing may cause emotional burden . For staff overseeing these sites, limited evidence is available to guide community growth, which is known to be a time- and resource-intensive task .
Facebook Advertisement Campaign Advertisement Performance Measures From August to December 2017, 5 weeklong Facebook campaigns were launched with the objective of increasing opt ins to LungCancer.net. Each campaign consisted of 3 unique advertisements that contained an image, a text, and a call to action . The visuals included 6 static images and 1 image in the Graphics Interchange Format (signaled with the “†” symbol in ). The text included messages crafted by community managers and quotes from members. The target audience was adults (18 years or older) with an interest in lung cancer–related content and/or Facebook pages. No other demographic variables were used to define the audience within the Facebook Ads Manager system. The budget for each advertisement was US $25 per day. Facebook utilizes a bidding cost system, and actual expenditures for each test averaged within 4% of the desired budget, with the exception of 1 outlying test, which was 19% below the budget.
The performance of each advertisement was evaluated using metrics rooted in advertisement engagement frameworks . According to McGuire’s Model of Persuasion, eliciting action begins with advertisement exposure and moves across a continuum of cognitive and behavioral responses . Exposure in this campaign is operationalized as impressions (number of times the advertisement appears in News Feeds) and reach (number of individuals exposed to the advertisement). Frameworks proposed by Neiger et al and Platt et al were used to define low-to-high behavioral responses. As the goal of this campaign was to increase opt ins to the LungCancer.net community, low user engagement was defined as interacting with the advertisement through clicks (ie, reacting to the post, clicking a post link, or liking the LungCancer.net Facebook page), medium user engagement was defined as sharing or commenting on the advertisement, and high user engagement was defined as opting in or signing up for the LungCancer.net community. After each campaign, metrics were pulled for each advertisement, and advertisements with the lowest opt in cost were run with new advertisements during the next weeklong campaign. Advertisements with the lowest opt in cost during each weeklong campaign are signaled with the “‡” symbol in .
Audience Demographics Advertisement Engagement Results displays engagement results. During the first campaign (August 24-30), advertisement B attracted the greatest level of engagement, including the greatest reach (10,556 people), number of impressions (12,569), reactions (221), link clicks (131), page likes (11), and opt ins (81 new community members) and the lowest opt in per cost rate (US $1.99 per opt in). This advertisement featured an image of lungs with the text “connect with others who understand what life with lung cancer is like.” Advertisements B and C were then used in the second campaign (August 31-September 6) alongside 1 new advertisement. In week 2, advertisement B again outperformed other advertisements and was subsequently implemented in week 3 (October 5-11). During the third campaign, advertisement F, featuring the same image as advertisement B with new text “After reading some of the comments on LungCancer.net, no more self-pity, no more discouragement, as I read these comments of strength and survival and determination I feel revived” attracted the greatest number of reactions (194), comments (19), link clicks (176), and opt ins (82) at the lowest cost (US $1.10 per opt in). In the fourth campaign (November 9-15), advertisement H, with the same text as advertisement F but a simpler lung image, attracted the greatest engagement including 179 reactions, 14 page likes, and 60 opt ins at US $1.47 per opt in. In the fifth campaign (December 7-17), advertisement H was outperformed by an advertisement featuring the same image with the text, “A place to share the good, the bad, and everything in between” (advertisement I). Advertisement I attracted 114 link clicks, 22 page likes, and 50 opt ins at US $1.89 per opt in.
Over the course of the 5 campaigns, the sum reach was 91,835 people, and 863 members opted in to the LungCancer.net community (ie, demonstrated high engagement; ). Females between 55 and 64 years represented the largest population reached by the campaign (31,401/91,835; 34.29%), whereas females aged 65 years and older represented the largest population that opted in to the LungCancer.net community (307/863; 35.57%). Given that US $1742 was invested across the 5 campaigns, approximately US $2.02 was spent per opt in, and just over 1 cent was spent per exposure to the campaign.
Principal Findings Limitations and Future Research Conclusions This study provides a foundation for research to optimize the reach of online health communities. Facebook was a feasible, cost-effective recruitment channel for this online community, and evaluation of other advertisement designs may provide further evidence for promising engagement strategies. Online communities are vital to health promotion efforts as multiple populations seek low-cost, easily accessible health resources. Focusing on expanding the reach of such communities could have major implications for the health of future populations.
Our findings demonstrate the feasibility of utilizing Facebook advertising as a cost-efficient tool to grow online health communities. Across the 5 campaigns, 863 new members opted in to the LungCancer.net community, yielding an opt in rate (opt ins/reach) of 0.94% (863/91,835) and a cost/opt in rate of US $2.02. Although the cost-effectiveness of Facebook advertisements varies widely in recruitment literature , our cost is but slightly higher than the average cost per click of US $1.32 for health care advertisements on Facebook . Although Facebook advertisements were a cost-efficient community growth tool in this study, other research provides mixed results regarding the effectiveness of Facebook advertising . Some agree that Facebook is an efficient way to draw diverse audiences to health promotion interventions . Others have found Facebook to be a useful tool to increase advertisement reach, yet the actual rate of results per reach remains low . This may indicate that Facebook advertisements are more efficient than traditional approaches (eg, physician referral, direct mail, and email) for online community growth outside research recruitment, where strict eligibility criteria often narrow the target audience . Additional research is needed to test this hypothesis and optimize strategies to grow online health communities. Although these findings do not provide for specific design recommendations to increase engagement, we found some support for promising features of advertisements that match suggestions in previous literature: use of direct quotes/testimonials ; explicit reference to social support available in the community ; and simple lung images that are likely to be easily interpreted as relevant to those seeking lung cancer communities.
Although this research provides foundational knowledge regarding the feasibility of Facebook advertisements to grow the LungCancer.net community, the findings are limited to the advertisement images and text used. Additional research is needed to systematically compare engagement with different images, texts, channels, and times of year to identify strategies associated with optimal community growth. Research is also needed to identify the impact that community growth through Facebook advertisements has on community engagement. Users who respond to a Facebook advertisement already demonstrate online engagement and may be more likely to contribute to an online health community than members recruited through other traditional strategies. Finally, given suggestions that Facebook advertising can effectively engage hardly reached populations in health education and intervention , additional research is needed to identify the sociodemographic characteristics of those engaged. Data presented here demonstrate a campaign that engaged primarily ageing female populations, representative of the current LungCancer.net site visitors (61% female and 55 years and above).
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Shelter‐Based Integrated Model Is Effective in Scaling Up Hepatitis C Testing and Treatment in Persons Experiencing Homelessness | ab7294e7-eb47-4219-900e-623e692bc38d | 8710795 | Patient Education as Topic[mh] | Study Population and Study Design Study Procedures Assessment of Clinical Variables Statistical Analysis This prospective study was conducted by a multidisciplinary team at four large homeless shelters, two in San Francisco, CA, and two in Minneapolis, MN, from August 1, 2018, to January 30, 2021. These homeless shelters provided supportive services on a daily basis to more than 300 residents in San Francisco and between 170 to 350 residents in Minneapolis. The services provided included shelter, meals, case management, and some level of medical care ranging from basic triage to specialist consultation. Following informed consent, adults 18 years of age and older seeking shelter services and who were either treatment naive or had not received HCV treatment within the prior 12 weeks were enrolled. In addition, clients experiencing homelessness who were HCV positive and who accessed low‐threshold temporary shelters and safety‐net liver specialty care were also recruited. Patients with significant medical or psychiatric conditions that prevented consenting or participation in the study were excluded.
Clients who agreed to HCV testing and who met study eligibility criteria were enrolled, completed a questionnaire, and underwent point of care HCV testing (OraQuick HCV Rapid Antibody Test; OraSure Technology, Bethlehem, PA). Participants who tested negative for HCV antibody were provided information about HCV and its prevention. ( , ) Those who tested positive for HCV antibody received a confirmatory HCV RNA test and a standardized 30‐minute HCV education. This in‐person comprehensive education was delivered using a PowerPoint slide format and was led by a designated nurse, pharmacist, or advanced practice provider. In addition, pre‐education and post‐education questionnaires ( , ) were administered, and information on HCV risk factors and HCV awareness was captured. Standard‐of‐care HCV therapy was offered to all participants with detectable HCV RNA through insurance or patient‐assistance programs for HCV by the prescribing provider. Treatment was primarily delivered on‐site at the shelters or through coordination with their primary care provider, liver specialty provider, or addiction services. The treatment plan was made at the discretion of the treating provider and in accordance with clinical practice guidelines and insurance restrictions. The medication dispensation, therefore, varied and ranged from directly observed therapy (within addiction services), to weekly or monthly dispensing, to dispensation of all medication supply at the start of therapy. If needed, the study team assisted participants in obtaining insurance and facilitated linkage to primary care services. In San Francisco, a designated HCV registered nurse coordinator managed patients on treatment within the shelters in collaboration with shelter clinic providers, primary care providers, or the San Francisco safety‐net liver specialty clinic. In Minneapolis, a Doctor of Pharmacy embedded within shelter clinics managed patients on treatment in collaboration with shelter clinic staff and the Minneapolis safety‐net liver specialty clinic. Patients were followed throughout therapy and completed laboratory tests and questionnaires at the end of treatment and at 12 weeks following completion of therapy (SVR). Clinical data, including bloodwork, imaging, medical history, and adherence to medications, were collected from the participants’ medical records. A US $25 incentive was given for HCV testing, and a total of $75 was given following HCV education and completion of HCV therapy, including SVR blood work. Institutional review board approvals were obtained from the University of California San Francisco and Hennepin Healthcare Human Subjects Research Committee, and all participants provided written consent.
Active HCV infection was confirmed with a detectable HCV RNA, and response to therapy was evaluated by HCV RNA testing at the end of therapy and at SVR time points. Diagnosis of cirrhosis was made based on abdominal imaging or presence of fibrosis stage 4 (F4) on transient elastography (FibroScan), FibroSURE (Laboratory Corporation of America, Burlington, NC), or FIB‐4 index >3.25. ( ) In addition, fibrosis stage F0‐F3 was captured when transient elastography or FibroSURE was available. Cirrhotic decompensation was assessed by presence of ascites, hepatic encephalopathy, or history of variceal hemorrhage. Presence of HCC was captured from clinical records and by liver imaging. Nonprescription drug use, alcohol consumption, and receipt of substance‐use therapy before treatment, at the end of therapy, and SVR time point were assessed by self‐report. Alcohol consumption was categorized as (1) none or minimal (<1 drink per month), (2) moderate (more than none or minimal but no more than four drinks/day or 14 drinks/week in men, no more than three drinks/day or seven drinks/week in women), (3) heavy (more than four drinks/day for men, more than three drinks/day for women; or binge drinking [five or more drinks for men, four or more drinks for women, on the same occasion]). ( )
Descriptive analyses of cohort characteristics were performed to obtain frequency for categorical variables and median (interquartile range [IQR]) or mean (SD) for continuous variables. Patient characteristics between those with and without receipt of therapy and those who achieved SVR at 12 weeks and those who did not achieve SVR were compared using the chi‐squared test or the Fisher’s exact test if appropriate for categorical variables and the Mann‐Whitney test for continuous variables. Univariate and multivariable modeling was performed with the outcome measures of receipt of therapy and SVR as well as an a priori list of predictors and those predictors with a P < 0.2 on univariate analysis. The multivariable model for the outcome of receipt of therapy was adjusted for age, sex, race, illicit drug or alcohol use within the past year, receipt of HCV education, and severity of liver disease, while the outcome of SVR was adjusted for age, sex, and race. All analyses were performed in Stata 15 statistical software (Stata Corp LP, College Station, TX).
Participants HCV Screening Chronic HCV Therapy Chronic HCV Treatment Outcomes Factors Associated With Receipt of Therapy Factors Associated with SVR Characteristics of patients by known SVR status are summarized in Table . The median duration of treatment was similar in the SVR and no SVR groups at 58.5 and 58 days ( P = 0.1), respectively. In addition, there were no statistically significant differences in patient sociodemographic, clinical, and laboratory characteristics or length of stay at shelter between the SVR and no SVR groups, except those with SVR had significantly lower HCV RNA at treatment initiation (median log 10 , 6.1 vs. 6.8; P = 0.01) and a higher proportion were adherent to HCV medication by provider report (56.7% vs. 12.5%, P = 0.03). There were no differences in HCV genotype among those who did or did not achieve SVR. Importantly, all 5 patients with genotype 3 achieved SVR. Moreover, 9 of 11 patients with cirrhosis, including both patients with decompensation, had SVR. On ITT analysis, the only independent predictor of achieving SVR when controlling for age, sex, and race was adherence to HCV medication (OR, 14.5; P = 0.01). Although a higher viral load was associated with lower odds of achieving SVR (median log 10 OR, 0.4; 95% CI, 0.2‐1.2; P = 0.09), this did not reach statistical significance.
Participant recruitment strategies included advertisement for availability of HCV testing in the shelters as well as directly approaching 1,199 shelter clients. A total of 772 clients agreed to participate in the study; of these, 766 were deemed eligible. There were no significant differences ( P > 0.05) in age, sex, race, or shelter site among those who met or did not meet enrollment eligibility (data not shown).
During the study, a total of 120 HCV testing sessions were performed (72 in San Francisco and 49 in Minneapolis). Of the 766 participants, 162 (21.1%) tested positive for HCV antibody; of these, 107 (66.0%) had detectable HCV RNA. Patient characteristics overall and by HCV antibody status are summarized in Table . Those who were HCV antibody positive were older (median age 55.8 vs. 52.6 years), a higher proportion were men (75.3% vs. 66.2%), and of non‐Hispanic White race (39.1% vs. 24.4%) compared to those who were HCV antibody negative. In addition, patients who were HCV positive (vs. HCV antibody negative) were more likely to report having a health care provider (82.6% vs. 73.8%), receipt of prior HCV testing (79.6% vs. 45.2%), history of injection drug use (66.7% vs. 13.0%), history of substance‐use therapy (62.4% vs. 37.6%), and illicit drug use within the prior year (84.4% vs. 66.7%). The proportion of participants who reported receipt of prior HCV testing in San Francisco and Minneapolis was 63.4% and 38.6%, respectively; of these, 17.6% and 12.9%, respectively, reported testing positive. Overall, 60.5% of participants who reported having previously tested positive for HCV had active infection with detectable HCV RNA. There was no significant difference in willingness to engage in HCV therapy or in subsequent receipt of HCV therapy between those patients who were viremic who did or did not report prior knowledge of a positive HCV test (90.8% vs. 93.0% and 68.2% vs. 65.9%, respectively).
Of the 107 participants with detectable HCV RNA, 66 (61.7%) initiated standard of care HCV treatment (44 in San Francisco and 22 in Minneapolis). The treatment regimens prescribed were as follows: 43 patients (65.2%) received glecaprevir/pibrentasvir, 18 patients (27.3%) sofosbuvir/velpatasvir, 2 patients (3%) elbasvir/grazoprevir, and 1 (1.5%) patient each of sofosbuvir/ledipasvir, sofosbuvir/ledipasvir+ribavirin, and sofosbuvir/velpatasvir/voxilaprevir. Characteristics of participants with detectable HCV RNA by receipt of HCV therapy are summarized in Table . There were no significant differences with respect to sociodemographic status, substance use, history of psychiatric illness, or other medical comorbidities, laboratory, or clinical measures in those who initiated therapy compared to those who did not initiate treatment. However, a significantly higher proportion of patients who did not initiate therapy were from Minneapolis compared to San Francisco (56.1% vs. 43.9%, P = 0.03). In comparing the characteristics of patients with detectable HCV RNA by shelter location, a higher proportion of patients in San Francisco were non‐Hispanic White whereas a higher proportion of patients in Minneapolis were Black ( P = 0.03). There were no other significant differences in patient characteristics between the two sites (Supporting Table ). With respect to severity of liver disease, of the 106 patients with detectable HCV RNA and available data, 19 (17.9%) had cirrhosis, 3 (2.8%) had liver decompensation, and 3 (2.8%) had evidence of HCC; the distribution of these patients was similar in the untreated and treated groups. When evaluating fibrosis stage in participants without cirrhosis by either noninvasive imaging (transient elastography, n = 20) or noninvasive serologic methods (FibroSURE, n = 26), 2 (3.1%) were F0, 15 (23.1%) were F1, 18 (27.7%) were F2, and 11 (16.9%) were F3 (Fig 1A) . Therefore, the overall severity of liver disease by all modalities was 26.2% mild (F0 and F1), 27.7% moderate (F2), and 46.1% advanced fibrosis or cirrhosis (F3 and F4). The distribution of liver disease severity by shelter location is shown in Fig. B. Although the proportion of patients with cirrhosis was not statistically different between San Francisco and Minneapolis ( P = 1.0), a higher proportion of patients from Minneapolis had F0‐F1 and conversely, a higher proportion in San Francisco had F2‐F3 severity ( P = 0.006). The severity of liver disease did not differ significantly between those who did or did not receive therapy in this study.
Of the 66 patients who were initiated on HCV therapy, 54 patients (81.8%) achieved SVR, 8 (12.1%) did not achieve SVR, and the SVR status was unknown in 4 patients (6.1%) (Fig. ). The median time to initiation of therapy was 56 (IQR, 32‐107) days overall and longer in Minneapolis compared to San Francisco (76 days vs. 43 days, P = 0.04). At the start of HCV therapy, 10 patients had a history of prior HCV treatment (3 with pegylated interferon [PEG‐IFN]/ribavirin, 4 with DAA, and 3 with unknown prior treatment regimen). Of those with prior PEG‐IFN/ribavirin therapy, 1 patient was treated with sofosbuvir/velpatasvir and achieved SVR and 2 were treated with glecaprevir/pibrentasvir, of whom 1 achieved SVR and 1 did not. Of the 4 with prior DAA therapy, 3 had received sofosbuvir/ledipasvir; 1 of these patients was treated with sofosbuvir/velpatasvir and did not achieve SVR, and 2 were treated with glecaprevir/pibrentasvir or elbasvir/grazoprevir and both achieved SVR. One patient was previously treated with glecaprevir/pibrentasvir and treated with sofosbuvir/velpatasvir in the study and achieved SVR. Among those with unknown prior treatment regimen, 2 achieved SVR and 1 patient had an unknown SVR status. There were no significant differences in rate of SVR among those with known SVR status by treatment site (82.5% in San Francisco, 95.5% in Minneapolis; P = 0.2). However, on intention to treat (ITT) analysis that included all patients, a higher proportion of patients in Minneapolis achieved SVR compared to San Francisco (95.5% vs. 75.0%, P = 0.049). Of the 8 patients who did not achieve SVR, 7 were treatment nonresponders and 1 achieved response at the end of therapy but experienced virologic relapse after discontinuation of therapy. Four patients (1 with relapse and 3 with nonresponse) were retreated for HCV with sofosbuvir/velpatasvir/voxilaprevir; 2 of these patients subsequently achieved SVR, SVR status was unknown in 1 patient, and another patient was deemed noncompliant to retreatment.
Univariate and multivariate models of factors associated with receipt of therapy are shown in Table . On univariate analysis, shelter location was associated with receipt of therapy. On multivariate analysis, having been enrolled in a shelter in Minneapolis compared to San Francisco was negatively associated with receipt of therapy (odds ratio [OR], 0.3; 95% confidence interval [CI], 0.1‐0.7; P = 0.01), while having identified a health care provider was positively associated with receipt of therapy (OR, 4.1; 95% CI, 1.2‐14.1; P = 0.02). These associations were independent of.
This is the first prospective study to our knowledge that implemented HCV testing and treatment within homeless shelters. Among the 766 participants screened, 21% tested positive for HCV antibody, the majority (66.0%) of whom were actively infected with detectable HCV RNA, supporting the previously reported high prevalence of HCV in the homeless population. ( , , , ) In addition, similar to prior studies, ( , ) history of injection and illicit drug use represented a main risk factor for HCV in this population. Importantly, nearly 18% had evidence of cirrhosis, and among those with available data, another 17% had advanced stage fibrosis (F3). This highlights the potential impact of delayed HCV therapy despite ongoing engagement in care among the homeless and emphasizes the need for optimized HCV care delivery in this vulnerable population. ( ) Using an integrated and homeless shelter‐based intervention, we were able to engage 62% of eligible patients in HCV therapy and achieve HCV cure in over 80% of this difficult‐to‐reach and vulnerable population. We observed significant gaps in the HCV care cascade among those experiencing homelessness. First, half of all enrolled participants experiencing homelessness had reported prior HCV testing, likely due to screening in high‐risk groups, such as those with recent illicit drug use as reported by 70% of our study population. Second, there were geographic differences in receipt of prior HCV testing, with San Francisco clients experiencing homelessness reporting a higher testing rate compared to Minneapolis (63.5% vs. 38.6%, P < 0.001). This is not surprising as important public health community‐based initiatives, such as End Hep C SF, in San Francisco have resulted in increased HCV awareness and rates of HCV testing in the city. ( ) Third, although nearly 80% of those who tested positive for HCV antibody following enrollment reported receipt of HCV testing in the past and over 80% reported having a health care provider, the majority of the patients who were actively infected had not engaged in HCV therapy. The disparity between the rates of HCV screening and linkage to HCV therapy in this vulnerable population is similar to that reported in other homeless populations. ( ) Our integrated and shelter‐based approach that included HCV education was highly effective in engaging patients in HCV therapy; over 60% of patients with active infection received standard‐of‐care HCV therapy, which is significantly higher than reports in homeless populations infected with HCV. ( , , , ) Characteristics, including demographics and comorbidities, varied within the treatment cohort. However, participants who reported having a health care provider were 4 times more likely to receive therapy compared to those who did not. Research has shown that a high proportion of patients who remain engaged in health care also engage in HCV therapy. ( ) Our findings also highlight that primary care or health care provider engagement in the HCV cascade of care is critical to reducing HCV burden in this highly marginalized population. The majority of patients in our study achieved HCV cure, with the only independent predictor of response being adherence to HCV therapy. Real‐world studies in underserved populations report high rates of SVR in the era of DAA therapy, similar to that reported in the general population. ( , , , ) In addition, lack of patient compliance with treatment is associated with lower treatment response rates, ( ) as observed in our study. Although the overall rate of SVR of 81.8% in our prospective study is similar to that reported in a retrospective study of patients with a history of homelessness, ( ) it was lower than another study of patients (SVR of 97%) who were either currently homeless or had marginalized housing. ( ) Aside from population differences, the latter study selected patients based on treatment readiness as determined by adherence to appointments and collaborative decision making among care providers. ( ) Although treatment willingness and readiness were also assessed in our study, the threshold for treatment initiation was low, especially in the San Francisco sites. This may have influenced differences in treatment initiation rates observed between the cities, resulting in inadvertent patient drop‐off before treatment initiation as well as SVR rates. Indeed, on ITT analysis, the SVR rates were lower at 75.0% in San Francisco compared to 95.5% in Minneapolis where patient selection was more restrictive. Moreover, patients in San Francisco were more likely to initiate treatment within a shorter time compared to Minneapolis. These geographic differences likely reflect potential provider or system factors that may influence treatment initiation despite similarly implemented and integrated infrastructure for HCV treatment within shelters and similar rates of patient acceptance of HCV therapy following education across sites. Furthermore, at the time of the study, access to HCV therapy through insurance varied between sites, with requirements of at least 6 months of substance use sobriety in Minneapolis. Applying for exemption from this requirement on a case‐by‐case basis may have resulted in further delays in treatment initiation. Alternatively, there was a higher rate of task shifting with respect to HCV therapy from specialty to primary care and community engagement in low‐barrier treatment within San Francisco that may have influenced rates of treatment initiation, especially among the most challenging groups, such as those with active substance use, psychiatric illness, and instability of shelter access. This study has several limitations. First, patients who agreed to participate in the study and received education may have been more motivated to engage in HCV care. Second, the majority of the patients enrolled in this study were English speaking, and therefore we cannot generalize our results to non‐English speaking individuals. Third, our treatment response rates may not be generalizable to all homeless populations. There were site differences in HCV treatment initiation and response rates on ITT. While it is unclear what led to the delay in HCV treatment initiation in some patients at the Minneapolis site, this factor coupled with other barriers unique to Minneapolis may have led to an unintentional selection bias favoring patients that were more likely to adhere to treatment appointments and laboratory testing but not necessarily medication dosing, accounting for the differences in ITT SVR rates between sites. Lastly, adherence to medication was not captured using pill count due to lack of feasibility. Nevertheless, we were able to implement an effective model of shelter‐based HCV education, testing, and treatment in uniquely diverse patient populations from two geographically distinct locations, enhancing the generalizability of our findings to shelters in other urban settings. Despite numerous reported barriers to HCV therapy spanning patient, provider, and system factors in individuals experiencing homelessness, ( , , ) our integrated model of on‐site HCV testing, education, and treatment following extensive needs assessment involving stakeholders ( , ) was successful in enhancing HCV screening and linking to HCV therapy. Our study illustrates that tailored models of care that reduce barriers to patient engagement, including colocalization of HCV care within shelters or supportive housing and simplified steps for HCV treatment as recently proposed by guidelines, ( ) are critical to HCV elimination efforts in this highly vulnerable population.
Tables S1 Click here for additional data file.
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The chronic wound characterisation study and biobank: a study protocol for a prospective observational cohort investigation of bacterial community composition, inflammatory responses and wound-healing trajectories in non-healing wounds | 922e6b95-3330-4523-b349-56e607a3ce31 | 11487800 | Debridement[mh] | Chronic wounds are estimated to affect between 1% and 2% of the global population and reports rate that, in developed countries, 3%–5.5% of the total healthcare costs are associated with the treatment of chronic wounds. Chronic wounds are wounds that become stalled at some point in the normal healing trajectory. Although no international consensus exists on the specific duration required to define chronicity, these wounds are typically defined as wounds that have not healed within 4–12 weeks. Chronic wounds are often classified based on aetiology, with the most common types being: venous, diabetic, pressure or ischaemic ulcers. Chronic wounds cannot always be reliably classified based on one single driver. As noted by Falanga et al , chronic wounds have an inherent heterogeneity with a need for, but also a challenge towards, more precise classifications. The treatment of chronic wounds has improved over the last decades, but chronic wounds have a rising incidence due to an increase in the ageing population and accumulation of predisposing, lifestyle-related diseases—making the need for effective treatment even more urgent. In 2008, Bjarnsholt et al and James et al provided evidence of bacterial biofilm in clinical samples from chronic wounds. Subsequently, in a 2017 meta-analysis conducted by Malone et al , it was affirmed that bacterial biofilms can be identified in approximately 80% of samples from chronic wounds. Moreover, recent findings suggest the presence of scattered single-cell bacteria in chronic wounds. Limited research has explored patterns in the distribution of microbes in wound tissue based on species. However, a study by Fazli et al documented the uneven distribution of Staphylococcus aureus and Pseudomonas aeruginosa in separate formations. Though the exact mechanism is not understood, microbes including bacterial biofilms are believed to negatively affect wound healing by exacerbating or capturing the wound in an inflammatory state. Host immune response activation is considered the key factor distinguishing microbial wound colonisation from infection, and previously used criteria such as the term ‘critical colonisation’ (eg, >10 5 bacteria per gram tissue) are no longer considered valid. The debate continues regarding the utility of surface culture to guide treatment but for selected species, eg, P. aeruginosa , positive cultures have previously been found to correlate with larger, potentially older wounds and less favourable outcomes following, for example, split-thickness skin grafting (STSG). Moreover, investigations suggest that a high proportion of facultative anaerobes can negatively affect healing, and other findings have concluded positive correlations between microbial community dynamics and healing. Guidelines emphasise that clinical signs of infection can be masked due to a compromised immune system and decreased circulation, and clinical assessment is challenging. This finding is supported by a study using dual RNA sequencing (RNA-Seq) on debrided material from chronic wounds, which revealed that the observed patterns of host gene expression and inflammation frequently diverged from the clinical infection severity classifications attributed. Research on the wound microbiome’s relationship with healing remains limited, often constrained by sample size, incomplete clinical data or a lack of investigation into both host response and microbial burden and to our knowledge, no studies have investigated the potential correlation between the skin microbiome of patients with chronic wounds and the outcome following STSG. RNA-Seq data and proteomics can offer insights into the molecular mechanisms underlying chronic wounds and transcriptomic changes over time.
A better understanding of chronic wounds on the molecular and cellular levels is crucial to improve our understanding of the role of bacteria in chronic wounds, with the goal of improving treatment and classification. An improved understanding of the infectious microenvironment can improve the application of laboratory and animal models, and important knowledge can be obtained and translated to other chronic infections.
Our hypothesis is that bacterial community composition and activity in chronic wounds play a pivotal role in influencing host gene expression and inflammatory profiles. These, in turn, affect the wound-healing trajectories. Therefore, the primary objective of this study is to identify gene expression patterns in samples from chronic wounds that can be linked to inflammatory activity and healing outcomes and to correlate them with bacterial community characteristics.
The primary objective of the chronic wound characterisation (CWC) study is to investigate bacterial community composition, inflammatory responses and the influence of bacteria on wound-healing trajectories and outcomes following STSG. To achieve this, we will assess healing trajectories including clinical factors potentially contributing to delayed healing, aiming to explore the role of microbes in wound healing and the mechanisms associated. The primary objective will be investigated through the following: Investigate microbial clonal diversity, coexistence and adaptation over time and their association with healing outcomes. Determine the transcriptional levels of bacteria and human cells in chronic wounds, and how it varies with time, treatment and healing. Determine the spatial and temporal distribution of bacteria and differences in distribution based on species. Compare the presence of bacteria and inflammatory cells between STSG, that is, the autografted donor skin, and wound tissue. Investigate the correlation between the wound and skin microbiome and healing in patients with chronic wounds. Develop a non-invasive method to sample surface microbes in chronic wounds and investigate their spatial distribution through cultivation. Establish a biobank consisting of longitudinal and clinical data to make advanced analyses and provide a comprehensive understanding of chronic wounds.
The predictive cohort The in-depth cohort Secondary outcomes (both cohorts) The primary endpoint for the predictive cohort is the assessment of wound healing, based on any available clinical evaluations and photographic analysis within 12 months following inclusion. Wound healing will be assessed as the percentage change in wound area (measured in cm 2 ), comparing the two-dimensional area at follow-up to that at inclusion.
The primary outcome for the in-depth cohort is the assessment of wound healing following STSG. This assessment is based on photographic analysis within approximately 4 months after inclusion.
Treatment with antibiotics. Revascularisation procedures. Incidence of STSG. Incidence of amputation. Mortality rate.
Study design Definition of wound chronicity Participants Materials Sampling Sample handling, preparation and analysis Analysis of clinical photographs Clinical data Statistics Sample size Previously published and unpublished RNA-Seq data on diabetic foot ulcers from the Costerton Biofilm Center, Department of Immunology and Microbiology, University of Copenhagen, has shown that the wound transcriptomes cluster into three distinct groups, with a coefficient of biological variation (CV) of 0.4, according to their inflammatory profiles. We expect the samples from this study to fall into the same groups. Thus, the sample size calculation is based on DGE analysis between these biologically relevant clusters and carried out using the R package RNASeqPower. Assuming an average coverage depth of 5 reads per transcript, a within-group CV of 0.4 across all groups, and a target fold-change of 1.5, approximately 34 patients per group are needed to achieve a power of 0.8 and a false positive rate of 0.05. The previous calculation applies to the predictive group, as we expect the in-depth cohort to be considerably smaller due to the more demanding inclusion criteria. For the latter cohort, we based our sample size calculation on a generic DGE analysis of two groups which could include: healed versus non-healed wounds at approximately 4 months following STSG, the centre versus the edge of the wound, wound versus transplant or wound before STSG versus wound at follow-up. We approximate that around 20 patients will be included. With an average coverage depth of 5, CV of 0.4 (as is typical for human clinical studies) and aiming for a target false positive rate of 0.05, we will achieve a power of 0.7 to detect a fold change of 2.
The CWC study is a prospective, observational cohort study planned across multiple sites. Inclusion and sampling will be performed at the Copenhagen Wound Healing Center, Department of Dermatology and Wounds, Bispebjerg and Frederiksberg Hospital, and Section of Infections and Amputations, Department of Orthopedic Surgery, Herlev and Gentofte Hospital. The CWC study employs a multifaceted approach that includes meticulous and longitudinal sampling, advanced diagnostics and comprehensive collection of clinical data, including assessments of healing.
Chronic wounds are defined as those deemed chronic or non-healing by the treating physician, acknowledging the lack of an international consensus on the definition of chronicity. This approach reflects the clinical reality and will be supplemented with clinical data, such as wound duration and wound aetiology, as detailed in the section on clinical data.
Study participants must be ≥18 years old and legally competent, be referred to one of the included departments with at least one chronic wound below knee level and understand Danish language in spoken and written form. Moreover, to be eligible for the predictive cohort , the patient needs to undergo mechanical debridement, either through simple curettage or surgical debridement. Sampling will be performed during routine debridement, and patients will be followed up using health records. Assessments will be conducted from available data within approximately 12 months after inclusion. For the in-depth cohort , patients must be planned for and undergo surgical debridement followed by STSG. Subsequently, they will be seen and sampled during two follow-up visits planned independently of their inclusion in the CWC study. The follow-up visits are expected to be conducted within approximately 4 months following inclusion. Exclusion criteria for both groups are pregnancy, dementia or individual reasons that make the patient unsuitable for inclusion, as defined by a responsible wound care physician. The last exclusion criterion is set since, in the in-depth group, patients will undergo multiple punch biopsies. Individual factors, such as the location, shape or size of the chronic wound, should be considered to ensure that biopsies can be performed without causing unnecessary harm. An overview of the inclusion and exclusion criteria for the predictive and in-depth cohorts can be seen in . All participants receive oral and written information and consent to voluntary inclusion prior to sampling. The study design is non-interventional in terms of treatment, and patients can withdraw their consent at any time point. A comparative overview of the design and overall investigations planned for the in-depth and predictive cohorts is presented in .
Patients or the public will not be involved in the design, conduct, reporting or dissemination plans of our research.
BD ESwab Collection Kit, Copan Italia SpA. Nylon filters (1213812, pore size 5 µm, GVS North America, USA). Tape strips: 2×16 consecutive D-Squame disc tapes (D100, Monaderm, Monaco). D-Squame pressure instrument (D500, 225 g/cm 2 , Monaderm, Monaco).
For the predictive cohort, the following samples will be obtained: A photo of the wound: the photo is obtained after the application of a sticky marker containing a scale (0–30 mm) (Dansk Telemedicin A/S) in close proximity to the wound, intended for size calibration and patient identification. For RNA-Seq+16 s ribosomal RNA (rRNA) sequencing: the material removed on debridement. For microbial culturing: one ESwab conducted after debridement as a tight swab, covering the whole wound surface. All the sampling will be conducted after the removal of any compression and/or bandages. If any ointments from previous applications are covering the wound these will be removed using sterile gauze and/or sterile saline. For the in-depth cohort, the patients will be seen and sampled at inclusion (in relation to STSG) and at follow-up visits ( ). In relation to STSG, prior to surgical debridement In relation to STSG, during surgical debridement and STSG Sampling done at follow-up visits Sampling at follow-up visits will depend on the status of the wound progression. Samples obtained from all patients If there is transplant/healed skin after STSG If there is still a non-healed wound For microbial culturing: The whole wound area is sampled using an ESwab. If the wound is at least 5 mm long on at least one sideIf debridement is performed For RNA-Seq+16s rRNA sequencing: the material removed on debridement. For microbial culturing: the whole wound area is sampled using an ESwab.
Amicrobial culturing: Two consecutive nylon filters (‘Imprints’) placed in the same area of the wound for 30 s each and transferred to culture plates (laboratory testing has been performed in a pilot study on the transfer and replication of microbial 2D patterns using different types of filters. The best-performing filter was subsequently evaluated using a pig skin model). The area is secured using a silicone mould. The silicone mould is made by cutting out the centre of the silicone in the same size as the filters. A photo is obtained after the application of the silicone mould to the woun. One ESwab performed as a tight swabbing motion, covering the whole wound surface. One ESwab from a ~10×5 cm area of the patient’s thigh marked for grafting. This ESwab will be conducted from dry skin, and the tip of the ESwab will be moistened using sterile saline prior to swabbing. For RNA-Seq+16s rRNA sequencing: Tape strips 2×16 consecutive disc tapes from two different sites; 2–3 cm above and 2–3 cm below the wound. All tape strips for the in-depth cohort will be obtained as follows: tape strips are applied onto the targeted skin area, and a sterile marker will be used to mark the edges of the first disc to ensure consecutive sampling from the same area. A D-Squame pressure instrument will be used to apply uniform pressure for 10 s. Subsequently, the tape strips will be transferred to sterile tubes using sterile gloves and tweezers to maintain aseptic conditions throughout the process. In cases of limb amputation, the tape strips which are supposed to be collected below the wound will be collected on the adjacent side of the lower leg. Biopsies 6 mm punch biopsy from the edge of the wound, if possible, with a brim of epithelium. 6 mm punch biopsy from the centre of the wound (ie, wound on all sides of the punch biopsy instrument). A photo is obtained to visualise where the biopsies are taken. Each biopsy will be divided with maintained organisation using a sterile surgical scalpel. Half of each biopsy will undergo RNA-Seq+16s rRNA sequencing, and the other half will be analysed for proteomics. For proteomics:For microscopy: 4–6 mm punch biopsy from the edge of the wound, if possible, with a brim of epithelium. 4–6 mm punch biopsy from the centre of the wound (ie, wound on all sites of the punch biopsy instrument).
For RNA-Seq+16s rRNA sequencing: Debrided material. A piece of the skin graft, approximately 0.5 cm 2 obtained after meshing. For proteomics: Debrided material. A piece of the skin graft, approximately 0.5 cm 2 obtained after meshing. For microbial culturing: Two consecutive nylon filters placed in the same area of the wound bed for 30 s each and transferred to culture plates. The area is secured using a silicone mould. Three ESwabs:One ESwab rotated once around the edge of the wound. One ESwab from the whole wound surface.
Sampling at follow-up visits will depend on the status of the wound progression.
A photo of the wound. The photo is obtained after the application of a sticker: 16 consecutive tape strips from 2 to 3 cm above the wound/healed transplant.
For microbial culturing: The skin graft/healed skin is sampled using an ESwab. For RNA-Seq: 16 consecutive tape strips from the healed area.
For microbial culturing: The whole wound area is sampled using an ESwab.
. The area is secured using a silicone mould. One ESwab from the same area as the filters. The area is secured using a silicone mould as described above.
Samples for cultivation, identification of isolates, storing and Whole Genome Sequencing (WGS) Samples for microscopy Samples for RNA-Seq+16s rRNA sequencing RNA extraction Samples for proteomics Debrided material, biopsies and skin graft: the samples are immediately frozen using liquid nitrogen and later transferred to storage at −80°C until analysis for proteomics. The samples will be shipped to a company for analysis on Olink Target 96 Inflammation panel (Olink Proteomics). The panel is a high-throughput proteomics assay designed to measure 92 inflammation-related protein biomarkers in a single sample. By profiling a broad range of inflammation-related proteins, it is possible to gain insights into the underlying biological pathways involved in the inflammation processes and identify key proteins. A simplified, schematic illustration for sampling and follow-up for both cohorts is presented in .
Cultivation of filters: each filter is placed on culture plates for 3 hours and hereafter removed. From each patient, at each time point, one filter will be cultivated using a chocolate plate (SSI, Denmark) and an anaerobic plate (SSI, Denmark). It is decided to focus on blood agar plates and chocolate plates and not to include blue agar plates (selective for gram-negative species). After several studies we have experienced that blue agar plates does not give additional information when the two other plates are included. The plates will be incubated for 48 hours in a humidified 5% CO 2 incubator at 37°C and 10 days in an anaerobic bench (anaerobic plates). Plates are used for qualitative culture. Cultivation of ESwabs: the ESwab is placed at 5°C and cultured within 36 hours using routine methods. The first streak is made using the ESwab as inoculator. Identification of isolates: individual colonies are isolated based on morphology and identified using Matrix-Assisted Laser Desorption Ionization Time-Of-Flight (MALDI-TOF) (Bruker, Billerica, Massachusetts, USA). If isolates score below 2.0, they will be analysed again with a maximum of 3 attempts per isolate. Isolates are stored at –80°C. WGS: depending on the results from the MALDI-TOF identification, WGS analysis will be conducted on a subset of the isolates.
Sample preparation: the biopsies will be placed immediately in formalin and stored at 4°C for at least 24 hours prior to paraffin embedding following standard operating procedures. All biopsies will be divided with organisation maintained by sectioning each sample into three levels. From each section, six cuts are done (4 um) and placed on three glass slides. From each sample, one section is cut and used for H&E staining. Peptide nucleic acid fluorescence in situ hybridisation (PNA-FISH): specific PNA-FISH probes for 16s rRNA will be used to image the bacterial colonisation. Confocal laser scanning microscopy (CLSM): at least two sections from each set of biopsies will be screened using a CLSM with a 20× and a 63× objective.
Handling Debrided material, biopsies and the sample from the skin graft: samples will be placed in a tube containing RNAlater stabilisation solution for approximately 48 hours at 5°C before RNAlater stabilisation solution is removed, and the sample is frozen at −80°C until RNA and DNA extraction. Tape strips: the tapes are handled using sterile gloves and forceps and put into sterile tubes, two and two, immediately placed on dry ice before being transferred to −80°C until RNA extraction and sequencing.
Tissue samples: approximately 20 mg tissue is used for the extraction of RNA. RNA is extracted with Trizol+Chloroform, resuspended in nuclease-free water and stored at −80°C until further processing. Tape strips: RNA will be extracted from tapes with Trizol+Chloroform extraction. RNA from tapes will be pooled by rinsing all tapes with the same Trizol aliquot to increase the RNA concentration. Purified RNA will be stored in nuclease-free water and stored at −80°C. Sequencing: the concentration of isolated RNA is measured with an Agilent Bioanalyzer. For library preparation, the NEB Ultra II directional RNA library prep kit is used. Samples are sequenced on an Illumina NovaSeq6000 with a target depth of 100M reads per sample. DNA extraction for 16s rRNA: the DNA will be isolated from the samples used for RNA extraction during Trizol extraction procedure. The isolated DNA will be used for 16s rRNA analysis.
Wound area assessment for both the predictive and the in-depth cohort is performed manually using Image-J software (Fiji V.2.3.1) and calibrated using the sticky marker (Dansk Telemedicin A/S). The wound outline is traced using the freehand selection tool, and the area is calculated. If wounds are concomitant, the areas are combined. A record of the traced area, along with the calculated wound area, is then reviewed by a senior orthopaedic surgeon. Based on the tracked area, further grouping into healed versus unhealed wounds with percentage evaluation can be performed. In select cases, it will be possible to use clinical evaluation of healing, in which case it will be noted including the basis for evaluation.
Collection of clinical data Patient-Reported Information Extraction from the Electronic Medical Chart (Sundhedsplatformen, EPIC) and Online Database for Medication Prescription (Fælles Medicinkort/FMK Online) Manual Extraction from the Electronic Medical Chart (Sundhedsplatformen, EPIC) Information on the specific wound care treatments and interventions provided during the study period will be manually extracted
Clinical data for this study will be collected from three primary sources: Patient-Reported Information,.
At the time of inclusion, patients are asked to provide an estimate of the duration of their wound, categorised into years, months or weeks.
Systematic extraction, facilitated by a Python script, will be used to gather the following data: Age. Gender. Smoking Status. Alcohol Consumption. Body mass index. Assessment of distal perfusion: includes measurements such as Ankle-Brachial Index and Toe-Brachial Index. Diagnosis list: based on International Classification of Diseases 10th Revision (ICD-10) codes, including specific categories for diabetes, pressure ulcers, venous/varicose ulcers, diabetic ulcers and ischaemic ulcers. Surgical history: documented using the Nordic Medico-Statistical Commitee (NOMESCO) Classification of Surgical Procedures. Medicine prescription. Death within 1 year following inclusion.
The statistical analyses to be carried out in this exploratory research study involves both supervised and unsupervised approaches for the two patient groups: the predictive and the in-depth cohort. For the predictive cohort, the supervised analyses of bacterial community composition obtained from the 16S rRNA amplicon sequencing will involve permutational multivariate analysis of variance (ANOVA) analysis of beta diversity to assess differences between the healed and non-healed groups. Additionally, alpha diversity will be measured to determine the microbial diversity within each sample, and its association with wound-healing outcome will be tested using an ANOVA test. In the unsupervised analysis, principal component analysis will be employed on the centred log-ratio transformed relative abundances to reveal underlying groupings in the bacterial community compositions. Furthermore, a Dirichlet multinomial mixture model will be used for community typing, and the community types’ association with healing outcome will be tested using Fisher’s exact test. The same analyses will be performed on the in-depth cohort; however, comparisons will be made among the different wound locations, the wound and the transplanted skin and the wound at different time points following STSG. Raw dual RNA-Seq data will be processed into host transcriptomes (ie, a feature count matrix) and active bacterial community data in both patient groups. For the predictive cohort, supervised analysis of the host transcriptomes will involve differential gene expression (DGE) analysis of the healing outcome groups followed by functional analysis such as gene ontology or gene set enrichment analysis. On the other hand, the unsupervised analysis will begin with dimensionality reduction techniques, such as Principal Component Analysis (PCA), to characterise the heterogeneity of chronic wound transcriptomes, followed by data-driven clustering and functional analysis. Similarly, for the in-depth cohort, the focus will be on DGE testing between different wound locations, the wound and the skin graft and the wound following up on the STSG. Again, unsupervised analyses will involve dimensionality reduction techniques to uncover subgroups or patterns within the data. The correlation between the active bacterial community compositions and the present taxa elucidated from 16S amplicon sequencing will be explored in both cohorts. In addition, the correlation between protein abundances and the corresponding gene expression levels will also be calculated for the in-depth cohort for samples where both proteomics and transcriptomics data are available. In order to integrate omics data layers with clinical information, a supervised analysis will involve using Lasso logistic regression to select important predictors of healing outcomes. On the unsupervised side, Multi-Omics Factor Analysis (MOFA) will be employed to identify shared sources of variation across all data layers : the host’s transcriptome and proteome, the bacterial community compositions (active and overall present taxa), and clinical data. Finally, the association of MOFA factors with healing outcomes may also be tested, to investigate the relationship between the data types and healing outcomes.
study has been approved by the Board of Health Ethics, Capital Region of Denmark, under protocol number H-20032214. No expected harmful effects are anticipated from the predictive cohort . The in-depth cohort will be subjected to invasive sampling in the form of punch biopsies, which will be conducted by the responsible surgeon in relation to surgical debridement followed by STSG. The safety of the biopsy procedure will be assessed, and if the location and/or size of the wound is deemed to make the biopsies unsafe, the patient will not be included in the study. The study findings will be disseminated through publications intended for submission to journals undergoing peer review. Additionally, the outcomes will be presented at national as well as international conferences in microbiology, wound healing and infection.
The CWC study received ethical approval on 28 October 2020. The predictive cohort began inclusion and sampling in February 2021. The in-depth cohort initiated inclusion in August 2021, with the first patient included and sampled in October 2021. As of January 2024, inclusion and sampling in the CWC study are still ongoing.
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Peripheral K | 94579f8d-3720-4832-8f03-6ed9d1745b28 | 5456027 | Physiology[mh] | Chronic pain associated with visceral hypersensitivity to physiological stimuli is a hallmark of functional gastrointestinal (GI) disorders including irritable bowel syndrome (IBS). Persistent pain and decreased thresholds to bowel distension are a function of altered neuronal excitability of both sensitized peripheral sensory input and maladaptive central pain processing. The K V 7 family (K V 7.1–K V 7.5) of voltage-gated potassium channels encoded by KCNQ1–5 are responsible for mediating the M current and can regulate excitability in a variety of central and peripheral neurons involved in pain pathways. – At thresholds below action potential initiation and near resting membrane potential, K V 7 channels have a significant influence on the resting membrane potential and contribute to a membrane-potential clamping effect that stabilizes neuronal excitability and acts to restrict repetitive firing. In acute inflammatory pain, G q/11 -coupled receptor-mediated attenuation of the M current can evoke depolarization and increased excitability of nociceptors. Thus, genetic or pharmacological inhibition of K V 7 leads to increased action potential firing to excitatory stimuli, whereas K V 7 openers, including the anticonvulsant drug retigabine, produce membrane hyperpolarization and reduced excitability. , , Retigabine is selective for K V 7.2–7.5 over the cardiac K V 7.1 subunit and is clinically efficacious in reducing partial seizures in epilepsies; however, significant centrally mediated adverse effects have restricted its more widespread utility both as an anticonvulsant and putative analgesic. In the periphery, enhanced K V 7 channel activity has been identified as the main site of action for retigabine in the attenuation of somatic inflammatory pain as well as in bone cancer models where retigabine inhibits mechanical allodynia and thermal hyperalgesia. , In addition to retigabine, tannic acid can also enhance the M current by acting on K V 7.2/K V 7.3 channels in small diameter neurons. Conversely, K V 7 channel inhibitors act to sensitize peripheral neurons such as XE-991, which sensitizes C-fibres to noxious heat stimulation and induces chronic spontaneous activity of Aδ fibres, and camphor or menthol, which act on K V 7.2/K V 7.3 expressed on cutaneous nerve endings. , Of particular relevance to this study is the action of inhibitory K V 7 mediators under inflammatory conditions, with which visceral pain is often associated. For example, protease activated receptor 2 (PAR-2) has been implicated in mediating colonic visceral hypersensitivity and in IBS, which is characterized by abdominal pain. , In rat nociceptive sensory neurons, PAR-2 inhibits the M current via phosphoinositide phospholipase (PLC) activation. Similarly, inhibition of Mas-related gene receptor D (MrgprD; which is known to be up-regulated during intestinal inflammation ), by its ligand ß-alanine also inhibits K V 7.2/K V 7.3 activity. As such, a peripherally restricted K V 7 opener may represent a useful analgesic therapy for treating visceral pain associated with inflammation. Indeed when injected into the peritoneal cavity, retigabine reduces visceral pain behaviours in mice following intracolonic application of capsaicin; however, the exact site of action is still to be resolved. Moreover, following neuropathic injury, increased expression of repressor element 1-silencing transcription factor in dorsal root ganglia (DRG) down-regulates expression of KCNQ2 leading to neuropathic hyperalgesia. Selective ablation of KCNQ2 from the mouse forebrain results in visceral hyperalgesia suggesting that there may also be a contribution of CNS K V 7 channels to visceral pain. Here, we sought to identify and confirm the role of peripheral K V 7 channels in visceral nociception by studying nerve fibres innervating the GI tract of both mouse and human. K V 7 channel subtype expression is tissue specific, with K V 7.1 highly expressed by cardiac myocytes as well as in epithelial and smooth muscle tissue, while K V 7.2 to K V 7.5 are found in the nervous system. In spinal sensory pathways, rodent small diameter transient receptor potential vanilloid subfamily type 1-positive nociceptors express K V 7.2, K V 7.3 and K V 7.5, whilst retigabine can reduce the excitability of unmyelinated peripheral human axons. In the GI tract, K V 7.2, K V 7.3 and K V 7.5 have all been identified in the smooth muscle of mouse colon suggesting a role in regulating gut motility. Whether such motor events are additionally mediated by K V 7 expression within the enteric nervous system itself remains to be determined. With regard to the extrinsic innervation (vagal and spinal pathways) of the GI tract, K V 7 channels regulate excitability in the predominantly non-nociceptive population of vagal sensory neurons that innervates the upper gut. However, the contribution of K V 7 channels to the excitability of nociceptive sensory neurons innervating the colon is unknown. Therefore, the aims of this study were to (1) determine the expression of K V 7.3 and K V 7.5 subtypes in mouse and human lower GI tract, (2) determine the expression of K V 7 channel subtypes in DRG sensory neurons innervating the lower GI tract of mouse, (3) investigate the ability of K V 7 channel opener retigabine to inhibit visceral afferent excitability to acute inflammatory and noxious stimuli (including bradykinin and mechanical distension) and (4) validate the ability of retigabine to attenuate human visceral afferent excitability using a novel human ex vivo model of visceral nociception.
Mouse and human tissues Immunohistochemistry KCNQ subunit expression by single-cell qualitative real-time polymerase chain reaction in colonic sensory neurons Isolation of mouse and human colonic tissues Extracellular electrophysiological recordings of mouse and human afferent fibre activity Electrophysiological protocols Drugs Data analysis In multi-unit experiments, peak changes of electrophysiological nerve activity were determined by subtracting baseline firing (3 min before drug application) from increases in nerve activity following nucleotide superfusion. The visualization of single-cell qRT-PCR expression data was created using R and the ggplot2 package. Statistical significance was set at P < 0.05, and data are displayed as mean ± SEM.
Experiments were performed using male C57BL/6 mice (10–12 weeks of age) and in accordance with the UK Animal (Scientific Procedures) Act 1986. The collection and use of human tissues was performed with approval of the East London and The City HA Local Research Ethics Committee (NREC 09/H0704/2). Fresh, non-inflamed ileum ( N = 4) and colon ( N = 6 for immunohistochemistry and N = 6 for electrophysiology) were obtained under full written consent from 4 to 6 patients (four male, median age: 58, range: 33–67 years) undergoing surgery at Barts and The London NHS Trust and Whipps Cross University Hospitals (London, UK). All specimens were obtained from patients with non-obstructive tumours that were not occuring in the context of inflammatory bowel disease. Specimens were taken with the permission of the histopathologist following macroscopic examination and were a minimum of 10 cm away from tumour, resection margins or lymphatic drainage field.
For sectioning studies, fresh mouse colon, as well as human ileum and colon, were post-fixed overnight (at 4℃) in 4% paraformaldehyde. Sections (10 µm) of cryoprotected tissue (30% sucrose w/v in 0.1 M phosphate-buffered saline (PBS)) were cut, incubated with blocking buffer (Dako, UK) for 1 h, before primary antibodies were applied overnight (4℃): K V 7.3 (1:500, AP1-930, Thermo Scientific, UK), K V 7.5 (1:500, PA1-941, Thermo Scientific, UK), CGRP (1:400, ab36001, Abcam, UK) and synaptophysin (1:200, MO776, Dako, UK). Tissues were washed (PBS; 3 × 5 min) and species-specific secondary antibodies conjugated to Alexa Fluor fluorescent dyes (1:400, Thermo Fisher Scientific, UK) applied for 1 h, before washing (PBS; 3 × 5 min), mounting (Vectashield® hard set mounting media, Vector Laboratories, USA) and coverslipping. For whole-mount studies, fresh mouse colon was cut open along the mesenteric border, pinned flat and fixed overnight (4℃) in 4% paraformaldehyde. Following fixation, mucosa was removed, washed (PBS; 3 × 10 min), blocked (blocking buffer; 1 h) and incubated with primary antibodies (K V 7.3, K V 7.5 and CGRP; all at 1:300) for 48 h at 4℃. Tissues were washed (PBS; 2 × 10 min), and species-specific secondary antibodies conjugated to Alexa Fluor fluorescent dyes (1:200, Thermo Fisher Scientific, UK) applied for 2 h. After a final wash (PBS; 3 × 10 min), tissues were mounted (50% glycerol in PBS) and coverslipped. Controls with no primary antibody were used in all experiments to check for non-specific secondary antibody binding. A Leica DM4000 epifluorescence microscope was used to visualize immunoreactivity of sections while whole mounts were examined with a Zeiss LSM710 confocal microscope. CGRP is commonly used as a marker of spinal sensory afferents , and is a nociceptive marker in rodents. At present, there is no biochemical marker that can distinguish between intrinsic and extrinsic neurons in the GI tract. Intrinsic CGRP expression has been shown in some species where colchicine has been used to sequester CGRP, while others have demonstrated extrinsic CGRP and used it as a marker of extrinsic fibres showing co-labelling with TRPA1 34 and TRPV4.
Sensory neurons innervating the distal colon of mouse were labelled with Fast Blue, individually harvested and collected for single-cell qualitative real-time polymerase chain reaction (qRT-PCR) of KCNQ subunits as previously described in literature. In brief, 6 to 8 injections (0.2 µl each) of retrograde neuronal tracer Fast Blue (2% in saline; polysciences GmbH) were injected into the wall of the distal colon under anesthetic (isoflurane; 4% induction and 1.5% maintenance) via laparotomy. Three to five days after recovery, thoracolumbar (TL; T10-L1) and lumbosacral (LS; L6-S1) DRG were removed and enzymatically dissociated as described previously in literature. Briefly, dissected ganglia were washed in PBS and incubated in Lebovitz L-15 Glutamax (Invitrogen) media containing 6 mg/ml bovine serum albumin (Sigma-Aldrich) and 1 mg/ml collagenase type 1 A (Sigma-Aldrich) for 15 min at 37℃ (in 5% CO 2 ). Ganglia were subsequently incubated in L-15 media containing 1 mg/ml trypsin (Sigma-Aldrich) and 6 mg/ml bovine serum albumin for 30 min. After gentle trituration using fire-polished pipettes, dissociated cells were pelleted by centrifugation (350X g) and resuspended in L-15 Glutamax media containing 24 mM NaHCO 3 , 38 mM Glucose, 2% penicillin/streptomycin and 10% fetal bovine serum. Cells were plated onto poly-D-lysine/laminin-coated coverslips (BD Biosciences, UK) and incubated at 37℃ in 5% CO 2 . Individual cells were collected by pulled glass pipette within 24 h of plating. Glass tips containing single cells or no-template bath solution controls were collected in tubes containing 9 µl of preamplification mastermix (5 µl CellDirect 2x reaction buffer (Invitrogen, UK), 2.5 µl 0.2X primer/probe mix, 0.1 µl SUPERase-in (Ambion, USA) and 0.2 µl Superscript III Reverse Transcriptase/Platinum Taq mix (Invitrogen, UK)) in TE buffer (Applichem, Germany) and subjected to thermal cycling (50℃ for 30 min, 95℃ for 2 min, then 21 cycles of (95℃ for 15 s, 60℃ for 4 min); Bio-Rad C1000 Thermal Cycler) to produce cDNA products. Following dilution with 91 µl TE buffer, TaqMan quantitative polymerase chain assays were performed on the cDNA products for each gene of interest (TaqMan Assay ID: Bdkrb2 , Mm00437788_s1; KCNQ1 , Mm00434640_m1; KCNQ2 , Mm00440080_m1; KCNQ3 , Mm00548884_m1; KCNQ4 , Mm01185500_m1 and KCNQ5 , Mm01226041_m1; glyceraldehyde-3-phosphate dehydrogenase, Mm99999915_g1; Applied Biosystems, USA) using the following thermal cycle (50℃ for 2 min, 95℃ for 10 min, then 40 cycles of (95℃ for 15 s, 60℃ for 1 min); Bio-Rad C1000 Thermal Cycler and CFX96 Real-Time System). All single-cell reverse transcription polymerase chain reaction products expressed glyceraldehyde-3-phosphate dehydrogenase, the expression of which acted as an internal positive control, whilst bath control samples did not. The determination of gene expression was evaluated by quantification cycle (Cq) values less than the threshold of 35 being considered as positive. Overall, the expression of genes of interest was examined in 45 TL colonic sensory neurons ( N = 3) and 45 LS colonic sensory neurons ( N = 3).
Mouse and human GI tissues were isolated, and electrophysiological activity of nerves innervating these tissues recorded using previously described methods. , Human tissues removed during surgical resection, and in excess to histopathological assessment, were immediately placed in ice-cold carbogenated Krebs buffer (in mM: 124 NaCl, 4.8 KCl, 1.3 NaH 2 PO 4 , 2.5 CaCl 2 , 1.2 MgSO 4 .7H 2 O, 11.1 glucose and 25 NaHCO 3 ) supplemented with nifedipine (10 µM) and atropine (10 µM) to block smooth muscle activity. In a bespoke recording chamber, the colonic wall was pinned mucosa-side down, the mesenteric blood vessels supplying the gut wall were identified and the surrounding mesentery dissected away. Mesenteric nerves running in close proximity to blood vessels were identified, dissected and isolated, and the tissue superfused with Krebs buffer (7–8 ml/min; 32℃–34℃). Mice were humanely killed by cervical dislocation of the neck, the distal colon gross dissected and removed along with all associated neurovasculature. In a recording chamber, the colon was superfused (7–8 ml/min; carbogenated Krebs buffer) and cannulated enabling luminal perfusion (0.1 ml/min; carbogenated Krebs buffer). In murine experiments, the Krebs buffer was additionally supplemented with 3 µM indomethacin to reduce endogenous prostanoid production. The lumbar splanchnic nerve was identified and isolated in preparation for electrophysiological recordings.
In both mesenteric nerve recordings from human tissues and lumbar splanchnic nerve recordings from the distal colon of mouse, isolated nerves were drawn into a borosilicate glass suction electrode (Harvard Apparatus, UK) prefilled with Krebs buffer. Nerve activity was recorded on a Neurolog headstage (Digitimer Ltd, UK), amplified at a gain of 5000X, band pass filtered (100–2000 Hz) and data acquired (20 kHz sampling rate; micro1401, Cambridge Electronic Design, UK) to a desktop computer running Spike2 (Cambridge Electronic Design, UK) software. Action potentials were counted using an online spike processor (Digitimer Ltd, UK), and the threshold level for spike processing was set to the smallest identifiable spike (typically∼100 µV).
For all electrophysiological experiments, a minimum of 15 min of baseline spontaneous activity was recorded prior to conducting any experimental protocol. In mouse distal colon recordings, four bradykinin (1 µM) bath superfusions (20 ml) were applied at ∼30 min intervals. Prior to, and in the presence of the third consecutive bradykinin application, retigabine (1 or 10 µM) or vehicle control (0.1% dimethyl sulfoxide (DMSO)) was applied by bath superfusion (20 ml volume). In separate experiments, phasic distension of the colon was performed by blocking the luminal out-flow and applying an elevated pressure head enabling rapid increases in intraluminal pressure (0–80 mmHg). Pain behaviours in vivo are evoked by such noxious pressures and have been previously used to investigate colonic nociceptor function. Specifically, repeat rapid phasic distensions (0–80 mmHg, 60 s at 9 min intervals) were applied until the response stabilized (typically after 5–6 distensions), at this point increasing concentrations of retigabine were superfused prior to, and during, every third phasic distension sequentially. In one set of experiments, responses to 0.1 µM, 1 µM and 10 µM retigabine were determined, and in a second set of experiments, responses to 30 µM and 100 µM retigabine were investigated. In separate phasic distension experiments, the K V 7 blocker XE991 (10 µM) was superfused prior to, and during, the seventh phasic distension. In human tissues, the ability of bradykinin to repeatedly activate human colonic afferents was investigated. Bradykinin (1 µM) was applied, at an interval of 45 min, in a ‘step-up’ manner where bath superfusion of 10 ml of 1 µM bradykinin was supplemented with a direct application of 100 µM bradykinin (0.9 ml) to the recording chamber. In total, four sequential applications of bradykinin were made in parity with murine recording protocols. We next investigated the effect of retigabine on bradykinin-stimulated human colonic afferents. During the third repeat bradykinin application, retigabine (100 µM) was co-applied to the bath. Following the third bradykinin stimulation, 45 min of washout time was allowed before the fourth and final bradykinin stimulation.
Stock concentrations of atropine (10 mM; ethanol), bradykinin (10 mM; H 2 0), indomethacin (3 mM; DMSO), retigabine (100 mM; DMSO), XE991 (100 mM; DMSO) and nifedipine (10 mM; DMSO) were all purchased from Sigma Aldrich (UK) and prepared as described. All compounds were diluted to working concentrations in buffer on the day of experimentation.
K Expression of KCNQ transcripts in extrinsic sensory neurons innervating the mouse colon Co-expression of KCNQ channel subtypes with bradykinin receptor subtype B Retigabine inhibits bradykinin-induced afferent activation in mouse colon Retigabine inhibits visceral afferent sensitivity to noxious distension K Retigabine inhibits bradykinin-induced afferent activation in human colon V 7 subtypes are expressed in neurons and mesenteric fibres of mouse bowel tissues GI function is regulated by a vast network of enteric neurons organized into two plexuses: the myenteric plexus located between the longitudinal and circular muscle layers, and the submucosal plexus located between the mucosa and the circular muscle. Afferent fibres of extrinsic spinal sensory neurons are also present in these tissues with projections to multiple layers of the bowel wall. The functional K V 7 heteromultimers known to underpin neuronal M currents are combinations of K V 7.2/K V 7.3 and K V 7.3/K V 7.5. – Thus, we first examined the expression of K V 7.3 and K V 7.5 in mouse colonic tissues. We hypothesized that K V 7 channel subtypes were expressed by enteric neurons, and in extrinsic sensory fibres contributing to nociception. In order to investigate this, we performed whole-mount immunohistochemistry against multiple K V 7 channel subtypes and the sensory neuronal marker CGRP, in separate layers of the mouse distal colon. Immunostaining revealed both K V 7.3 and K V 7.5 expression in the enteric neurons of the myenteric plexus, which co-localized with neuronal varicosities as identified by CGRP ( and ( )). In the mesentery associated with the colon, extensive punctate expression of both K V 7.3 and K V 7.5 was observed along mesenteric blood vessels, which also co-expressed CGRP ( and ( )). For both K V 7 subtypes, examples of K V 7-negative, CGRP-positive nerve fibres were also present ( ), suggesting heterogeneity of K V 7 expression. Taken together, these observations identify K V 7.3 and K V 7.5 in both enteric neurons and extrinsic sensory fibres of the mouse distal colon. The expression of K V 7 by extrinsic mesenteric fibres, which are important nociceptors in the gut, suggests that K V 7 may be involved in visceral pain pathways.
To confirm the expression of K V 7 channel subtypes observed by immunohistochemistry in extrinsic sensory fibres innervating the distal colon, we performed single-cell qRT-PCR for KCNQ1–5 transcripts in DRG neurons retrogradely labelled from the mouse distal colon. We examined individual Fast Blue-positive sensory neurons harvested following primary culture of TL (T10-L1) and LS (L6-S1) DRG, corresponding to the splanchnic and pelvic neurons specifically innervating the distal colon, respectively. In total, 15 single cells were collected per spinal region (e.g., TL or LS) per mouse ( N = 3). In colonic sensory neurons isolated from the TL region (representative of the splanchnic innervation), analysis of qRT-PCR products revealed greater expression of KCNQ5 transcripts (86.7 ± 3.8%) compared to KCNQ2 (57.8 ± 2.2%), KCNQ3 (42.2 ± 2.2%) and KCNQ4 (28.9 ± 5.9%; ). KCNQ1 transcripts were observed infrequently (6.7 ± 0.0%) and were only present in one cell from each of the three animals labelled. By contrast, colonic pelvic sensory neurons isolated from LS DRG exhibited significantly lower expression of KCNQ4 transcripts (8.9 ± 2.2%; P < 0.05, unpaired t -test) compared to TL sensory neurons, whilst levels of KCNQ2 (48.9 ± 9.7%), KCNQ3 transcripts (40.0 ± 3.8%) and KCNQ5 (68.9 ± 5.9%; ) did not significantly differ. No LS colonic sensory neuron expressed KCNQ1 transcripts ( and ( )). Although KCNQ5 transcripts were present in the vast majority of TL and LS neurons investigated, the co-expression of KCNQ2 and KCNQ3 transcripts (as a proportion of all cells investigated) was greatest in TL (TL, 33.3 ± 3.8% vs. LS, 24.4 ± 2.2%). From this data, it is clear that KCNQ2 , KCNQ3 and KCNQ5 are expressed in a large proportion of colonic sensory neurons; however, it is difficult to interpret whether differences in TL versus LS expression profiles are functionally meaningful. Overall, however, the high expression of KCNQ5 in extrinsic sensory neurons agrees with protein expression data described in . Similarly, moderate expression of KCNQ3 follows the pattern of protein expressed observed in mouse colonic mesentery ( ).
2 in mouse colonic sensory neurons The algogenic peptide bradykinin is an important endogenous inflammatory mediator that can activate pain pathways by binding two G-protein coupled receptor subtypes: B 1 and B 2 . The acute effects of bradykinin are predominantly mediated by the constitutively expressed bradykinin B 2 receptors , which are capable of altering sensory neuronal excitability by multiple downstream intracellular pathways, including phosphorylation of TRP channels, , increased Ca 2+ -activated Cl − conductance and suppression of Ca 2+ -dependent K + , and M currents. In order to test the coupling of K V 7 channels to bradykinin-evoked excitation in visceral sensory neurons, we first examined the co-expression of bradykinin receptor B 2 ( Bdkrb2 ) with KCNQ1–5 transcripts in single-cell qRT-PCR products. In TL colonic sensory neurons, Bdkrb2 mRNA transcripts were observed in 64.4 ± 5.9% of cells, and of these Bdkrb2 -positive neurons, 72.9 ± 4.8% also expressed KCNQ2 , 41.0 ± 2.3% expressed KCNQ3 transcripts and 100.0 ±0.0% expressed KCNQ5 transcripts ( ). Similar proportions of LS colonic sensory neurons expressed Bdkrb2 transcripts (64.4 ± 12.4%); however, co-expression with KCNQ2 (54.3 ± 7.2%), KCNQ3 (37.4 ± 10.4%) and KCNQ5 (85.6 ± 7.5%) transcripts were partially reduced compared to the TL populations ( ). Of both the TL and LS colonic sensory neurons that did express Bdkrb2 transcripts, 24% to 31% expressed transcripts for only one KCNQ subtype (exclusively KCNQ5 ), the rest of Bdkrb2 -positive colonic sensory neurons co-expressed multiple KCNQ channel subtypes, principally KCNQ3 and KCNQ5 .
We next investigated whether the opening of K V 7 channels was sufficient to alter acute chemosensitivity to bradykinin in peripheral terminals of sensory neurons innervating the distal colon. Bradykinin was the chosen chemical stimulus as activation of the B 2 receptor on sensory neurons is known to induce store release of Ca 2+ that then binds to K V 7-associated calmodulin resulting in closure of K V 7 channels and increased neuronal excitability. Specifically, multi-unit ex vivo extracellular electrophysiological recordings of the lumbar splanchnic nerve were made after cannulation of the mouse distal colon enabling stimulation by repeat bath superfusion of bradykinin. Application of bradykinin (1 µM) led to an increase in afferent firing to 22.3 ± 2.8 spikes/s above baseline discharge levels with a duration of approximately 20 min ( N = 5). Repeat bradykinin applications were made at ∼30 min intervals. A significant desensitization in response to the second bradykinin (1 µM) response was observed (5.4 ± 1.21 spikes/s). Importantly, subsequent responses to the third (5.1 ± 0.9 spikes/s) and fourth (6.2 ± 1.9 spikes/s) bradykinin applications remained consistent in vehicle experiments ( P = 0.54, N = 5, repeated measures (RM) analysis of variance (ANOVA); ) enabling investigation of the effect of the K V 7 channel opener retigabine on peripheral visceral afferent responses to bradykinin after the second application of bradykinin. Pre-incubation, and co-application, of 10 µM retigabine with 1 µM bradykinin abolished virtually all afferent firing (0.0 ±1.5 spikes/s; P < 0.05, N = 5, two-way RM ANOVA with Dunnett’s post hoc). This effect was completely reversible following washout with the response to subsequent superfusion of 1 µM bradykinin comparable to pre-retigabine responses (5.4 ± 0.7 spikes/s; ). In the example trace shown in , 10 µM retigabine was additionally superfused during the washout to the final bradykinin application, again completely inhibiting the elevated afferent firing. In separate experiments, the application of 1 µM retigabine also had an inhibitory effect on afferent firing to bradykinin; however, this was smaller and did not significantly differ from vehicle controls (4.0 ± 0.6 spikes/s; P > 0.05, N = 5, two-way RM ANOVA with Dunnett’s post hoc). Collectively, these data confirm that opening of K V 7 reverses bradykinin-induced action potential firing in peripheral visceral afferent fibres and highlights the importance of K V 7 channels in mechanisms underlying visceral inflammatory pain.
Next we investigated the ability of K V 7 channels to alter visceral mechanosensitivity in peripheral afferent terminals of the mouse distal colon. We applied repeat phasic distensions (0–80 mmHg for 60 s at an interval of 9 min; a pressure shown to activate all known mouse mechanosensitive nociceptors ), to determine the effect of the K V 7 channel opener retigabine on afferent mechanoreceptors. Phasic distension led to an increase in splanchnic nerve afferent firing and rapid adaptation of the response during the 1-min distension ( ). Upon relief of the pressure, firing rates quickly returned to baseline levels. As with previous studies, tachyphylaxis of the response to repeat distension was observed over the subsequent distensions, with responses typically stabilizing by the fifth to sixth distension. Varying concentrations of retigabine (0.1–100 µM) were superfused before and during the seventh distension, leading to concentration-dependent inhibition of the peak firing rates to subsequent phasic distension ( N = 4–6, ). Although the response to the first distension directly after application of retigabine was inhibited, the maximal attenuation of response occurred by the third distension after application (e.g. ∼30 min post-retigabine; ). At the concentration of retigabine (10 µM) where complete block of the bradykinin-evoked excitation occurred; ), there was a 48.5 ± 3.2% inhibition of response to noxious distension. Application of the K V 7 channel blocker XE991 (10 µM) led to a moderate increase (29.1 ± 9.2%) in response to noxious distension, highlighting the presence of a tonic K V 7 current during non-inflamed states. In addition to regulating the response to phasic distension, there was a trend for decreased spontaneous discharge in the presence of retigabine (e.g. pre-retigabine, 3.89 ± 2.52 spikes/s vs. post-retigabine 10 µM, 0.87 ± 0.20 spikes/s, N = 6, P = 0.27, paired t -test), although this was not observable in all experiments due to very low spontaneous discharge in some preparations. By contrast, addition of XE991 (10 µM) tended to potentiate spontaneous discharge (pre-XE991, 3.70 ± 1.10 spikes/s vs. post-XE991 10 µM, 9.57 ± 2.51 spikes/s, N = 5, P = 0.05, paired t -test). These data suggest that K V 7 channels are present on visceral nociceptors responsive to mechanical stimuli and that opening of K V 7 channels is sufficient to make the afferent terminal less excitable by such stimuli.
V 7 channels are expressed in neurons of human bowel tissue In order to investigate the contribution of K V channels to human GI function, we performed immunohistochemistry on human bowel tissues (ileum and colon). In normal human ileum, K V 7.3 expression was found in the neuronal varicosities of the mucosal villi and showed significant co-expression with the neuronal marker synaptophysin (an integral membrane protein present in presynaptic vesicles of neurons; ). Synaptophysin-positive nerve varicosities also co-labelled with K V 7.3 in the intracellular spaces between villi ( ). In the submucosal plexus of human ileum, K V 7.3 immunostaining was punctate and observed around the cell bodies rather than within the neurons themselves. Structures that stained positively for K V 7.3 co-labelled with synaptophysin ( ) and CGRP ( ). In human colon, K V 7.5 expression was observed in the lamina propria between mucosal crypts. Structures that positively stained for K V 7.5 were identified as synaptophysin-positive fibres innervating the colonic mucosa ( ). Neuronal varicosities, identified by co-expression with synaptophysin, expressed K V 7.5 in nerve fibres surrounding the colonic myenteric plexus ( ). K V 7.5 also co-localized with CGRP in the myenteric plexus suggesting expression by sensory afferent pathways known to terminate at myenteric ganglia. Collectively, these data identify significant expression of K V 7 channels subtypes in both intrinsic and extrinsic neuronal populations of the human GI tract.
In order to further understand the role of K V 7 channels in human spontaneous inflammatory pain and to translate our murine findings into man, we utilized a model of human visceral afferent activity and a similar experimental paradigm as above. Using surgically resected bowel tissues, we recorded mesenteric nerve activity to repeat application of bradykinin. A concentration of 1 µM bradykinin was used as this has previously been shown robustly excite human visceral afferent fibres in ex vivo electrophysiological recordings. , Bath superfusion of 1 µM bradykinin and a bolus of 10 ml bradykinin applied directly to the nerve under investigation led to a robust increase in human mesenteric afferent firing averaging 20 min in duration ( ). Following a washout interval of 45 min, a subsequent repeat bradykinin concentration was applied, although the response was partly diminished, it did not significantly differ from the first (first bradykinin, 2.14 ± 0.30 spikes/s vs. second bradykinin, 1.40 ± 0.36 spikes/s, P = 0.13, N = 7, paired t -test, ). To investigate the role of K V 7 in human visceral afferent sensitivity to bradykinin, we applied 100 µM retigabine in the presence of the third bradykinin application. This led to a significant decrease of the afferent response, reducing both the duration of the overall response (991 ± 522 s vs. retigabine, 178 ± 102 s) and change in peak firing (pre-retigabine, 1.40 ± 0.36 spikes/s vs. retigabine, 0.36 ± 0.23 spikes/s, P < 0.05, N = 7, one-way ANOVA with Dunnett’s post hoc) compared to the response to bradykinin prior to retigabine being applied. Following the washout period of 45 min, bradykinin was again applied to the bath and a partial recovery in response to levels of afferent activation comparable to control applications was observed (retigabine vs. post-retigabine, 0.89 ± 0.17 spikes/s, P > 0.05, N = 7, one-way ANOVA with Dunnett’s post hoc, ). Collectively, these findings demonstrate a functional role for K V 7 channels in modulating the sensitivity of peripheral terminals of visceral sensory neurons innervating the human lower GI tract.
Here we demonstrate that K V 7 channel subtypes are expressed by gut-projecting sensory neurons and that modulation of these channels can greatly alter afferent sensitivity to mechanical and inflammatory stimuli. We have shown in mouse that opening K V 7 channels attenuates visceral afferent sensitivity to noxious distension of the colon and to the algogenic inflammatory mediator bradykinin; the latter an effect that translates to human visceral afferents. Conversely, blockade of K V 7 channels results in hypersensitivity to mechanical stimuli. As such, K V 7/M-channel activity represents an integral regulator of visceral afferent sensitivity downstream of multiple transduction mechanisms and is likely to contribute to dampening of peripheral pain pathways from visceral organs in humans. The molecular constituents of the neuronal M current are heteromultimeric combinations of K V 7 channel subtypes, principally K V 7.2, K V 7.3 and K V 7.5. As a slowly deactivating, non-inactivating sub-threshold current, the M current exerts a stabilizing influence on neuronal excitability, with small changes in currents capable of significant alterations in excitability. , In the central nervous system, mutations in KCNQ2 and KCNQ3 can manifest as familial neonatal epilepsies with unprovoked partial or generalized convulsions. , Peripherally, K V 7 channels are expressed by sensory neurons of the DRG and contribute to the regulation of nociceptor excitability in models of both inflammatory and neuropathic pain. , , , , Whilst its role in peripheral pain pathways is well established, the contribution of K V 7 in peripheral visceral nociception and the site of action of retigabine however remains largely unknown. Here we show in mouse GI tissues that K V 7 channel subunits (K V 7.3 and K V 7.5) are expressed in extrinsic sensory afferents (as identified by the presence of CGRP) associated with blood vessels in the colonic mesentery. Such vascular mesenteric afferents are important nociceptors known to transduce noxious stimuli including ischaemia, inflammation and traction of the mesentery. , We also confirm the presence of mRNA transcripts for those subunits contributing to the M current, including KCNQ2 , KCNQ3 and KCNQ5 , in the soma of individual sensory neurons innervating the distal colon via either the lumbar splanchnic or pelvic nerves. By using the potent algogen bradykinin in rodent ex vivo recordings of visceral afferent activity, we show that the K V 7/M-channel opener retigabine could abolish bradykinin-evoked responses thus supporting the KCNQ expression observed by single-cell qRT-PCR and immunohistochemistry in visceral nociceptors innervating the colon. We observed significant co-expression of mRNA transcripts for B 2 ( Brdrb2 ), the primary receptor responsible for bradykinin sensitivity in visceral afferents, , , with KCNQ /M current relevant subtypes: KCNQ2 , KCNQ3 and KCNQ5 . Bradykinin is algogenic in human blister base applications and evokes nocifensive behaviours in rodents such as licking and paw lifting. – B 2 receptor-mediated activation undergoes tachyphylaxis or desensitization , , via NO-cGMP pathway and receptor down regulation, – in agreement with our observations following sequential bradykinin application. Indeed, bradykinin acts to depolarize and sensitize sensory nociceptors via multiple downstream signalling cascades including PKA- and PKC-dependant phosphorylation of TRPA1, PKC-mediated phosphorylation of transient receptor potential vanilloid subfamily type 1, reduced Ca 2+ -dependent K + conductance , and also increased Ca 2+ activated Cl − conductance. The latter mechanism contributing to membrane depolarization in sensory neurons due to elevated Cl − concentrations driven by constitutive activity of Na-K-Cl co-transporter. , In addition, bradykinin inhibits M currents in sensory neurons via a PLC-IP 3 -Ca 2+ pathway. In the present study, the ability of retigabine to completely suppress bradykinin-evoked firing would suggest that K V 7 modulation is a critical mechanism by which visceral afferent sensitivity may be regulated; however, it is likely that multiple rheostatic mechanisms act in consort to enable neuronal depolarization. Indeed, opening of M channels, blockade of Ca 2+ activated Cl − channels and genetic ablation of the persistent sodium channel Na V 1.9 are all capable of attenuating nocifensive behaviours to intraplantar injection of bradykinin. , As such, it is highly likely that K V 7 channels represent one mechanism by which visceral afferents regulate firing and by which retigabine is able to reduce visceral pain behaviours in mice evoked by algogens capsaicin or acetic acid; mirroring recordings from the saphenous nerve where retigabine blocks responses to inflammatory mediators. Whilst bradykinin-mediated excitation is clearly closely coupled to M current inhibition, M current modulation is a downstream effector that is utilized by multiple extracellular signalling pathways to alter sensory neuronal sensitivity to stimuli. Receptors, such as purinergic P2Y 1,2 , histamine H 1 , bradykinin B 2 and substance P NK 1 are all capable of inducing PLC cascades, which are likely to lead to inhibition of M current in sensory neurons and that have well-characterized roles in potentiating visceral pain mechanisms. , Indeed, activation of PAR-2 by exogenous proteases and mrgprD by β-alanine both inhibit M current causing depolarization leading to nociception. , We also observe a strong modulatory effect of K V 7 on mechanosensitivity in visceral afferents with retigabine capable of inhibiting both spontaneous discharge and evoked firing during phasic distension of the distal colon in a concentration-dependent manner. In contrast to saphenous recordings, peripheral nerve damage was not required for retigabine to attenuate responses to mechanical stimuli. Application of XE991 led to increased spontaneous discharge and also increased (∼30%) firing to distension in naïve animals, suggesting the presence of a background, and therefore suppressant, K V 7 current in these preparations. Whilst slowly adapting (C and Aδ) mechanoreceptors innervating the paw have been shown to be resistant to K V 7 block by XE991, rapidly-adapting (Aβ) mechanoreceptors may alter their firing characteristics to release a volley of action potential in response to mechanical probe. Linopirdine, a generic K V 7 channel blocker potentiates excitability of rapidly adapting mechanoreceptors and D-hair (Aδ) receptors to mechanical probe; the former in a K V 7.4-dependent manner. This heterogeneity in K V 7 function in afferents innervating the paw seems to be less relevant to those innervating the viscera where fibres are typically less diverse in conduction velocity and fibre ending type. , K V 7 is likely to not only be able to modulate neuronal responses to chemical and mechanical stimuli but also to do so across the vast majority of afferents innervating the distal colon; a concept supported by the significant expression of K V 7 observed by single-cell quantitative polymerase chain. The presence of KCNQ4 mRNA transcripts in some TL versus LS neurons could imply a role in mechanosensitivity given its association with touch sensation in mice and humans, although further studies are required to confirm this. Multiple ion channels have been associated with mechanotransduction in visceral afferents, including TRP , and ASIC channels. , We show that M-channel openers are capable of inhibiting depolarizing mechanical stimuli in a process that is likely to be independent of secondary intracellular signalling cascades. In order to translate our findings to humans, we confirmed expression of K V 7.3 and K V 7.5 in GI tissues. Specifically, both were observed co-localizing with synaptic vesicle membrane protein synaptophysin and the neuropeptide CGRP. Synaptophysin is found within the submucosal and myenteric plexi, longitudinal and circular muscle and co-localizes with the pan-neuronal marker PGP9.5. Therefore, co-labelling of K V 7 with synaptophysin and CGRP in human ileum and colon indicates expression within neuronal populations either extrinsically or intrinsically innervating the GI tract. Interestingly, the presence of K V 7 in the mucosa and submucosal plexus of the ileum is also suggestive of a role in chemosensing and possibly in response to inflammatory stimuli. Whilst the GI smooth muscle of the colon expresses K V 7 channels, we observed minimal labelling in either the circular or longitudinal muscle layers. Nevertheless, we cannot totally exclude the possibility that the inhibitory effects of retigabine are at least partially driven by direct activity of K V 7 on smooth muscle. Using afferent recordings in human gut, , we confirm that peripherally expressed K V 7 channels can modulate human colonic afferent firing to noxious stimuli. In parity to mouse recordings discussed above, responses to repeat applications of bradykinin to the serosal surface of the colon were significantly attenuated in the presence of retigabine. These build on previous findings showing that retigabine, by increasing K V 7 channel conductance, can alter excitability in isolated human sural nerve by confirming that K V 7 opening can counteract disease-relevant stimuli applied to the native tissue with the endogenous nerve transduction architecture in place. In conclusion, we show that K V 7 is expressed, and that opening of K V 7 channels can inhibit chemical and mechanical responsiveness in mouse and human colonic afferents. We also confirm expression of KCNQ mRNA in gut-projecting DRG sensory neurons from both splanchnic and pelvic extrinsic pathways innervating the distal colon. As such K V 7 channels contribute to modulating the excitability of afferents innervating the distal colon and are likely to be a key mechanism in acute excitation to inflammatory mediators such as bradykinin or increased sensitivity to normally innocuous mechanical stimuli in pathologies where visceral hypersensitivity is a principal symptom, such as IBS and inflammatory bowel disease. Our data support the hypothesis that a peripherally restricted K V 7 channel opener capable of limiting centrally mediated side effects may represent a therapeutic opportunity for GI disease, especially where inflammation is an important component. As such, the data presented here offer promising evidence for the usefulness of K V 7 channels as novel GI targets.
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The impact of the consensus statement on abusive head trauma in infants and young children | 649153a9-2150-477d-ae22-744b7919b605 | 8126591 | Pediatrics[mh] | It is ironic that in the current coronavirus disease 2019 (COVID-19) environment — where at the time of writing this article there had been more than 9.9 million cases and 237,000 deaths from COVID-19 in the United States — the science, evidence and facts are still considered controversial . We have been working at “warp speed” to develop a vaccine for COVID-19 together with 165 organizations around the world to halt the pandemic, but the measles–mumps–rubella (MMR) vaccine–autism controversy still lingers and similar anti-vaccination groups are also agitating against the COVID-19 vaccine . A recent Gallup poll revealed that 1 in 3 Americans is reluctant to get vaccinated, even if a vaccine is approved by the U.S. Food and Drug Administration (FDA) and is available at no cost . We are well aware of the devastating effects of the MMR vaccine–autism controversy, first espoused by Wakefield et al. in a Lancet article. The article was subsequently retracted and the science thoroughly debunked . As a direct fallout, Dr. Wakefield lost his medical license to practice, for serious professional misconduct . The expectation was that the bad science would be laid to rest. However, the controversy lives on, further enabled by powerful social media influencers and fringe proponents of this MMR vaccine–autism myth in the medical science world. These have included films such as Vaxxed and news articles in reputed journals and mainstream media platforms . It has found mention on The Oprah Winfrey Show and was even discussed during a 2016 presidential political debate . These theories, in effect, have acquired a zombie-like state, where they are sustained by conspiracy theorists even after being debunked. The consequences have been devastating, with resurgence of measles outbreaks all over the globe. Measles was supposedly eradicated in USA but has made a comeback with multiple deaths . We are seeing a similar template being followed with the abusive head trauma (AHT) diagnosis. There have been high-profile news reports on the Cable News Network (CNN) and in The New York Times , along with governmental reports such as that by the Swedish Agency for Health Technology Assessment and Assessment of Social Services (SBU), that have discredited the diagnosis of AHT and turned it into a legal controversy . There is similarly a small group of physicians that has created a Potemkin village of unsubstantiated alternative diagnoses, without any factual basis . However, as noted in an editorial, the Wakefield report is considered a fraud, whereas the SBU report is considered a limited review of AHT with an artificial view of traumatic shaking and of limited clinical value in the real world of clinical practice . The deficiencies of the SBU report have been highlighted in many reviews .
In response, the Child Abuse Imaging Committee of the Society for Pediatric Radiology (SPR), under the guidance of Dr. Tom Slovis, collaborated with a broad-based team of multidisciplinary multinational authors with expertise in abusive head trauma to develop a consensus statement on abusive head trauma . These authors were experienced in their specialty; had numerous publications, presentations and grants; and were actively engaged in clinical work including managing children with AHT. The questions considered in the document include : What are the causes of head injury in infants and young children? Why has AHT terminology evolved (shaken baby syndrome, battered child, abusive head trauma, etc.)? What are the presenting features of AHT? How is the diagnosis of AHT made? What unsubstantiated alternative diagnoses are being proffered in the court (e.g., cerebral sinovenous thrombosis, hypoxic–ischemic injury, lumbar puncture, dysphagia, birth-related injury and benign enlargement of subarachnoid spaces)? What is the role of the multidisciplinary child protection team in the determination of AHT? What issues allow the misconceptions to be perpetuated in the courtroom? What can be done to provide the court accurate information about the state of medical knowledge in AHT? Endorsements Impact This consensus statement has been supported and endorsed by multiple organizations worldwide . We are pleased to report that two additional organizations have endorsed the statement. These are the Society of German-speaking Pediatric Radiologists (GPR) and the Pediatric Society of New Zealand. This makes a total of 17 multinational multidisciplinary organizations that have endorsed the consensus statement on AHT in young infants and children (Table ).
We have had a tremendous response from the clinical community worldwide, as reflected by their endorsements and positive feedback. At this writing, the consensus statement had been downloaded more than 13,000 times and was in the 98th percentile of all articles being tracked by Altmetric; it had been cited 63 times and had an Altmetric score of 108 . At this writing, the consensus statement was ranked number 1 of 1,520 outputs from the Pediatric Radiology journal. It has been cited in and influenced legal rulings . This confirms that there was a critical need for this professional-society-led consensus document. Furthermore, this statement has the potential to significantly impact the lives of vulnerable children and their caregivers.
Abusive head trauma is an established medical diagnosis with well-defined clinical criteria, but it represents a complex medicolegal challenge. The acceptance, endorsement and application of the consensus statement on AHT, successfully around the world by multidisciplinary organizations, demonstrates the utility of consensus statements that are coordinated and developed by professional societies.
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Proceedings of the 28th European Paediatric Rheumatology Congress (PReS 2022) | 9bb3e8e7-4e1f-4575-87a7-022243fdba18 | 9466322 | Internal Medicine[mh] | O01. Modeling HLH & MAS susceptibility identifies the characteristics of hyperinflammatory CD8 T-cells O02. Characteristics and disease course of patients with systemic juvenile idiopathic arthritis without arthritis in the German AID-NET cohort O03. Impact of interferon signalling on response to canakinumab treatment in systemic juvenile idiopathic arthritis as revealed by whole blood RNA sequencing O04. Systemic juvenile idiopathic arthritis associated lung disease in Europe O05. Analysis of pyrin inflammasome activation defines surf patients from FMF and other recurrent fevers O06. MIS-C phenotypes vary between SARS-COV-2 variants O07. Development of the oncoreum score for differential diagnosis between childhood cancer with arthropathy and juvenile idiopathic arthritis 8. NLRP3 splicing variants as a regulatory mechanism of inflammasome priming in auto-inflammation O09. Candidate gene sequencing in systemic juvenile idiopathic arthritis implicates rare variation in hereditary periodic fever and familial hemophagocytic lymphohistiocytosis genes M. Correia Marques, D. Rubin, E. Shuldiner, E. Schmitz, E. Baskin, A. Patt, M. Ombrello on behalf of Incharge Consortium Landy 1 , V. Dang 2 , J. Varghese 2 , P. Tsoukas 3 , S. Canna 2 1 University of Pittsburgh, Pittsburgh, 2 The Children’s Hospital of Philadelphia, Philadelphia, United States, 3 Hospital for Sick Children, Toronto, Canada Correspondence: S. Canna Introduction: Life-threatening hyperinflammatory Syndromes like Hemophagocytic Lymphohistiocytosis (HLH) and Macrophage Activation Syndrome (MAS) often result from the interactions of multiple susceptibility factors and environmental insults. Recent studies in both HLH and MAS patients demonstrate CD8 T-cell activation profiles in peripheral blood. Objectives: We wished to test how two independent and synergistic susceptibility factors, perforin insufficiency and excess IL-18, drove pathology in a spontaneous murine model of HLH/MAS. Methods: We bred and examined transgenic mice bearing knock-out alleles in perforin ( Prf1 +/- and -/- ) as well as transgenic expression of mature murine IL-18 ( Il18tg ) by “clinical” measures, cytokine levels, flow cytometry, bulk RNA- and TCR-sequencing, and in functional studies. Results: Mice bearing dual susceptibility factors (DS mice, Prf1 -/- Il18tg ) develop spontaneous HLH/MAS - even Prf1 +/- Il18tg mice develop spontaneous HLH-like immunopathology - in a manner dependent on IFNg. Detailed flow cytometric organ phenotyping reveals a dense expansion of CD8 T-cells bearing high levels of the IL-18 receptor as well as multiple markers associated with exhaustion (PD-1, Lag-3, CD39), yet overproduce IFNg. Such “hyperinflammatory CD8 T-cells” are present in the reticuloendothelial organs (spleen, bone marrow, and liver) but not lymph nodes, peripheral blood, or thymus. RNAseq analyses of hyperinflammatory CD8 T-cells alongside well-described exhausted cells reflects a pattern of activation distinct from acute effector, effector-memory, tissue-resident memory, or exhaustion. Multiple features suggest cells that have received recent antigen stimulation but are terminally-differentiated. TCR sequencing of bulk splenic CD8 T-cells shows oligoclonal expansion in DS mice. Attempts to circumvent CD8 T-cell activation, using inducible deletion of Il18r1 only on CD8 T-cells, or by promoting allelic exclusion by fixing the T-cell receptor of DS mice, shows that IL-18 responsive, oligoclonal CD8 T-cells circumvent these efforts to hinder their ability to expand. In vitro studies show that the effects of IL-18 require recent TCR stimulation, but do not inhibit activation-induced cell death. Conclusion: Both perforin deficiency and excess IL-18 seem to exert preferential effects on post-thymically activated CD8 T-cells in reticuloendothelial organs. These are precisely the sites where hemophagocytosis is most commonly observed. These data suggest a requirement for TCR stimulation reminiscent of the infectious triggers common to HLH and MAS, demonstrate these cells remarkable resilience, and identify potentially-targetable nodes of T-cell activation. Patient Consent: Not applicable (there are no patient data) Disclosure of Interest : E. Landy: None declared, V. Dang: None declared, J. Varghese: None declared, P. TsoukasSpeaker Bureau with: Clinical Viewpoints
C. Hinze 1 , H. Wittkowski 1 , E. Lainka 2 , J.-P. Haas 3 , T. Kallinich 4 , D. Föll 1 on behalf of for the AID-registry investigators 1 Pediatric Rheumatology and Immunology, University Hospital Münster, Münster, 2 Pediatrics II, University Hospital of Essen, Essen, 3 German Centre for Paediatric and Adolescent Rheumatology, Garmisch-Partenkirchen, 4 Paediatric Rheumatology, Charity-University Medicine, Berlin, Germany Correspondence: C. Hinze Introduction: In systemic juvenile idiopathic arthritis (SJIA), arthritis is often absent during the initial presentation and may not develop at all in some patients (pts). It is unclear how SJIA pts with and without arthritis differ otherwise. Objectives: To evaluate the clinical characteristics and disease courses in pts diagnosed with SJIA and compare those between pts who had or developed chronic arthritis and those who did not. Methods: The German AID-Net cohort enrolled pts between 2009 and 2018, some of them retrospectively. Pts with physician-diagnosed SJIA were analyzed in regards to clinical and laboratory parameters at study inclusion and during the disease course. Pts were considered in the arthritis group if arthritis was recorded at the time of enrolment and/or if it was recorded during the disease course for a duration of at least 6 weeks. Distributions and frequencies of clinical parameters were compared between the groups. Results: The study included 262 pts with SJIA, with a median (interquartile range) age at inclusion of 10.2 (6.0-14.4) yrs, a median age at diagnosis of 7.6 (3.7-12.0) yrs and a median follow-up of 4.1 (1.4-8.1) yrs. At baseline, 147 pts (56%) had arthritis recorded, and of the remaining 115 pts (44%), 30 later developed arthritis (11% of all patients) after 11 (2-20) months, i.e., 85 (32%) pts never had arthritis according to the case definition. Demographic data and clinical parameters that differed significantly between the groups (arthritis versus no arthritis) at baseline are shown in the table. The following parameters did not show significant differences in those for whom the parameters was available: sex, time from onset to diagnosis, adenopathy, macrophage activation syndrome (MAS) (11%:14%), leukocyte count, CRP, S100A8/A9, interleukin-18 or CXCL9 at the time of highest S100A12 level. Medication use was more variable in SJIA patients with arthritis, and the following medications were used more frequently in SJIA with arthritis versus never arthritis (chi square p<0.05): methotrexate (81%:48%), anakinra (47%:27%), etanercept (34%:2%), ciclosporin A (27%:12%), azathioprine (19%:4%), adalimumab (18%:1%), and leflunomide (11%:0%). Concerning the outcomes during the disease, significant differences were seen in the proportion of active disease at the last study visit (25%:13%), and the proportion of pts on glucocorticoids (GC) (23%:11%). There were no significant differences concerning MAS (13%:8%) or death (3 [2%]:0 [0%]). Conclusion: In this German registry-based cohort of patients with physician-diagnosed SJIA, about one third of pts did not have chronic arthritis. SJIA pts without arthritis on average were older and inflammatory signs were more frequent at baseline, while MAS was similarly frequent. The pharmacologic therapy was substantially more variable in SJIA with arthritis. During the follow-up, SJIA pts without arthritis tended to have better outcomes, i.e., less frequently active disease, less GC use, and no significant difference in MAS occurrence. Limitations include overall shorter follow-up for SJIA pts without arthritis. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
T. Hinze, C. Hinze 2 , C. Kessel 2 , A. Huge 2 , C. Farady 3 , S. McCreddin 4 , K. Gandhi 5 , D. Foell 2 1 Department of Pediatric Rheumatology and Immunology, 2 University of Münster, Münster, Germany, 3 Novartis Pharma AG, Basel, Switzerland, 4 Novartis Ireland Limited, Dublin, Ireland, 5 Novartis Pharmaceuticals Corporation, East Hanover, NJ, United States Correspondence: T. Hinze Introduction: Active systemic juvenile idiopathic arthritis (SJIA) is characterized by marked innate immune overactivation and dysregulation of immune-related peripheral blood gene expression. Canakinumab (CAN), an interleukin (IL)-1β blocking antibody has shown to be effective in patients with SJIA. We had previously observed that patients with a higher serum protein IL-18:CXCL9 ratio responded better to CAN than those with a lower ratio. However, it is unclear which factors specifically govern differences in treatment response. Objectives: The objective of this study was to identify differences in peripheral blood gene expression patterns between patients with active SJIA who subsequently showed sustained complete response to CAN and those that did not. Methods: Whole blood RNA samples from (1) CAN-naïve patients from an open-label randomized CAN trial (NCT02296424) in active disease (AD), (2) CAN-treated patients enrolled in a randomized controlled CAN trial (NCT00891046) in inactive disease (ID), and (3) paediatric healthy controls (HC) were studied via whole RNAseq on the Illumina NextSeq 2000 platform, assessing 58395 genes overall. For a proof-of-principle analysis, gene expression patterns were compared across SJIA patients with AD (n=10), ID (n=10), and HC (n=10). CAN-naïve patients were further categorized according to their subsequent treatment response. We specifically considered patients with subsequent sustained complete response (SCR) (n=5), i.e., inactive disease or ACR100 response within 4 weeks of treatment, no subsequent flares or macrophage activation syndrome [MAS] during the study, and non-responders (NR) who did not have an ACR30 response (n=4). Differential gene expression was analysed using the R package deseq2. Adjustment of p-value for multiple comparisons was achieved via a false discovery rate (FDR) <0.05 or <0.1. Results: The proof-of-principle analysis demonstrated marked differential gene expression when comparing AD versus both ID and HC, assuming an FDR<0.05 (AD versus HC: 1872 genes up, 326 down; AD versus ID: 2269 up, 618 down). The prominently upregulated genes included multiple neutrophil-related genes (e.g., CD177, CD93, HK3, MMP9, NLRC4, PADI4, NCF4, NCF1B, PSTPIP2, S100A8, S100A9). When comparing 5 patients with SCR and 4 with NR, assuming an FDR <0.1, 98 genes were differentially regulated (14 up in SCR, 84 up in NR). The 14 genes up in SCR included several type 1 interferon (IFN)-regulated genes, including ERAP2, RSAD2 and SIGLEC1. The 84 genes up in NR included multiple erythropoiesis-related genes, including EPB42, GYPC, PRDX2, RHAG, SPTA1, SPTB, TFR2, TMOD1, TFRC, and TRIM58. Conclusion: As expected, there was marked dysregulation of peripheral blood gene expression in patients with active SJIA, prominently including the overexpression or overrepresentation of neutrophil-related genes. Relative overexpression of peripheral blood type 1 IFN-stimulated genes may correlate with an excellent response to CAN. In contrast, relative overexpression of erythropoiesis-related genes, which in turn may correlate with ineffective erythropoiesis/occult MAS and IFN-γ activity, may correlate with a poor response to CAN. In summary, these findings support the notion that dysregulation of the type 1 IFN-IL-18-IFN-γ axis may play a role in the treatment response to CAN in SJIA. Patient Consent: Not applicable (there are no patient data) Disclosure of Interest : T. Hinze Grant / Research Support with: Novartis Pharma AG, C. Hinze: None declared, C. Kessel Grant / Research Support with: Novartis, Consultant with: Novartis, Sobi, A. Huge: None declared, C. Farady Employee with: Novartis Pharma AG, S. McCreddin Employee with: Novartis Ireland Limited, K. Gandhi Employee with: Novartis Pharmaceuticals Corporation, D. Foell Grant / Research Support with: Novartis, Consultant with: Novartis
C. Bracaglia 1 , F. Minoia 2 , C. Kessel 3 , S. Vastert 4 , M. Pardeo 1 , A. Arduini 1 , O. Basaran 5 , N. Kipper 6 , M. Kostik 7 , M. Glerup 8 , S. Fingerhutova 9 , R. Caorsi 10 , A. Horne 11 , G. Filocamo 2 , H. Wittkowski 3 , M. Jelusic 12 , J. Anton 13 , S. Khaldi-Plassart 14 , A. Belot 14 , G. Horneff 15 , S. Palmer Sarott 16 , E. Cannizzaro Schneider 16 , P. Dolezalova 9 , A. Ravelli 17 , S. Ozen 5 , F. De Benedetti 1 on behalf of MAS/sJIA working party 1 Division of Rheumatology, IRCCS Ospedale Pediatrico Bambino Gesù, Roma, 2 Fondazione IRCCS Ca’ Grande Ospedale Maggiore Policlinico, Milano, Italy, 3 Department of Pediatric Rheumatology & Immunology, WWU Medical Center (UKM), Munster, Germany, 4 Pediatric Rheumatology & Immunology, University Medical Center Utrecht, Utrecht, Netherlands, 5 Department of Pediatrics, Division of Pediatric Rheumatology, 6 Department of Pediatrics, Division of Pediatric Pulmonology, Hacettepe University, Ankara, Turkey, 7 Saint-Petersburg State Pediatric Medical University, Saint-Petersburg, Russian Federation, 8 Department of Pediatrics, Aarhus University Hospital, Aarhus, Denmark, 9 Centre for Paediatric Rheumatology and Autoinflammatory Diseases, Department of Paediatrics and Inherited Metabolic Disorders, 1st Faculty of Medicine, Charles University and General University Hospital in Prague, Prague, Czech Republic, 10 Department of pediatrics and Rheumatology, IRRCS Istituto G. Gaslini, Genova, Italy, 11 Department of pediatric rheumathology, Karolinska University Hospital and Department of pediatrics, Karolinska Institute, Stockholm, Sweden, 12 Department of Paediatrics, University of Zagreb School of Medicine, University Hospital Centre, Zagreb, Croatia, 13 Pediatric Rheumatology, Hospital Sant Joan de Déu, Universitat de Barcelona, Barcelona, Spain, 14, 15 Pediatrics, Asklepios Clinic Sankt Augustin, Sank Augustin, Germany, 16 Paediatric Rheumatology, University Children’s Hospital Zurich, Zurich, Switzerland, 17 IRRCS Istituto Giannina Gaslini and Università degli Studi di Genova, Genova, Italy Correspondence: C. BracagliasJIA-LD in Europe. Methods: Patients with diagnosis of sJIA with LD, including pulmonary alveolar proteinosis (PAP), interstitial lung disease (ILD) and pulmonary arterial hypertension (PAH), followed in European paediatric rheumatology centres were identified through a survey sent to the members of the MAS/SJIA Working Party. Results: Data from 34 sJIA-LD patients, diagnosed in 15 European paediatric rheumatology centres between 2007 and 2022, were collected. 33 patients were Caucasian and 1 was African-American; 21 were female. The median age at sJIA onset was 6 years and LD occurred after a median time of 2 years. 19 patients had a chronic persistent sJIA course, 14 had a polycyclic course and only 1 patient had a monocyclic course; 29 (85%) had active sJIA at time of LD diagnosis. During the disease course, 28 (82%) patients developed MAS, 12 (35%) of whom had MAS at sJIA onset and 19 (56%) had full-blown MAS at time of LD diagnosis; 23 (68%) patients had >1 MAS episode. 28 (82 %) patients were treated with at least one IL-1 or IL-6 inhibitor before LD diagnosis: 15 with canakinumab, 24 with anakinra and 13 with tocilizumab; 13 (38%) patients experienced drug adverse reaction to a cytokine inhibitor: 9 to tocilizumab and 4 to anakinra. 24 (70%) patients developed ILD, 6 (18%) PAP and 4 (12%) PAH. 15 (44%) patients presented acute digital clubbing; 16 (47%) patients developed hypoxia and 9 (26%) developed pulmonary hypertension. A chest CT scan was performed in all patients with evidence of septal thickening, peri-bronchovascular thickening and ground glass opacities in the majority of patients (26, 18 and 18 respectively). In 17 patients a bronchoalveolar lavage was performed and 12 underwent a lung biopsy. The histopathological pattern was alveolar proteinosis in 5 patients, endogenous lipoid pneumonia in 3, vasculitis in 1 and fibrosis in 1. Half of the patients (17) required ICU admission and 6 (18%) died. All the patients were treated with glucocorticoids (GCs) at time of diagnosis, and 26 received IL-1 or IL-6 inhibitor after the diagnosis (13 canakinumab, 20 anakinra, 14 tocilizumab). Conclusion: Lung involvement is an emerging life-threatening complication of sJIA and patients are also diagnos: Yes, I received consent Disclosure of Interest : C. Bracaglia Consultant with: Sobi, Novartis: None declared, A. Arduini: None declared, O. Basaran: None declared, N. Kipper: None declared, M. Kostik: None declared, M. Glerup: None declared, S. FingerhutovaobiP. Dolezalova: None declared, A. Ravelli: None declared, S. Ozen Consultant with: Sobi, Novartis, Speaker Bureau with: Sobi, Novartis, F. De Benedetti Consultant with: Abbvie, Sobi, Novimmune, Novartis, Roche, Pfizer
S. Palmeri 1 , A. Bertoni 2 , R. Papa 2 , F. Penco 2 , A. Corcione 2 , R. Caorsi 2 , C. Matucci-Cerinic 1 , M. Bustaffa 2 , F. Schena 2 , P. Bocca 2 , S. Volpi 1,2 , A. Rubartelli 2 , M. Gattorno 2 , I. Prigione 2 1 Department of Neuroscience, Rehabilitation, Ophthalmology, Genetics, Maternal and Child Health, University of Genova, 2 Center for Autoinflammatory Diseases and Immunodeficiencies, IRCCS Istituto Giannina Gaslini, Genova, Italy Correspondence: S. Palmeri Introduction: The best known of recurrent fevers, familial Mediterranean fever (FMF), is genetically determined and its pathogenetic mechanism has already been extensively investigated, revealing a role of the pyrin inflammasome (1). However, cohorts of patients with undifferentiated, genetically negative forms of relapsing fever (SURF) are still poorly studied. These patients are genetically negative, and their clinical picture resemble FMF, including the good response to colchicine. However, the underlying inflammatory mechanisms of SURF are not yet known (2). Objectives: To assess the in vitro activation of the pyrin inflammasome in a cohort of patients with SURF, compared with FMF and Periodic Fever, Aphthous Stomatitis, Pharyngitis, Adenitis (PFAPA) patients; to stratify the response between in vivo colchicine-treated and untreated individuals; to dissect the in vitro response to colchicine in the same cohort of patients. Methods: Peripheral blood mononuclear cells (PBMC) of a subset of SURF (N=15, colchicine-treated and untreated), FMF(N=8), PFAPA (N=8) and HD (N=13) were tested for ASC speck formation and IL-1beta production in response to different stimuli. Clostridium difficile toxin A (TcdA) and PKN1/2inhibitor (UCN-01) were used to trigger pyrin inflammasome. Colchicine was added in vitro to evaluate the pyrin inflammasome inhibition. We used a flow cytometric method to measure the percentage of ASC speck formation in monocytes and ELISA assay to quantify the secretion of IL-1beta on cell supernatants. We performed non-parametric Mann-Whitney test (MW) for the analysis of differences between groups. Results: in SURF patients, we did not observe spontaneous activation of the inflammasome in the absence of clinical flare. FMF, SURF, PFAPA and HD displayed no differences in TcdA-induced activation of pyrin inflammasome (% of ASC speck formation). However, in vivo colchicine-untreated SURF patients showed a reduced response to TcdA, with a normalization of values in SURF patients after treatment (% of ASC speck formation, MW, p<0,05 ; IL1-beta (pg/mL) MW, p<0,05 ). In contrast with FMF, SURF, PFAPA patients and HD showed the following features: i) UCN-01 mediated-pyrin dephosphorylation was not sufficient to trigger Pyrin inflammasome activation; ii) the in vitro colchicine administration caused a huge inhibition of TcdA-induced pyrin inflammasome activation. Conclusion: we applied functional in vitro tests for pyrin inflammasome activation analysis to a clinically homogeneous group of subjects with SURF. These preliminary data show that SURF patients differ from FMF, PFAPA, and HD. SURF subjects showed a different response to TcdA, with a normalization after colchicine therapy, suggesting an involvement of pyrin inflammasome in the physiopathology of SURF. References: 1) Magnotti F. et al. “Fast diagnostic test for familial Mediterranean fever based on a kinase inhibitor.” Annals of the rheumatic diseases vol. 80,1 (2021): 128-132. 2) Papa R. et al. “” Journal of clinical medicine vol. 10,9 1963. Trial registration identifying number: Patient Consent: Yes, I received consent Disclosure of Interest : None declared
G. Mastrangelo 1 , E. Go 1,2,3 , P. Tsoukas 1,2 , H. Lu 1,2 , A. Hoi Hin Cheng 1,2 , R. S. Yeung 1,2,4 on behalf of on behalf of SickKids MIS-C Working Group 1 Division of Rheumatology, Department of Paediatrics, The Hospital for Sick Children, University of Toronto, 2 Cell Biology Program, The Hospital for Sick Children Research Institute, Toronto, Canada, 3 Division of Pediatric Rheumatology, Riley Hospital for Children, Indianapolis, United States, 4 Department of Immunology and Institute of Medical Science, University of Toronto, Toronto, Canada Correspondence: G. Mastrangelo Introduction: Multisystem Inflammatory Syndrome in Children (MIS-C) is a serious complication associated with COVID-19, presenting as a hyperinflammatory disorder characterized by fever and multiorgan dysfunction. Whether the MIS-C phenotype varies accordingly to the SARS-CoV-2 variants is still unclear. Objectives: We aim to compare MIS-C clinical features, treatments, and outcomes across the various waves of COVID-19, dividing the patient population into three cohorts according to the Alpha/Beta/Gamma, Delta, and Omicron MIS-C waves. Our secondary objective is to evaluate if the clinical phenotype (shock, Kawasaki Disease (KD), fever with hyperinflammation) varies across the three cohorts. Methods: We performed a prospective cohort study of 252 patients with MIS-C, at a tertiary care pediatric center from March 2020 to March 2022. Clinical and laboratory features, complications, treatments, and outcomes were evaluated. The association with SARS-CoV-2 variants and MIS-C cohorts was assumed based on local epidemiology and sequencing data, representing the predominant strain across the three-time periods. The starting date of each MIS-C wave for study purposes was set at two weeks after the first case of COVID-19 from that respective variant in the community, as the actual time lag for developing MIS-C is within 2-6 weeks after the acute infection. Descriptive statistics were performed to assess differences between the three MIS-C cohorts and clinical phenotypes. Results: Of the 252 patients (150 with Alpha/Beta/Gamma variants, 59 with Delta, 43 with Omicron), the median age was 5.2 years, 58.7% were male, and 50% had SARS-CoV-2 exposure. The three cohorts showed a significant difference in MIS-C phenotype distribution (p=0.003). Fever and hyperinflammation was the predominant phenotype (20%) in the Alpha/Beta/Gamma cohort; shock represented the majority (39%) in the Delta cohort; and the KD phenotype was prevalent (67%) in the Omicron cohort. Cardiac and gastrointestinal involvements were the most common features in all the cohorts, whereas, neurological involvement was the least prevalent. The Omicron cohort had more mucocutaneous involvement and renal abnormalities compared to the others. The main difference between the various waves was reflected in measures of complications and outcome with the MIS-C cohort associated with the Delta variant capturing the most severe phenotype with a higher incidence of shock (39%), MAS (22%), and PICU admission (34%). The proportion of children developing coronary artery lesions was similar in all groups. Among all the three MIS-C cohorts, the majority of patients received either IVIG alone or together with upfront steroids. Pulsed high-dose steroids and anticoagulation therapy were more commonly used among children in the Delta MIS-C cohort, findings in keeping with the prevalence of the MIS-C shock phenotype in this group. Conclusion: The MIS-C phenotype varies accordingly to the SARS-CoV-2 variants, and patients with the Delta variant had a more severe phenotype with a greater proportion of complications. This is the first study that compares MIS-C phenotypes stratified by virus variant waves, including the Omicron wave. These findings provide new insights into disease phenotype and SARS-CoV-2 variants and may have important implications for diagnosis and managementCivino 1 , F. Bovis 2 , M. Ponzano 2 , S. Magni-Manzoni 3 , S. Sorrentino 4 , L. Vinti 5 , M. Romano 6 , N. Santoro 7 , G. Filocamo 8 , C. Gorio 9 , F. Santarelli 10 , M. Cattalini 11 , F. Diomeda 12 , G. Alighieri 13 , E. Prete 14 , G. Stabile 15 , R. Rondelli 16 , V. Conter 17 , A. Pession 18 , A. Ravelli 19,20 on behalf of ONCOREUM Study group 1 Reumatologia e Immunologia Pediatrica, Ospedale “Vito Fazzi”, Lecce, 2 Dipartimento di Scienze della Salute, Sezione di Biostatistica, Università di Genova , Genova, 3 Reumatologia Pediatrica, Ospedale Pediatrico Bambino Gesù IRCCS, Roma, 4 Dipartimento di Oncoematologia Pediatrica, IRCCS Istituto ‘Giannina Gaslini’, Genova, 5 Dipartimento di Oncoematologia Pediatrica, Ospedale Pediatrico Bambino Gesù IRCCS, Roma, 6 Divisione di Reumatologia, ASST G. Pini-CT, Milano, 7 Oncoematologia Pediatrica, Azienda Ospedaliero Universitaria Consorziale Policlinico di Bari, Bari, 8 Fondazione IRCCS Cà Granda Ospedale Maggiore Policlinico, Milano, 9 Unità di Oncoematologia Pediatrica e TMO, “Spedali Civili”, Brescia, 10 Dipartimento di Pediatria, Ospedale Pediatrico “Regina Margherita”, Torino, 11 Clinica Pediatrica, Università degli Studi di Brescia e ASST Spedali Civili, Brescia, 12 Clinica Pediatrica, Università degli Studi di Bari Aldo Moro, Bari, 13 UO Neonatologia-UTIN, Azienda Ospedaliera “A. Perrino”, Brindisi, 14 Dipartimento di Ematologia, Azienda Ospedaliera “Cardinale G. Panico”, Tricase (Lecce), 15 Consorzio Interuniversitario Cineca, Casalecchio di Reno, 16 Oncoematologia Pediatrica, IRCCS Azienda Ospedaliero-Universitaria di Bologna, Bologna, 17 Oncoematologia Pediatrica, Fondazione MBBM, Università Milano Bicocca, Monza, 18 Clinica Pediatrica, IRCCS Azienda Ospedaliero-Universitaria di Bologna, Bologna, 19 Direzione Scientifica, IRCCS Istituto Giannina Gaslini, 20va, Italy Correspondence: A. Civino Introduction: Pediatric cancer with musculoskeletal symptoms at onset can mimic rheumatic diseases, particularly juvenile idiopathic arthritis (JIA). This could lead to inappropriate steroid treatment or immunosuppressive therapy with a delay in the diagnosis. Objectives: To develop and validate a weighted score, named ONCOREUM Score, that aids physicians in timely differentiation of cancer with arthropathy from JIA. Methods: Data were extracted from the ONCOREUM Study, a multicenter, prospective, cross-sectional study aimed to compare patients with cancer and arthropathy with those affected by JIA. Patients were younger than 16 years and were newly diagnosed with cancer at 25 Italian pediatric hemato-oncology centers or with JIA at 22 Italian pediatric rheumatology centers. Details concerning study design have been described in detail previously. 1 A multiple imputation by chained equations approach with 10 imputations was first performed. Then, 80% of patients were assigned to the developmental data set and 20% to the validation data set. Three statistical approaches were applied to develop the ONCOREUM Score, two based on multivariable analysis of different sets of variables (Models 1 and 2) and one based on a Bayesian Model Averaging method (Model 3). The β coefficients estimated in the models were used to assign points to the scores. Discriminating performance was evaluated by calculating sensitivity, specificity and AUC in the validation sample. Results: The study dataset included 772 patients, 95 with cancer and arthropathy and 677 with JIA. The highest AUC in the validation data set was yielded by Model 1, which was selected to constitute the ONCOREUM Score. Sensitivity, specificity, and AUC of the cutoff in the validation sample were 81.3%, 96.4%, and 0.89, respectively. The formula used to calculate the score includes 9 variables weighted according to the estimated coefficients, as follows (clinical features are included in the model only if present): ONCOREUM score = - 4.1 + 8.4 x limb bone pain + 4.7 x weight loss + 5.1 x thrombocytopenia - 5.2 x morning stiffness - 3.7 x joint swelling - 5.0 x small hand joint involvement + 4.1 x monoarticular involvement + 2.4 x hip involvement + 1.2 x male sex. The score ranges from - 18 to 21.8 and the optimal cutoff obtained through ROC analysis was – 2, with patients being classified at higher risk of having cancer and arthropathy if score is ≥ - 2, and at higher risk of having JIA if score is < - 2. Conclusion: The ONCOREUM score is composed of 9 clinical features that can easily be recorded at initial encounter with the patient. It is a powerful tool that may facilitate early differentiation of malignancies with arthropathy from JIA and timely referral of the child to the appropriate pediatric specialist. 1. Civino A, Alighieri G, Prete E et al. Musculoskeletal manifestations of childhood cancer and differential diagnosis with juvenile idiopathic arthritis (ONCOREUM): a multicentre, cross-sectional study. Lancet Rheumatol. 2021; 3 (7): e507-e516. Trial registration identifying number: ONCOREUM study group members Italian Association of Paediatric Haematology and Oncology (AIEOP) Centres Massimo Eraldo Abate (Bologna), Annalisa Arlotta (Parma), Catia Atzeni (Cagliari), Tamara Belotti (Bologna) Patrizia Bertolini (Parma), Barbara Bigucci (Rimini), Andrea Biondi (Monza), Roberta Burnelli (Ferrara), Maurizio Caniglia (Perugia), Ilaria Capolsini (Perugia), Anna Maria Caroleo (Genova), Maria Giusepina Cefalo (Roma), Monica Cellini (Modena), Simone Cesaro (Verona), Adele Civino (Tricase, Lecce), Elisa Coassin (Aviano), Antonella Colombini (Monza), Valentino Conter (Monza), Carmela De Fusco (Napoli), Raffaela De Santis (San Giovanni Rotondo), Andrea Di Cataldo (Catania), Elena Fabbri (Rimini), Franca Fagioli (Torino), Monica Ficara (Modena), Ilaria Fontanili (Parma), Alberto Garaventa (Genova), Chiara Gorio (Brescia), Saverio Ladogana (San Giovanni Rotondo), Franco Locatelli (Roma), Chiara Mainardi (Padova), Maurizio Mascarin (Aviano), Chiara Messina (Padova), Concetta Micalizzi (Genova), Rossella Mura (Cagliari), Daniela Onofrillo (Pescara), Roberta Pericoli (Rimini), Andrea Pession (Bologna), Cristina Pizzato (Treviso), Fulvio Porta (Brescia), Carmelo Rizzari (Monza), Andrea Roncadori (Bologna), Roberto Rondelli (Bologna), Elisa Rossi (Bologna), Giovanna Russo (Catania), Nicola Santoro (Bari), Giulia Stabile (Bologna), Elisa Tirtei (Torino), Assunta Tornesello (Lecce), Federico Verzegnassi (Trieste), Luciana Vinti (Roma) Italian Paediatric Rheumatology Study Group (IPRSG) Centres Giovanni Alighieri (Tricase), Martina Amatruda (Roma), Patrizia Barone (Catania), Luciana Breda (Chieti), Michela Cappella (Reggio Emilia), Marco Cattalini (Brescia), Adele Civino (Tricase, Lecce), Rita Consolini (Pisa), Elisabetta Cortis (Orvieto), Sergio Davì (Genova), Alessandro De Fanti (Reggio Emilia), Fabrizio De Benedetti (Roma), Giovanni Filocamo (Milano), Romina Gallizzi (Messina), Maria Francesca Gicchino (Napoli), Francesco La Torre (Brindisi), Bianca Lattanzi (Ancona), Loredana Lepore (Trieste), Maria Cristina Maggio (Palermo), Silvia Magni-Manzoni (Roma), Andrea Magnolato (Trieste), Manuela Marsili (Chieti), Silvana Martino (Torino), Angela Miniaci (Bologna), Alma Nunzia Olivieri (Napoli), Serena Pastore (Trieste), Maria Antonietta Pelagatti (Monza), Rosa Anna Podda (Cagliari), Eleonora Prete (Tricase), Angelo Ravelli (Genova), Francesca Ricci (Brescia), Donato Rigante (Roma), Micol Romano (Milano), Francesca Santarelli (Torino), Francesca Soscia (Orvieto)Erkens 1,2 , M. van Haaren 2 , R. Sanchez Rodriguez 2 , J. Calis 2 , S. Vastert 1,2 , J. van Loosdregt 2 1 Pediatric Rheumatology, Wilhelmina Children’s Hospital, 2 Center for Translational Immunology, UMC Utrecht, Utrecht, Netherlands Correspondence: R. Erkens Introduction: The activation of the NLRP3 Inflammasome is both transcriptionally and post-translationally regulated. Recently, it has been demonstrated that various isoforms of NLRP3 are expressed in human macrophages and that isoforms that lack certain exons due to alternative splicing are not able to form a functional inflammasome by being unable to bind to NEK7, an essential mediator of NLRP3 activation. Inflammasome activation seem to play an important role in systemic Juvenile Idiopathic Arthritis (SJIA) pathophysiology, characterized by high levels of IL-18 and IL-1 pathway activation. Here, we assessed whether NLRP3 splicing changes upon neutrophil and monocyte activation and investigated the role of alternative splicing in the auto-inflammatory setting of SJIA. Objectives: To explore the effects of alternative RNA splicing products of NLRP3 in neutrophils and monocytes in healthy individuals and SJIA patients on the activation of the NLRP3 inflammasome. Methods: We assessed NLRP3 isoform expression using third generation mRNA Nanopore sequencing, qPCR and western blot. The compared conditions included ex-vivo and 3 hours 100ng/ml LPS stimulated neutrophils and monocytes isolated from peripheral blood of healthy donors and SJIA patients. Inflammasome activation was assessed by measuring IL-1β production using ELISA after LPS priming and subsequent nigericin activation. Results: We identified various NLRP3 RNA isoforms in both neutrophils and monocytes using Nanopore sequencing and qPCR. Full length NLRP3 and NLRP3 δexon5 isoforms were found to be the most dominant. In monocytes and neutrophils, LPS mediated priming induces NLRP3 expression and a shift to full length NLRP3 expression due to a decrease in NLRP3 δexon5. Ex-vivo neutrophils and monocytes of biological and steroid naïve patients with active SJIA express relatively more full length NLRP3 compared to samples from healthy donors. After 3 days of treatment with anakinra, exon 5 inclusion decreased in both neutrophils and monocytes of the SJIA patients, but remains increased in neutrophils compared to healthy controls. These data suggest that neutrophils of active SJIA patients are more primed for NLRP3 inflammasome activation. Conclusion: Priming of neutrophils and monocytes induces profound changes in NLRP3 isoform expression. These changes after LPS priming might increase the potential of the cell to form an active inflammasome upon a second stimulation. Similarly, we demonstrated that neutrophils and monocytes from patients with active SJIA express more full-length NLRP3, able to form inflammasomes, compared to healthy controls. This observation could contribute to the increased inflammasome activation found in SJIA. Taken together, these results suggest an important level of regulation of inflammasome activation by alternative splicing. Further research is necessary to elucidate the pathophysiology of inflammasome activation in auto-inflammation in general as this opens a window for new therapeutic options. Patient Consent: Yes, I received consent Disclosure of Interest : R. Erkens: None declared, M. van Haaren: None declared, R. Sanchez Rodriguez: None declared, J. Calis: None declared, S. Vastert Consultant with: Sobi and Novartis, J. van Loosdregt: None declared
Translational Genetics and Genomics Section, NIAMS, National Institutes of Health, Bethesda, United States Correspondence: M. Correia Marques Introduction: Systemic juvenile idiopathic arthritis (sJIA) is a genetically complex inflammatory condition. It can be marked by severe systemic inflammation that resembles the hereditary periodic fever syndromes (HPF). Sometimes that inflammation leads toThe HPFs and familial forms of HLH (fHLH) are caused by rare genetic mutations, and it has been hypothesized that genetic variants of HPF or fHLH genes are involved in the pathophysiology of sJIA. Several studies have examined this question, but none have had the statistical power to provide an unequivocal answer. Objectives: We used targeted sequencing of HPF and fHLH genes in a large patient cohort to determine whether rare variation in these genes contributes to the risk of developing sJIA. Methods: Targeted sequencing of HPF ( MEFV, MVK, NLRP12, NLRP3, NOD2, PSTPIP1, TNFRSF1A ) and fHLH ( LYST, PRF1, RAB27A, STX11, STXBP2, UNC13D ) genes was performed in sJIA cases and control subjects from the International Childhood Arthritis Genetics Consortium cohort using Illumina Nextera Custom Capture Assays and Illumina sequencers. Sequence reads were aligned to human genome assembly hg19 with the Burrows-Wheeler Aligner. Data processing and quality control was performed using the Genome Analysis Toolkit. Variants were filtered to retain rare (minor allele frequency < 0.01), protein-altering variation that mapped to Ensembl canonical transcripts. The distribution of rare variants among sJIA cases was compared to the distributions among INCHARGE healthy controls or simulated data from the 33,370 Non-Finnish European (NFE) reference subjects from the Exome Aggregation Consortium (ExAC) using rare variant association testing (RVT). RVT was performed in R using the data-adaptive sum test and the sequence kernel association test (SKAT). Significance was evaluated at a threshold of p < 0.05 after 100,000 permutations. Results: Targeted sequencing was performed in 525 sJIA cases and 366 control subjects. Six sJIA cases were discovered to have a genetic diagnosis of either an HPF (n=4) or fHLH (n=2) and were excluded. We also found that 39 cases and 1 control subject were ancestrally dissimilar from the larger cohort by principal component analysis, leading to their exclusion. Sequencing of the remining 480 sJIA cases identified 78 rare fHLH gene variants and 62 rare HPF gene variants. RVT comparing the distribution of rare variants of sJIA cases with that of the ExAC NFE population revealed significant rare variant associations between sJIA and LYST, STXBP2, UNC13D and MEFV. We also discovered recurrent mutations of STXBP2, UNC13D, and MEFV among the sJIA cohort that were not observed in the ExAC population . A sub-analysis of 123 sJIA cases with known MAS status (32 with MAS, 91 without MAS) identified a significant association between rare variation of UNC13D and the development of MAS in sJIA (SKAT p=0.024). Conclusion: The observations of this study connect HPF and fHLH genes to the pathophysiology of sJIA. The distributions of rare genetic variants of LYST, STXBP2, UNC13D and MEFV were statistically different in children with sJIA than in the general population. We also identified novel, recurrent mutations in 3 of these 4 genes in children with sJIA. These results highlight the potential value of studying rare genetic variation in sJIA. To expand this approach, we have established a collaborative infrastructure to perform an exome sequencing-based study of rare variation across all protein-coding genes in sJIA10. CD14+ Monocyte-Derived oxidised mitochondrial DNA amplifies the inflammatory interferon type 1 signature in Juvenile dermatomyositis O11. A novel in vitro model to study precision targeting of IFN-mediated responses 12. Predicting immunoglobulin resistance in Kawasaki disease in multi-ethnic populations in europe: a multicenter cohort study O13. TIF1-GAMMA and NXP2 autoantibodies in children with JDM are underrepresented when assessed by immunoblot compared to immunoprecipitation O14. Comparing the use of 1g/kg intravenous immunoglobulin (IVIG) dose against the standard dose of 2g/Kg IVIG in patients with juvenile dermatomyositis (JDM): a retrospective cohort study O15. Measuring IFNA2 levels by a single-molecule array in clinical practice of childhood-onset SLE patients does matter; results from a single center longitudinal study O16. Panel sequencing links rare, possibly damaging genetic variants to a subset of patients with juvenile-onset SLE with distinct clinical phenotypes and outcomes O17. Genetical and phenotypical findings of childhood-onset systemic lupus erythematosus O18. Patient-specific and disease-related determinants for cardiovascular disease (CVD) risk stratification in the apple (atherosclerosis prevention in paediatric lupus erythematosus) clinical trial cohort M. Wilkinson 1,2,3 , D. Moulding 4 , T. C. R. McDonnell 5 , M. Orford 4 , C. Wincup 1,5 , J. Y. J. Ting 3 , G. W. Otto 2,6 , R. Restuadi 2,3 , D. Kelberman 2,7 , S. Castellano 2,6 , S. Eaton 4 , C. Deakin 1,2,3 , E. C. Rosser 1,5 , L. R. Wedderburn 1,2,3 on behalf of UK Juvenile Dermatomyositis Research Group and the Centre for Adolescent Rheumatology Versus Arthritis 1 Centre for Adolescent Rheumatology Versus Arthritis at UCL UCLH and GOSH, UCL, 2 NIHR GOSH BRC, 3 Infection, Immunity and Inflammation Research and Teaching Department, 4 Developmental Biology and Cancer Research & Teaching Department;, UCL GOS Institute of Child Health, 5 Centre for Rheumatology Research, UCL, 6 Genetics and Genomic Medicine Research & Teaching Department, UCL GOS Institute of Child Health, 7 Genetics and Genomic Medicine Research & Teaching Department, UCL, London, United Kingdom Correspondence: M. Wilkinson Introduction: JDM is a rare childhood autoimmune myositis that presents with proximal muscle weakness and associated skin changes. There is an unmet need to develop new targeted treatments. Objectives: This study aimed to identify dysregulated biological processes up-stream of the known, pathological interferon (IFN) type 1 signature in JDM by RNA-sequencing and develop functional assays to confirm these pathways. Methods: Peripheral blood samples were obtained from JDM patients [pre-n=10 on-n=11 treatment] and age/sex-matched child healthy controls [n=8]. CD4 + , CD8 + , CD14 + and CD19 + cells were sorted by flowcytometry from PBMC, and RNA was extracted and RNA-sequenced. Mitochondrial morphology and mitochondrial superoxide was assessed in CD14+ monocytes by fluorescence microscopy using MitoTracker and MitoSox dyes quantified by volume, JDM [n=6] and control [n=9]. Oxidised mitochondrial DNA (oxmtDNA) from CD14+ monocytes was measured by western dot-blot, JDM [n=10] and control [n=11]. Healthy control PBMC samples [n=6] were cultured with IFN-α or oxmtDNA (+ LL37) with or without TLR-9 antagonist or n -acetyl cysteine (NAC). Post-culture, IFN type 1 gene expression was measured by qPCR. Results: RNA-seq confirmed a strong IFN type 1 signature pre-treatment, and genes involved in mitochondrial function were abnormally expressed in both pre- and on-treatment CD14+ monocytes vs. controls, suggesting that mitochondrial dysfunction is not corrected by current treatment strategies. Investigating abnormal mitochondrial biology in JDM CD14+ monocytes by microscopy, we identified that the mitochondria were significantly more fragmented in JDM vs. control (p=0.0044) and evidence of megamitochondria. Analysis of the RNA-seq data showed that the oxidative phosphorylation pathway the gene expression of superoxide dismutase ( SOD1 ) were downregulated in JDM pre- and on-treatment vs. controls. We showed an increase in mitochondrial superoxide in CD14+ monocytes JDM vs. control (p=0.0005, p=0.017). By western dot-blot there was an increase in oxmtDNA in CD14+ monocytes JDM vs. control (p=0.0178). In vitro , oxmtDNA and IFN-α induced a comparative up-regulation IFN1 genes compared to unstimulated control (MX1 (p<0.0001, p<0.0001); RSAD2 (p=0.1508, p=0.001)). Both TLR-9 antagonist and NAC were able to down-regulate IFN type 1 genes after 24hr of oxmtDNA stimulation, suggesting that both could translate to therapeutic targets (TLR-9 (MX1, p=0.0001; RSAD2, p=0.0374); NAC (MX1, p<0.0001; RSAD2, p=0.00014)). Conclusion: Here, we show that dysregulated mitochondrial biology in JDM CD14+ monocytes is associated with increased oxidised mitochondria DNA (oxmtDNA) and which amplifies the interferon type 1 signature which characterises JDM, which represents a therapeutically targetable mechanism in JDM and potentially other IFN type 1-driven autoimmune diseases. Patient Consent: Yes, I received consent Disclosure of Interest : M. Wilkinson Grant / Research Support with: CureJM, NIHR GOSH BRC fellowship grants, non-renumerated collaboration with Novartis, D. Moulding: None declared, T. McDonnell: None declared, M. Orford: None declared, C. Wincup: None declared, J. Ting: None declared, G. Otto: None declared, R. Restuadi: None declared, D. Kelberman: None declared, S. Castellano: None declared, S. Eaton: None declared, C. Deakin: None declared, E. Rosser: None declared, L. Wedderburn Grant / Research Support with: NIHR (Senior Investigator award and the NIHR GOSH BRC), Myositis UK, Versus Arthritis and Great Ormond Street Childrens Charity. This research was supported by the NIHR Biomedical Research Centre at Great Ormond Street Hospital for Children NHS Foundation Trust (GOSH). The JDCBS is also supported by grants from Cure JM, Versus Arthritis (21593, 21552), Great Ormond Street Childrens Charity (W1143), Myositis UK, the NIHR GOSH BRC and the Remission Charity. Non-renumerated collaboration with Novartis
S. R. Veldkamp 1 , M. Reugebrink 1 , B. Lerkvaleekul 1,2 , E. P. A. H. Hoppenreijs 3 , S. Kamphuis 4 , W. Armbrust 5 , J. M. van den Berg 6 , P. C. E. Hissink Muller 7 , J. Wienke 1 , M. H. A. Jansen 2 , A. van Royen-Kerkhof 2 , F. van Wijk 1 1 Center for Translational Immunology, University Medical Center Utrecht, 2Utrecht, 3 Department of Paediatrics, Paediatric Rheumatology, Amalia Children’s Hospital, Radboud University Medical Centre Nijmegen, Nijmegen, 4 Paediatric Rheumatology, Sophia Children’s Hospital, Erasmus University Medical Centre, Rotterdam, 5 Department of Pediatric Rheumatology and Immunology, Beatrix Children’s Hospital, University Medical Center Groningen, Groningen, 6 Department of Pediatric Immunology, Rheumatology and Infectious Diseases, Emma Children’s Hospital, Amsterdam University Medical Centers, Amsterdam, 7 Department of Paediatric Rheumatology, Leiden University Medical Centre, Leiden, Netherlands Correspondence: S. R. Veldkamp Introduction: A dysregulated interferon (IFN) pathway is an important hallmark of JDM pathogenesis. Siglec-1, a macrophage/monocyte-restricted surface marker, was recently identified as a novel type I IFN-related activation marker in patients with JDM that correlated with clinical disease activity and could predict treatment response. Objectives: Our aim was to develop an in vitro model to study the inhibition of IFN-mediated responses with Siglec-1 as a read-out. Methods: PBMCs from healthy donors were stimulated in vitro for 0, 6 and 18 h with various TLR agonists (TLR-3, -4, -7, -9) or cytokines (IFNα/β/γ, TNFα, IL-1β). Pre-incubation for 1 h with various concentrations of JAK-inhibitors ruxolitinib (JAK1/2), baricitinib (JAK1/2), tofacitinib (JAK1/3), or filgotinib (JAK1), a TYK2-inhibitor (deucravacitinib) or an anti-IFNα/βR2 blocking antibody was performed to study their inhibitory effect on Siglec-1 induction. Furthermore, PBMCs were treated for 18 h with plasma of JDM patients or healthy donors (20% v/v), with or without anti-IFNα/βR2 blocking antibody. In all experiments, Siglec-1 expression was measured in CD14 + PBMCs by flow cytometry. Results: Siglec-1 expression was induced after 18 h by IFNα, IFNβ, and agonists of TLR-7 (imiquimod), TLR-9 (Class A CPG ODNs), and, most potently, TLR-3 (poly I:C). Induction by all these stimuli could be completely inhibited by IFNα/β receptor blockade. Pre-incubation with 1 μM ruxolitinib, baricitinib, or tofacitinib, or 10 μM filgotinib fully prevented induction of Siglec-1 by poly I:C. For full inhibition of IFNα-induced Siglec-1 expression, however, 10 μM of all JAK inhibitors was required. In contrast, deucravacitinib, a TYK2-inhibitor, was able to inhibit Siglec-1 induction by either poly I:C or IFNα at a concentration of 0.1 μM. Treating healthy donor PBMCs with JDM patient plasma induced Siglec-1 expression, while healthy donor plasma did not. The effect of the patient plasma could be inhibited by IFNα/β receptor blockade. Conclusion: Siglec-1 expression on monocytes was previously shown to be increased ex vivo in JDM patients and correlate with disease activity. These in vitro studies show that Siglec-1 expression can be induced in PBMCs by type I IFNs as well as certain TLR ligands, the latter of which mediate through type I IFN secretion. Siglec-1 induction can be inhibited to different extents by JAK/TYK inhibitors. Compared to the JAK-inhibitors, the TYK2-inhibitor deucravacitinib showed the strongest effect on inhibiting the IFN-mediated monocyte activation. This in vitro model has the potential to provide a biological basis for precision treatment of IFN-related systemic inflammatory diseases such as JDM and to study underlying mechanisms of derailed IFN responOuldali 1,2 , R. M. Dellepiane 3 , S. Torreggiani 3 , L. Mauri 4 , G. Beaujour 2 , C. Beyler 5 , M. Cucchetti 6 , C. Dumaine 2 , A. La Vecchia 7 , I. Melki 2 , R. Stracquadaino 7 , C. Vinit 2 , R. Cimaz 8 , U. Meinzer 1,2 1 Université Paris Cité, Center of Reserach on Inflammation, Inserm 1149, 2 National Reference Centre for Rare Paediatric Inflammatory Rheumatisms and Systemic Autoimmune diseases RAISE, Hôpital Robert Debré, APHP, Paris, Paris, France, 3 Pediatric Intermediate Care Unit, 4 Department of Paediatric Cardiology, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy, 5 Department of Paediatric Cardiology, Hôpital Robert Debré, APHP, Paris, Paris, France, 6 Department of Clinical Sciences and Community Health, Research Center for Adult and Pediatric Rheumatic Diseases, University of Milan, 7 University of Milan, 8Milan, Italy Correspondence: U. Meinzer Introduction: Early identification of high-risk patients is essential to stratify treatment algorithms of Kawasaki disease (KD) and to appropriately select patients at risk for complicated disease who would benefit from intensified first-line treatment. Several scores have been developed and validated in Asian populations but have shown low sensitivity in predicting intravenous immunoglobulin (IVIG) resistance in non-Asian populations. Objectives: We sought methods to predict the need for secondary treatment after initial IVIG in non-Asian populations. Methods: We conducted a retrospective, multicenter study including consecutive patients with KD admitted to two tertiary pediatric hospitals in France and Italy from 2005 to 2019. We evaluated the performance of the Kawanet-score and compared it with the performances of initial echocardiography findings, and of a newly proposed score combining the Kawanet-score and initial echocardiography findings. For each score, we assessed the AUC, sensitivity and specificity for predicting the need for second-line treatment. Results: We included 363 children with KD, 186 from France and 177 from Italy, of whom 57 (16%) required second-line therapy after the first IVIG dose. The Kawanet score, coronary artery dilation or aneurysm with maximal Z-score ≥2.0 at baseline, and abnormal initial echocardiography had a sensitivity of 43%, 55% and 65% and a specificity of 73%, 78%, 73%, respectively, for predicting the need for second-line treatment. The Kawanet-score was significantly improved by combining it with initial echocardiography findings. The best predictive performance (Sensitivity 76%, Specificity 54%) was obtained by combining the Kawanet-score with abnormal initial echocardiography, defined by the presence of either coronary artery maximal Z-score ≥2.0, pericarditis, myocarditis and/or ventricular dysfunction. This score predicted the need for second-line treatment in European, African/Afro-Caribbean and Asian ethnicity with a sensitivity of 80%, 65% and 100%, respectively, and a specificity of 56%, 51% and 61%, respectively. Conclusion: Our study proposes a score that we named the Kawanet-echo score, which allows early identification of children with KD who require a second-line treatment in multi-ethnic populations in Europe. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
H. D. Nguyen 1 , C. Papadopoulou 2 , D. Cancemi 1 , L. R. Wedderburn 1,2,3 , S. L. Tansley 4,5 on behalf of on behalf of the UK Juvenile Dermatomyositis Cohort and Biomarker Study 1 UCL Great Ormond Street Institute of Child Health, 2 NIHR Biomedical Research Centre at Great Ormond Street Hospital for Children, 3 Centre for Adolescent Rheumatology Versus Arthritis at UCL, UCLH and GOSH, London, 4 Royal United Hospitals, 5 The University of Bath, Bath, United Kingdom Correspondence: H. D. Nguyen Introduction: Juvenile Dermatomyositis (JDM) is a rare chronic autoimmune disease that causes proximal muscle weakness and skin rash in children and adolescents. Myositis specific and associated autoantibodies (MSA and MAA) are important prognostic biomarkers for JDM, yet the screening process of MSA and MAA is not standardised across healthcare centres, raising concerns about reliability or inter assay validity for this important prognostic tool. Although immunoprecipitation is considered the reference standard method to detect relevant autoantibodies, most autoantibody-testing laboratories use blotting-based immunoassays, for reasons of practicality and cost. A recent study suggested that immunoblot can be limited at detecting certain clinically important MSA subtypes 1 . Objectives: To compare relevant autoantibody frequencies detected by immunoprecipitation (IP) with autoantibody frequencies detected in immunoblots, and to determine the different levels of sensitivity of these techniques in detecting different myositis relevant autoantibody subtypes, in the UK Juvenile Dermatomyositis Cohort and Biomarker Study (JDCBS). Methods: A total of 472 JDM patients recruited to JDCBS were included. MSA/MAA status of each patient was determined by radio-labelled protein immunoprecipitation at the University of Bath (383 patients) or by immunoblot assays provided via the patients’ centre of care (89 patients). Results: The female distribution was 70.9% in immunoprecipitation cohort and 72.7% in immunoblot cohort. Regarding ethnicity, most patients in both cohorts identified as White: 77.1% of immunoprecipitation cohort and 65.9% of immunoblot cohort. Immunoprecipitation and immunoblot methods detected a myositis relevant autoantibody in 225 of 383 (58%) and 47 of 89 (53%) of samples respectively. The frequency of autoantibodies detected however, varied between the two cohorts. Anti-TIF1γ was identified in 70 patients (18.1%) in the immunoprecipitation cohort, followed by anti-NXP2 in 61 (15.8%), and anti-MDA5 in 23 (5.9%) patients. In contrast, in the immunoblot cohort, anti-MDA5 was the most prevalent autoantibody found in 15 (15.5%) patients, followed by anti-TIF1γ in 11 (11.3%), and anti-NXP2 in 9 (9.3%) of patients. Excluding anti-Ro52, which is not detected by immunoprecipitation, the identification of more than one autoantibody in a single patient was rare in both groups. Detection of more than one autoantibody was more prevalent in the immunoblot cohort (2.2%) versus immunoprecipitation (0.5%). Anti-Ro52 was present in 8 (9%) patients in the immunoblot cohort, and most commonly occurred in conjunction with anti-MDA5 (7 patients (7.9%)). Conclusion: The differing prevalence of autoantibodies in our cohort when analysed by immunoprecipitation and immunoblot raises concerns regarding the sensitivity and specificity of immunoblot to detect key autoantibodies relevant to patients with JDM. Poor sensitivity for immunoblot to detect anti-TIF1γ, in keeping with our data, has previously been reported but this is the first report of a potential reduction in sensitivity for anti-NXP2. The titre of anti-MDA5 has been shown to reduce with treatment 2 . The increase in prevalence of anti-MDA5 in the immunoblot cohort could relate to the analysis of earlier pre-treatment samples in this group, rather than false positive results. Further work is required to understand the reliability of immunoblot to detect myositis autoantibodies in JDM. References: 1. Tansley SL, Li D, Betteridge ZE, McHugh NJ. Rheumatology (Oxford). 59(8), 2109-2114 (2020). 2. Sato S, Kuwana M, Fujita T, Suzuki Y. Mod Rheumatol. 23, 496–502 (2012). Patient Consent: Yes, I received consent Disclosure of Interest : H. Nguyen Grant / Research Support with: NIHR Biomedical Research Centre at GOSH, C. Papadopoulou Grant / Research Support with: NIHR Biomedical Research Centre at GOSH, D. Cancemi Grant / Research Support with: NIHR Biomedical Research Centre at GOSH, L. Wedderburn Shareholder with: non-remunerated collaboration with Novartis, Grant / Research Support with: NIHR Biomedical Research Centre at GOSH; The JDCBS is supported by Cure JM, GOSCC, Myositis Remission, and the NIHR, S. Tansley: None declared
R. Ebrahim 1 , M. Al-Obaidi 2 , C. Papadopoulou 2 1 University College London, 2 Great Ormond Street Hospital, London, United Kingdom Correspondence: R. Ebrahim Introduction: Juvenile Dermatomyositis (JDM) is a rare childhood inflammatory disease affecting skin and muscle usually treated with corticosteroids alongside adjunctive therapies including intravenous immunoglobulins (IVIG). While typically dosed at 2g/kg on the recommendation of the Childhood Arthritis and Rheumatology Research Alliance (CARRA), national shortages in 2019 have meant JDM patients now receive a dose of 1g/kg IVIG. Objectives: This study aims to evaluate differences in remission odds, disease activity improvement and reduction of concomitant medication use in JDM patients receiving either dose. Methods: Data was collected from 48 JDM patients receiving IVIG for at least 6 months seen at London’s Great Ormond Street Hospital (GOSH) and part of the UK Juvenile Dermatomyositis Cohort and Biomarker Study (JDCBS). The primary outcomes were odds of remission at 0, 6 and 12 months post-IVIG initiation while disease improvement and reduction of concomitant medication were secondary outcomes measured at the same timepoints. Remission was defined using the Paediatric Rheumatology International Trials Organisation (PRINTO) criteria by meeting at least three of the following: CK ≤150U/I, CMAS >50/52, MMT8 >78/80, PGVAS ≤0.2/10. Logistic regression analysis was used to demonstrate effect of dose on remission odds while Friedman’s ANOVA and McNemar’s test measured disease activity improvement and reduction of medication. Results: Of the 48 patients, 41 were taking 2g/kg IVIG while only 7 were taking 1g/kg IVIG. There was no significant difference in remission odds after 6 (p=0.957) and 12 months (p=0.894) between IVIG doses after adjusting for confounders including age, sex, ethnicity, disease onset age (years) IVIG start age (years) time starting IVIG post diagnosis (weeks) Myositis Specific Antibodies (MSA) presence, MSA subtype, and treatment received. Improvements were noted at 6 and 12 months for both groups in skin, muscle and global disease activity, the most notable of which were significant improvements in the modified skin disease activity score in both the 2g/kg group (χ2=10.150, p=0.006) and 1g/kg group (χ2=7.176, p=0.028). There was a significant reduction in corticosteroid use after 12 months (p=0.035) but no significant difference between IVIG doses after 6 months (p=0.241) and 12 months (p=0.253). Conclusion: This study is the first to describe the effects of different IVIG doses in a large cohort of JDM patients and found that there is no significant difference in treatment outcomes and effect between JDM patients taking 1g/kg IVIG and 2g/kg IVIG. The results of this study further confirmed findings in the wider literature surrounding IVIG treatment in JDM with many patients achieving remission and improvement in muscle, skin and systemic disease. Meanwhile, the overall result of this study has important financial and clinical implications to reduce treatment cost and patients’ hospitalisation and thus presents an attractive proposition to healthcare providers to change their dose from 2g/kg IVIG to 1g/kg IVIG. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
M. J. Wahadat 1,2 , H. Qi 3 , C. van Helden-Meeuwsen 2 , E. Huijser 2 , L. van den Berg 1 , J. Göpfert 4 , M. Verkaaik 1 , M. Schreurs 2 , S. Kamphuis 1 , M. Versnel 2 1 Department of Paediatric Rheumatology, Sophia Children’s hospital, Erasmus University Medical Center, 2 Department of Immunology, 3 Department of Biostatistics, Erasmus University Medical Center, Rotterdam, Netherlands, 4 Department of Applied, Biomarkers and Immunoassays, NMI Natural and Medical Sciences Institute at the University of Tübingen, Reutlingen, Germany Correspondence: M. J. Wahadat Introduction: Type-I interferon (IFN-I) pathway activation plays a pivotal role in the pathogenesis of SLE and has been proposed as biomarker for disease activity. IFN-I pathway activation can be measured by determining the expression of IFN-I stimulated genes or a so-called IFN signature. Ultrasensitive single-molecule array (Simoa) technology enables measurement of IFN protein concentrations at subfemtomolar concentrations. Parallel use of these measuring methods in longitudinal cohorts of childhood-onset SLE (cSLE) patients in relation to disease activity could help in translating the most relevant technique for use in clinical practice. Objectives: To determine the association of serum IFNa2 levels and whole blood IFN-I stimulated gene expression with disease activity and study their potential to mark specific disease activity states in a longitudinal cohort of cSLE patients. Methods: Serum IFNa2 levels were measured in 338 samples from 48 cSLE patients and 67 healthy controls using an IFN-a2 Simoa assay (Quanterix) on an HD-X analyser. A 5 gene IFN-I signature was measured by RT-PCR in paired whole blood samples. Disease activity was assessed by the clinical SELENA-SLEDAI (cSLEDAI) and BILAG-2004. Low disease activity was defined by the Low Lupus Disease Activity State (LLDAS) and flares were characterized by the SELENA-SLEDAI flare index. Analysis was performed using linear mixed effect models. Results: A clear positive correlation was present between serum IFNa2 levels and the IFN-I gene signature (r=0.78, p<0.0001). Serum IFNa2 levels and the IFN-I gene signature showed the same significant negative trend in the first three years after diagnosis. In this timeframe, mean baseline serum IFNa2 levels decreased with 55.1% (delta 172 fg/mL, p<0.001) to a mean value of 164 fg/mL, which was below the calculated threshold of 219.4 fg/mL. In the linear mixed model, serum IFNa2 levels were significantly associated with both the cSLEDAI and the BILAG-2004 (p<0.001 and p<0.01), while the IFN-I gene signature did not show this association (p=0.35 and p=0.23). Moreover, 69.7% of the time points in LLDAS had a serum IFNa2 level under the calculated threshold, while only 31.9% of the time points in LLDAS reached an IFN-I gene signature below the calculated threshold. Both techniques were equally capable of marking disease flares (79.2% above threshold vs 87.5% above threshold). Conclusion: Serum IFNa2 levels measured by Simoa, but not the type-I IFN gene signature, are associated with disease activity scores and characterize disease activity states in cSLE patients. Hence, this technique has the potential to be implemented in clinical practice. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
A. Charras 1 , S. Haldenby 2 , E. Smith 1,3 , C. Roberts 1 , M. Beresford 1,3 , C. Hedrich 1,3 on behalf of On behalf of the UK JSLE Cohort Study 1 Department of Women’s and Children’s Health, Institute of Life Course and Medical Sciences, University of Liverpool, 2 Institute of Infection, Veterinary & Ecological Sciences, University of Liverpool, Centre for Genomic Research, 3 Alder Hey Children’s NHS Foundation Trust Hospital, Department of Paediatric Rheumatology, Liverpool, United Kingdom Correspondence: A. Charras Introduction: Juvenile-onset systemic lupus erythematosus (jSLE) affects 15-20% of lupus patients. Clinical heterogeneity across ethnicities, age groups and individual patients suggests a variable pathophysiology. Objectives: The aim of this study was to identify damaging high-penetrance mutations in genes associated with SLE or SLE-like diseases in a large national cohort (UK JSLE Cohort Study and Repository) and to compare demographic, clinical and laboratory characteristics in sub-cohorts of patients with “genetic” SLE versus other SLE patients. Methods: Based on a 2018 literature search targeting known Mendelian disease causes and risk alleles (using PubMed, OMIM and Ensembl), target enrichment and next generation sequencing was performed in 348 patients. Using in silico screening (SnpEFF) for ‘high impact’ variants with low minor allele frequencies (MAF <5%) followed by inheritance pattern analysis (using OMIM, ClinVar and SNPnexus), patients with predicted ‘high impact’ variants and autosomal dominant (AD) inheritance were included. Furthermore, patients with variants following autosomal recessive inheritance were included, if ≥1 additional “high impact” mutations were present on the second allele (compound heterozygote) or if ≥1 other SLE-associated gene in the same immunological pathway had a “high impact” mutation. Results were integrated with demographic, clinical and treatment-related datasets. Results: Variants predicted to be damaging were identified in approximately 3.5% of jSLE patients. Affected genes and associated pathways are summarised in the table. Compared to the rest of the cohort, patients with damaging gene variants (“genetic” SLE”) were younger and more frequently of African/Caribbean ancestry. Patients with “genetic” SLE had less overall organ involvement and associated damage, but neuropsychiatric involvement developed over time. Less aggressive first-line treatment was chosen in patients with “genetic” SLE, but more second- and third-line agents were used. “Genetic” SLE is associated with anti-dsDNA antibody positivity at diagnosis, and reduced ANA, anti-LA and anti-Sm antibody positivity at the last visit. Conclusion: “Genetic” jSLE associates with younger age at disease-onset, reduced persistent antibody positivity, less organ involvement, fewer disease flares and less damage, but the development of neuropsychiatric disease over time. Routine sequencing may allow patient stratification, risk assessment and targeted treatment, thereby increasing efficacy and reducing toxicity. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
P. Morán Álvarez 1 , C. Passarelli 2 , V. Messia 1 , M. Pardeo 1 , E. Marasco 1 , A. Insalaco 1 , F. De Benedetti 1 , C. Bracaglia 1 1 Ospedale Pediatrico Bambino Gesù, Rome, Italy, 2 Pediatric Rheumatology, Ospedale Pediatrico Bambino Gesù, Rome, Italy Correspondence: P. Morán Álvarez Introduction: Systemic lupus erythematosus (SLE) is a chronic systemic autoimmune disease which leads to inflammation and organ damage caused by immune complex deposition. Classically, childhood SLE (cSLE) has been considered as a polygenic autoimmune disease; however, a pediatric monogenic lupus-like phenotype is emerging due to the recent recognition of several related novel high-penetrance gene variants in the last years. This fact associated to the high degree of concordance among monozygotic twins, supports the importance of genetic background in the cSLE pathogenesis. Objectives: To identify the presence of variants in gene related to monogenic lupus and their relationship with clinical manifestations in cSLE or lupus-like phenotype. Methods: A descriptive, observational, cross-sectional study was carried out in children with a diagnosis of cSLE or with lupus-like. The genetic analysis (Sanger/Clinical Exome Sequencing) was performed from isolated DNA obtained from blood sample. Results: Forty-two children were included in the study. The genetic analysis detected at least one variant in 11 (26.1%) children, 5 (45.4%) with cSLE ( ADAR, TNFAIP3, RNASEH2B, SHOC2, IFIH1) and 6 (54.5%) with lupus-like phenotype ( TREX, DNASE1,RNASEH2B, C1S, TLR7, STAT5A, TNFRSF13B ). Of those who carry a genetic variant, the median age at disease onset was 11 years (range: 2-16) and 72.7% were female. Most of them were Caucasians (72.7%). Four (36.3%) and 3 (27.2%) out of 11 patients had a positive family history and/or a personal history for autoimmune diseases, respectively. Regarding the clinical manifestations at onset, musculoskeletal was the most frequent (8 patients, 72.7%), followed by hematological (6 patients, 54.5%), cutaneous (6 patients, 54.5%), constitutional with fever (5 patients, 45.45%), neurological (4 patients, 36.3%), renal (3 patients, 27.2%), cardiac (3 patients, 27.2%) and pulmonary (2 patients, 18.1%) manifestations. Related to immunological parameters, 10 (90.9%) were ANA positive, 5 (45.4%) anti-dsDNA, 4 (36.3%) ENA and 2 (18.1%) were antiphospholipid antibodies and lupus anticoagulant positive. Both C3 and C4 were low in 5 (45.4%) children and isolated C3 levels were low in 4 (36.3%) patients. Among the variants, we found that only two patients who carry a TREX variant showed normal C3 and C4 levels; one of them presented with lupus pernio as reported in literature. The same RNASEH2B (c.868G>A) variant was identified in two siblings with similar phenotype. The patient who carried the SHOC2 variant presented polyarthritis and serositis, while the patient with the TNFRSF13B variant onset with a glomerulonephritis. Those manifestations have already been described related to these gene variants. Conclusion: Around 25% pediatric patients with cSLE or lupus-like phenotype in our cohort showed at least one variant in gene related to monogenic-lupus and some of them had a phenotype similar to those already described. The evidence of these variants may suggest the genetics potential contribution to the cSLE pathogenesis. Further studies in larger cohorts are necessary to confirm these data. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
J. Peng 1,2 , G. Robinson 1,2 , S. Ardoin 3 , L. Schanberg 4 , E. Jury 1 , C. Ciurtin 2 1 Centre for Rheumatology Research, Division of Medicine, 2 Centre for Adolescent Rheumatology Versus Arthritis, Division of Medicine, University College London, London, United Kingdom, 3 Department of Medicine, Nationwide Children’s Hospital, Ohio State University, Columbus, 4 Duke Clinical Research Institute, Duke University School of Medicine, Durham, United States Correspondence: J. Peng Introduction: The risk of developing CVD through atherosclerosis in juvenile-onset systemic lupus erythematosus (JSLE) patients is significantly increased. Objectives: This study aimed to stratify and characterize JSLE patients at elevated CVD-risk using patient/disease-related factors and metabolomic data from patients recruited to the APPLE (Atherosclerosis Prevention in Paediatric Lupus Erythematosus) clinical trial, designed to assess atherosclerosis development. Methods: Unsupervised hierarchical clustering was performed to stratify patients by arterial intima-media thickness (IMT) measurements at baseline (N=151) and carotid (c)IMT progression over 36 months (placebo arm only, N=60). Baseline metabolomic profiles (~250 serum metabolites) were compared between clusters using conventional statistics, univariate logistic regression, sparse Partial Least-Squares Discriminant Analysis (sPLS-DA) and random forest classifier. An independent cohort (UCL-JSLE cohort, N=89) with matching metabolomics, immunophenotyping and proteomics, was used to validate the discovered CVD risk-related signatures from the APPLE cohort. Results: Baseline IMT stratification identified 3 clusters with high, intermediate, and low baseline IMT measurements and progression trajectories over 36 months, each having distinct racial/BMI/household education/income characteristics. Analysis of cIMT progression over 36 months identified 2 patient groups with high and low IMT progression. Unique metabolomic profiles differentiated high and low cIMT progression groups, with good discriminatory ability (0.81 AUC in ROC analysis) using the top 6 metabolites (Total cholesterol esters, Total cholesterol, Phospholipids in small LDL particles, Total cholesterol in small LDL particles, Free cholesterol in medium LDL particles and Total lipids in small LDL particles) selected from the analysis. cIMT progression over 36 months in the placebo group correlated positively with baseline disease activity (SLEDAI), damage score (SLICC), white blood cell count, serum complement C3, blood pressure (both systolic and diastolic) and BMI. Metabolomics signatures discovered from the APPLE cohort were applied to stratify JSLE patients in the validation cohort (UCL-JSLE), where 3 groups were identified with distinct metabolomics profiles indicating JSLE patients with high risk (N= 20), intermediate risk (N= 43) and low risk (N= 26) CVD-risk. Significant differences were observed in the frequency of classical monocytes (p=0.015) and nonclassical monocytes (p=0.005) when comparing high and low CVD risk group in the UCL-JSLE cohort. Conclusion: Complex analysis of IMT patterns and progression in the APPLE trial cohort identified novel key determinants that could guide further research for CVD-risk stratification in JSLE. Disclosure of Interest : None declared
O19. Outcomes of patients with juvenile idiopathic arthritis following biologic switching in the childhood arthritis and rheumatology research alliance registry O20. Orofacial manifestations of juvenile idiopathic arthritis from diagnosis to adult care transition: a population-based, cohort study O21. Joint-specific responses to tofacitinib in JIA: a post HOC analysis of the open-label period of a phase 3 clinical trial O22. Distinguishing Kawasaki disease from other febrile conditions in a US cohort with the Kawasaki disease gene expression profiling (KIDS-GEP) classifier O23. Challenges for Ukrainian patients with rheumatic diseases: how to survive O24. Disease activity in juvenile idiopathic arthritis from childhood to adulthood in the nordic JIA cohort O25. Anti-TNF agents impair seroprotection in paediatric patients with juvenile idiopathic arthritis and inflammatory bowel disease vaccinated against meningococcal ACWY: the 24 months post vaccination follow-up data O26. Factors affecting the scoring of physician global assessment in JIA – survey results from PR-COIN and PRINTO 27. Investigation the effects of Tai Chi in children with juvenil idiopathic arthritis: a randomized controlled trial M. Mannion 1 , S. Amin 2 , S. Balevic 3 , C. Correll 4 , T. Beukelman 1 , for the CARRA Registry Investigators 2 1 Pediatric Rheumatology, University of Alabama at Birmingham, Birmingham, 2 Childhood Arthritis and Rheumatology Research Alliance, Washington, DC, 3 Duke Clinical Research Institute, Duke University, Durham, NC, 4 Division of Rheumatology, Department of Pediatrics, University of Minnesota, Minneapolis, MN, United States Correspondence: M. Mannion Introduction: Tumor necrosis factor inhibitors (TNFi) are the most commonly used first biologics to treat juvenile idiopathic arthritis (JIA), but it is unknown what subsequent biologic medications are most effective after failure of an initial TNFi. Objectives: We compared the effectiveness of using a 2 nd TNFi versus a non-TNFi following failure of a 1 st TNFi in routine clinical practice. Methods: We included individuals with a primary diagnosis of non-systemic polyarticular course JIA who received a TNFi as their 1 st biologic medication and started a 2 nd biologic (index date) within 90 days of stopping the 1 st TNFi on or after enrollment in the Childhood Arthritis and Rheumatology Research Alliance Registry. Included patients had a 6 month follow up visit (+/- 3 months) after the index date and prior to February 29, 2020. Exclusion criteria included active uveitis on the index date and no Registry visit within 4 weeks of the index date. The primary outcome was inactive disease (ID) and minimal disease activity (MiDA) based upon the clinical juvenile arthritis disease activity score (cJADAS; cJADAS ID<2.5, cJADAS MiDA<5) at the 6 month visit. We used fully conditional specification multiple imputation to account for missing data. Propensity scores (PS) for likelihood of being in the 2 nd TNFi group were calculated by logistic regression (LR) with covariates from index date including active and limited joint count, physician and parent global assessments, childhood health assessment questionnaire, pain, erythrocyte sedimentation rate, time from diagnosis to index date, time from diagnosis to start of 1 st TNFi, sex, JIA category, methotrexate (MTX) use, glucocorticoid use, cJADAS, 1 st TNFi, index date calendar year, uveitis history. We used LR to compare outcomes following switch to a 2 nd TNFi versus non-TNFi between index date and 6 month follow up unadjusted and adjusted for PS quintile. Results: 216 individuals were included in the study, 84% receiving etanercept initially and most patients stopping for ineffectiveness (74%). 183 (85%) started a 2 nd TNFi and 33 (15%) started a non-TNFi. Adalimumab was the most common 2 nd biologic (71% overall, 84% of 2 nd TNFi) and tocilizumab was the most common non-TNFi 2 nd biologic (9% overall, 58% of non-TNFi). On the index date, 56% of patients had used MTX (55% 2 nd TNFi and 58% non-TNFi) and 12% of patients used glucocorticoids (11% 2 nd TNFi and 15% non-TNFi). There was no difference in meeting cJADAS ID or MiDA criteria by treatment group even after adjusting for PS quintile. Conclusion: Among polyarticular course JIA patients in a large North American registry following failure of a 1 st TNFi, switch to a 2 nd TNFi was more common than switch to a non-TNFi. There were no differences between those starting a 2 nd TNFi or non-TNFi in achieving cJADAS inactive disease or MiDA after 6 months. More research is necessary to determine which patients would benefit from change in treatment mechanismS. Amin: None declared, S. Balevic Grant / Research Support with: National Institutes of Health, US Food and Drug Administration, Patient-Centered Outcomes Research Institute, Rheumatology Research Foundation Scientist Development Award, Childhood Arthritis and Rheumatology Research Alliance, Purdue Pharma, Consultant with: UCB, C. Correll: None declared, T. Beukelman Consultant with: Novartis, UCB, ,. for the CARRA Registry Investigators: None declared
M. Glerup 1 , A. Tagkli 2 , A. Küseler 2 , A. Estmann 3 , C. Verna 4 , A. E. Bilgrau 5 , S. E. Nørholt 6,7 , T. Herlin 8 , T. K. Pedersen 2,6 , P. Stoustrup 2 1 MG and AT Contributed Equally to the Study and Shares the First Authorship. Department of Paediatrics and Adolescent Medicine, Aarhus University Hospital, 2 Section of Orthodontics, Aarhus University , Aarhus, 3 Department of Pediatrics, Odense University Hospital, Odense, Denmark, 4 Department of Pediatric Oral Health and Orthodontics and UZB University Center for Dental Medicine Basel, University of Basel, Basel, Switzerland, 5 Department of Mathematical Sciences, Aalborg University, Aalborg, 6 Department of Oral and Maxillofacial Surgery , Aarhus University Hospital , 7 Department of Dentistry and Oral Health, Aarhus University, 8M. Glerup Introduction: Juvenile idiopathic arthritis (JIA) involvement of the temporomandibular joint (TMJ) may cause orofacial symptoms and dysfunction that can persist into adulthood. Contemporary, population-based estimates for the prevalence of JIA-related orofacial manifestations are unavailable. Such estimates are important for patient counseling, critical for clinical decision-making, and planning of the transition to adult rheumatology care. Furthermore, much attention has been devoted to the identification of risk factors of poor outcome in JIA but knowledge about risk factors exclusive of TMJ involvement is limited. Objectives: 1) To estimate the cumulative incidences of orofacial conditions followed by temporomandibular joint (TMJ) arthritis in juvenile idiopathic arthritis (JIA) from diagnosis in childhood until transition to adult care. 2) To identify features in JIA associated with involvement of the temporomandibular joint (TMJ). Methods: A population-based cohort analysis was conducted, involving longitudinal data on orofacial health from 2000 to 2018. Regardless of TMJ status, the patients were referred to the Regional Craniofacial Clinic of Western Denmark for routine orofacial examinations according to a standardized protocol. All patients received tax-funded health care. Data collection included information about disease-specific background information, TMJ involvement, JIA-induced dentofacial deformity, and orofacial symptoms and dysfunction. The association between JIA disease parameters and the diagnosis of TMJ involvement at transition to adult care (16-18 years of age) was assessed using multivariate regression statistics. Results: 613 patients were followed with a mean clinical TMJ observation time of 4.0 years (range: 0-15.3 years; 0.25/0.75 percentiles: 0/6.7 years). From JIA onset to transition to adult care, the cumulative incidence of patients with JIA involvement of the TMJ was 30.0% during the 18 years of observation. Furthermore, 20.6% of the cohort had developed arthritis-induced dentofacial deformity. A substantial proportion of the cohort experienced several events of orofacial symptoms (23.5%) and dentofacial dysfunction (52%). In a multivariate analysis, the following baseline variables were significantly adversely associated with TMJ involvement: young age at diagnosis (<9 years), female gender, and ANA positivity. However, HLA-B27 positivity was associated with a lower risk of TMJ involvement. Of the orofacial symptoms and dysfunctions during the disease course TMJ pain during function, reduced translation, asymmetric mandibular mouth opening, and crepitation were significantly associated with TMJ involvement. Conclusion: Orofacial signs and symptoms were frequent findings in children and adolescence with JIA. Involvement of the TMJ was seen in 30% of the cohort. 20.6% of the total cohort developed JIA-related dentofacial deformity before transition into adult care. TMJ involvement and dentofacial deformity were associated with JIA diagnosis at age younger than 9 years, ANA positivity, and several events of orofacial symptoms and TMJ dysfunction. This is the first population-based study in the biologic era to document these frequent orofacial complications in children with JIA. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
R. Micheroli 1 , D. J. Lovell 2 , A. Martini 3 , L. Stockert 4 , H. Jo 5 , K. Kwok 5 , A. Diehl 4 , T. Killeen 6 , C. Ospelt 1 , H. I. Brunner 2 , N. Ruperto 3 on behalf of on behalf of PRINTO/PRCSG 1 University Hospital Zürich, Zürich, Switzerland, 2 Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, United States, 3 IRCCS Istituto Giannina Gaslini, Genova, Italy, 4 Pfizer Inc, Collegeville, PA, 5 Pfizer Inc, New York, NY, United States, 6 Pfizer Ltd, Tadworth, United Kingdom Correspondence: T. Killeen Introduction: Joint involvement in juvenile idiopathic arthritis (JIA) varies substantially. 1 Tofacitinib is an oral Janus kinase inhibitor for the treatment of active polyarticular-course JIA and active juvenile psoriatic arthritis. The effect of tofacitinib on specific joints has not been well studied. Objectives: Explore joint-specific responses to tofacitinib in the open-label phase of a clinical trial in patients with JIA. Methods: This was a post hoc analysis of week (W)18 data from the open-label treatment phase of a Phase 3 clinical trial 2 (N=225) in children with JIA aged 2–<18 years, where patients received oral tofacitinib (weight-based doses; ≤5 mg twice daily); 65% received concomitant methotrexate. Tenderness was defined as pain on motion and/or tenderness. Paired joint pathology scores (PJPS; range: 0 [neither side swollen and/or tender] to 4 [both sides swollen and tender] for each pair of joints) and PJPS % change from baseline (%Δ) were calculated; data were available for 34 joint pairs. Small joints of the hands and feet were grouped; for each grouping, PJPS was calculated as the mean of the included paired joints’ PJPS, and %Δ was calculated using the assigned % value (-100–100% by 25% increments) based on a combination of baseline and post-baseline scoring, which was calculated as mean of the individual paired joints’ PJPS. Results: Baseline joint involvement and PJPS were greatest in the ankle, knee and wrist, followed by the metacarpophalangeals and proximal interphalangeals (Table). PJPS decreased in all joints by W4, continuing up to W18. Greatest %Δ PJPS responses were observed in the ankle, knee and wrist; with metatarsophalangeals, toes and distal interphalangeals responding least (Table). Conclusion: Joint-specific responses to tofacitinib in JIA were strongest in the frequently involved ankle, knee and wrist, improving over time up to W18. This analysis was limited by its post hoc nature and lack of a placebo group. Trial registration identifying number: ClinicalTrials.gov (NCT02592434) References [1] Eng SWM, et al. PLOS Med 2019; 16: e1002750 [2] Ruperto N, et al. Lancet 2021; 10315: 1984-96 Acknowledgements: Study sponsored by Pfizer Inc. Medical writing support was provided by Karen Thompson, PhD, CMC Connect, and funded by Pfizer Inc. Patient Consent: Not applicable (there are no patient data) Disclosure of Interest : R. Micheroli Speaker Bureau with: AbbVie, Eli Lilly, Gilead Sciences and Pfizer Inc, D. J. Lovell Grant / Research Support with: Bristol-Myers Squibb, Janssen, Novartis, Pfizer Inc, Roche and UBC, Consultant with: AstraZeneca, Boehringer Ingelheim, GSK, Novartis, Pfizer Inc, Roche, Takeda and UBC, and DSMB chairperson for NIH, A. Martini: None declared, L. Stockert Shareholder with: Pfizer Inc, Employee with: Pfizer Inc, H. Jo Shareholder with: Pfizer Inc, Employee with: Syneos Health, K. Kwok Shareholder with: Pfizer Inc, Employee with: Pfizer Inc, A. Diehl Shareholder with: Pfizer Inc, Employee with: Pfizer Inc, T. Killeen Shareholder with: Pfizer Inc, Employee with: Pfizer Ltd, C. Ospelt Grant / Research Support with: Novartis Foundation for Biomedical Research, H. I. Brunner Grant / Research Support with: Bristol-Myers Squibb, MedImmune, Novartis and Pfizer Inc, Consultant with: AbbVie, AstraZeneca/MedImmune, Bayer, Biocon, Boehringer Ingelheim, Bristol-Myers Squibb, Eli Lilly, Janssen, Novartis, Pfizer Inc, Roche and R Pharm, Employee with: Cincinnati Children’s Hospital Medical Center, Speaker Bureau with: GSK, Novartis and Roche, N. Ruperto Grant / Research Support with: Bristol-Myers Squibb, Eli Lilly, F. Hoffmann-La Roche, GSK, Janssen, Novartis, Pfizer Inc and Sobi, Consultant, Speaker Bureau with: Ablynx, AstraZeneca/MedImmune, Biogen, Boehringer Ingelheim, Bristol-Myers Squibb, Eli Lilly, EMD Serono, F. Hoffmann-La Roche, GSK, Janssen, Merck Sharp & Dohme, Novartis, Pfizer Inc, R-Pharm, Sanofi, Servier, Sinergie and Sobi
D. Van Keulen 1 , C. Shimizu 2 , V. J. Wright 3 , D. Habgood-Coote 3 , D. Huigh 1 , A. H. Tremoulet 2 , R. Kuiper 1 , C. J. Hoggart 3,4 , J. Rodriguez-Manzano 3 , J. A. Herberg 3 , M. Kaforou 3 , D. Tempel 1 , M. Levin 3 , J. C. Burns 2 1 SkylineDx, Rotterdam, Netherlands, 2 Department of Pediatrics, Rady Children’s Hospital and University of California San Diego, La Jolla, California, United States, 3 Department of Infectious Disease, Imperial College London, London, United Kingdom, 4 Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York City, New York, United States Correspondence: D. Van Keulen Introduction: Timely diagnosis of Kawasaki disease (KD) is challenging but may become more straightforward with the Kawasaki Disease Gene Expression Profiling (KiDs-GEP). Objectives: To compare the KiDs-GEP classifier score between KD patients and febrile controls in a US cohort. Methods: Biobanked whole blood RNA samples from 100 KD patients and 400 febrile controls who were diagnosed at Rady Children’s Hospital in San Diego between 2010 and 2019 were retrospectively collected. All patients were under 18 years of age and blood samples were obtained within the first 12 days of illness and prior to treatment with IVIG. RNA expression of the collected blood samples was measured with qRT-PCR and analyzed with the KiDs-GEP classifier. Results: The KD patients had a median age of 3.05 years and 56.0% were male, which was comparable to the febrile control group (median age of 3.30 months and 56.3% male). KD patients had a significantly higher KiDs-GEP classifier score (mean 25.6 [IQR 24.0-27.1] than the febrile control patients (mean 21.0 [IQR 19.4-23.0], p<1x10 -10 ). The lowest classifier score was observed for patients with viral infections (mean 20.4 [IQR 19.1-22.1]), making them easiest to distinguish from KD patients with the KiDs-GEP classifier. Conclusion: The KiDs-GEP classifier score was significantly higher in KD patients than in febrile control patients. These results are consistent with our previous study and indicate that the KiDs-GEP classifier may be a useful tool to discriminate KD patients from febrile control patients. Patient Consent: Yes, I received consent Disclosure of Interest : D. Van Keulen Shareholder with: Option holder of SkylineDx, Employee with: Employee of SkylineDx, C. Shimizu: None declared, V. Wright: None declared, D. Habgood-Coote: None declared, D. Huigh Shareholder with: Option holder of SkylineDx, Employee with: Employee of SkylineDx, A. Tremoulet: None declared, R. Kuiper Shareholder with: Option holder of SkylineDx, Employee with: Employee of SkylineDx, C. Hoggart: None declared, J. Rodriguez-Manzano: None declared, J. Herberg: None declared, M. Kaforou: None declared, D. Tempel Shareholder with: Option holder of SkylineDx, Employee with: Employee of SkylineDx, M. Levin: None declared, J. Burns: None declared
Y. Vyzhga National PIROGOV Memorial Medical University, Vinnytsya, Vinnitsya, Ukraine Correspondence: Y. Vyzhga Introduction: We used to know that patients with rheumatic diseases – is potentially vulnerable group not just due to the aggressive diseases course, but as well due to irregularity of the medical care, access to drugs in dependence to country of origin. Objectives: The aim of my study was to analyse the impact of war on access and quality of medical care for children with rheumatic diseases in Ukraine. Methods: The social networks started to be the separate platform for direct communication between patients and doctors, as well that’s a way to share correct and updated professional information with them. I supervise my medical blog for more than 1 year, and by now my audience is more than 13 thousand of subscribers, among them more than 1.5 thousand – patients with rheumatic diseases, both adults and children, their parents. At the end of March, in 1 month after the war started, I proposed my subscribers to complete questionnaire in goggle-form, that counted 15 questions highlighted current medical and social situation. Results of the questionaries were analysed with description statistical methods. Results: Had been analysed 278 unique completed questionaries from the patients with rheumatic diseases and their parents. Among the patients mainly were children diagnosed with JIA (79.5 %), adults with RA (15.8 %), patients with SLE (1.4 %), dermatomyositis (0.7 %) and autoinflammatory syndromes (2.6 %). Geography of the patients before the war counted 15 regions from 24 available in Ukraine. 52,5 % of the responders were forced to leave their usual place of residence due to the war, among them – 42.7 % went abroad. Among the countries that mainly accepted our patients were: Poland, Germany, Czech Republic, Slovak, Italy, Belgium, Great Britain, France, Ireland, Portugal. 67.7 % of the patients that had gone abroad, recieved medical care from paediatric rheumatologists, 17.7 % were able to get medical support by general practitioners at the country of temporary residence. Among the patients that stayed in Ukraine, 74 % were able to receive consultation of general practitioner, without access to rheumatologist, 11 % were consulted by rheumatologist online. 47.8 % of the responders admitted worsening of the disease status or flare that occurred within the last month. Majority of the patients – 81 % were able to continue their DMARD therapy at least partially, 15 % of the patients temporarily interrupted their therapy due to the lack of the access to treatment. 33 % of the responders received immune-biologic therapy of rheumatic disease before the war, that represents general trend through the country. 65 % of the patients were able to continue it, among them 38% continued abroad. Among the patients settled abroad, 34 % received medical care thanks to PRINTO contacts, 22 % with patient’s organizations and societies support, 15 % - were assisted by volunteers and others – by general practitioners’ admissions. The separate aspect of the questionnaire counted psychological condition of the patients with its evaluation in 10 points score. According to parental evaluation, just 8 % of responders were not influenced by the war, all of them are infants. Absolut result was 7±2 points, that confirms absolute negative impact. Conclusion: War makes unreversible influence on humanitarian life aspects and when we discuss problems of the patients with rheumatic disease, its difficult to predict final outcomes. But its possible to improve cooperation and networking between European centres, that will help our patient to win in every life causality. I want to thank our association, every doctor and nurse, every member of our society, patients’ organization, European citizens, that didn’t allow us and our patient to feel themselves alonV. Rypdal 1,2 on behalf of Nordic Study Group of Pediatric Rheumatology (NoSPeR), M. Glerup 3 , M. Rypdal 4 , E. D. Arnstad 5,6 , K. Aalto 7 , L. Berntson 8 , A. Fasth 9 , T. Herlin 10 , S. Nielsen 11 , M. Rygg 12,13 , E. Nordal 1,2 1 Dep.of Pediatrics, University Hospital of North Norway, 2 Dep.of Clinical Medicine, UIT the Arctic University of Norway, Tromsø, Norway, 3 Dep.of Pediatrics, Aarhus University Hospital, Aarhus, Denmark, 4 Dep.of Mathematics and Statistics, UIT the Arctic University of Norway, Tromsø, 5 Dep.of Pediatrics, Levanger Hospital, Nord-Trøndelag Hospital Trust, Levanger, 6 Dep.of Clinical and Molecular Medicine, NTNU, Trondheim, Norway, 7 Dep.of Pediatrics, New Children’s Hospital, Helsinki University Hospital, Helsinki, Finland, 8 Dep.of Women’s and Children’s Health, Uppsala University , Uppsala, 9 Dep.of Pediatrics, Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden, 10 Dep. of Pediatrics, Aarhus University Hospital, Aarhus, 11 Dep.of Pediatrics, Rigshospitalet Copenhagen University Hospital , Copenhagen, Denmark, 12 Dep.of Clinical and Molecular Medicine, NTNU , 13 Dep.of Pediatrics, St. Olavs Hospital, Trondheim, Norway Correspondence: V. Rypdal Introduction: Inactive disease (ID) is the primary goal of the treat-to-target (T2T) strategy in juvenile idiopathic arthritis (JIA). The Juvenile Arthritis Disease Activity Score (JADAS) has cut-off values for ID, low disease activity (LDA), moderate disease activity (MDA), and high disease activity (HDA) for oligo- and polyarticular disease courses. Few studies have assessed the disease activity trajectories in a population-based setting from childhood into adulthood. Objectives: This study aimed to evaluate JADAS10 through a disease course of 18 years, and to investigate if there are identifiable clusters of JADAS10 development over time among the Nordic patients with JIA. Methods: Patients with JIA (n=510) from centers participating in the Nordic JIA study were included at disease onset and followed prospectively. There were three major study visits: the baseline, the 8-year, and 18-year visit. The patients were divided into poly- and oligoarthritis groups if >4 joints or ≤4 joints were affected, respectively. Descriptive statistics and K-means clustering were used to present differences in disease activity states throughout the follow-up period. McNemar’s test was used to determine if there were statistically significant changes in disease activity states from baseline to the 18-year visit. Results: At baseline, JADAS10 was available in 219 patients with a median score of 5.0 (IQR 2.0-11.0). At the 8-year visit, JADAS10 was available in 240 patients with a median score of 1.2 (IQR 0-4.2), and at the 18-year visit 268 patients had a JADAS10 score of median 2.2 (IQR 0.8-6.0). From baseline to the 8-year visit the proportion of ID increased ( p <10 -4 , table), however, ID significantly decreased from the 8-year to the 18-year follow-up both for the oligo- and the polyarthritis groups ( p =0.004 and 0.03, respectively). In addition, there was a significant increase of HDA in the oligoarthritis group ( p =0.02). We identified three distinct JADAS10 patterns over time among 105 patients with complete JADAS10 information in the 3 major visits. Cluster 1: patients with high disease activity early in the course and improvement during the period. Cluster 2: patients with remaining low disease activity, and Cluster 3: patients with early low disease activity with worsening during the disease course. Conclusion: We found increasing disease activity on long term follow-up after initial improvement the first years after onset of JIA. We identified a subset of children with increasing disease activity over time that may need tighter follow-up with a T2T-strategy. The impact of different JADAS cut-offs for oligo- and polyarticular JIA should further be investigatedM. Ohm 1 , J. W. van Straalen 2 , M. Zijlstra 3 , A. J. Sellies 2 , G. C. De Joode-Smink 2 , J. F. Swart 2 , S. J. Vastert 2 , J. M. van Montfrans 2 , M. Bartels 4 , A. van Royen-Kerkhof 2 , J. G. Wildenbeest 5 , C. A. Lindemans 2,6 , V. M. Wolters 3 , R. A. Wennink 7 , J. H. de Boer 7 , M. W. Heijstek 8 , F. M. Verduyn Lunel 9 , G. Berbers 1 , N. M. Wulffraat 2 , M. H. Jansen 2 1 Centre for Infectious Disease Control, National Institute for Public Health and the Environment, Bilthoven, 2 Department of Pediatric Immunology and Rheumatology, 3 Department of Pediatric Gastroenterology, 4 Department of Pediatric Hematology, 5 Department of Pediatric Infectiology and Immunology, Wilhelmina Children’s Hospital, University Medical Center Utrecht, 6 Stem cell transplantation, Princess Máxima Center for Pediatric Oncology, 7 Department of Ophthalmology, 8 Department of Rheumatology and Immunology, 9 Microbiology and Virology, University Medical Center Utrecht, Utrecht, Netherlands Correspondence: M. H. Jansen Introduction: In 2018, the meningococcal C vaccination was replaced by the meningococcal ACWY (MenACWY) vaccination in the Dutch Immunisation Programme. Therefore, we investigated the immunogenicity and safety of the MenACWY vaccine in paediatric patients with (auto)immune disease, here focussing on Juvenile Idiopathic Arthritis (JIA) and Inflammatory Bowel Disease (IBD). We now present the 24 month follow-up data and the results of the serum bactericidal antibody (SBA) assay. Objectives: To assess immunogenicity and safety of the MenACWY vaccine in paediatric patients with JIA or IBD and study the effect of biological DMARDs on seroprotection. Methods: In this prospective study, patients with immune disorders from the Wilhelmina Children’s Hospital Utrecht were sampled at baseline and 3, 12 and 24 months post vaccination. Serology was performed using the Fluorescent bead-based Multiplex ImmunoAssay (MIA) and the SBA assay. We assessed immunogenicity by determining geometric mean concentrations (GMCs) for polysaccharide-specific IgG concentrations. We assessed the proportion protected at 12 months post vaccination in a subset of the patients with the internationally-accepted correlate of protection (SBA titre ≥8). Safety was measured by questioning adverse events 3 months post vaccination. Alterations in disease activity were measured by the Clinical Juvenile Arthritis Disease Activity Score-27 (cJADAS-27) in JIA patients and the Paediatric Crohn’s Disease Activity Index (PCDAI) and Paediatric Ulcerative Colitis Activity Index (PUCAI) in IBD patients. Results: 229 patients were included (33% IBD and 67% JIA). 40% were male and median age was 16 years (IQR: 14 – 17). At baseline, 72% of the patients were using DMARDs, of which 48% biologicals (39% anti-TNF agents). Except for MenC at 3 months, GMCs were significantly lower for each serogroup at all post vaccination time-points in patients who were treated with anti-TNF agents. The proportions protected (SBA titre ≥8) for MenA, C, W, Y 12 months post vaccination were 94%, 96%, 85% and 96%, respectively. This was significantly lower for anti-TNF users for serogroup W but not for A, C and Y. The MenACWY vaccine did not increase disease activity and no severe adverse events were reported. Conclusion: The MenACWY vaccine is well tolerated in JIA and IBD patients but less immunogenic compared to healthy controls. GMC’s are significantly lower for anti-TNF users at 3, 12 and 24 months post vaccination and the proportion protected at 12 months post vaccination was significantly lower in anti-TNF users for serogroup W (but not A, C and Y). We therefore advice to measure antibodies in patients on anti-TNF 12 months post vaccination and to consider a booster vaccination accordingly. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
Tarkiainen 1,2 , M. Backström 3,4 , B. Gottlieb 5 , C. Trincianti 6 , T. Qiu 7 , E. Morgan 8 , D. Lovell 9 , N. Ruperto 10 , A. Consolaro 6,10 , P. Vähäsalo 11,12,13 1 University of Helsinki, 2 New Children’s hospital, Helsinki University Central Hospital, Helsinki, 3 Department of Pediatrics, The Wellbeing Services County of Ostrobothnia, Vaasa, Finland, Vaasa, 4 Pedego Research Unit, University of Oulu, Oulu, Finland, 5 Cohen Children’s Medical Center, New York, United States, 6 University of Genova, Genova, Italy, 7 Department of Biostatistics and Epidemiology, USA., Cincinnati Children’s Hospital Medical Center, Cincinnati, 8 Seattle Children’s Hospital, Seattle, 9 Cincinnati Children’s Hospital, Cincinnati, United States, 10 IRCCS Istituto G. Gaslini, Genova, Italy, 11 PEDEGO Research Unit, University of Oulu, 12 Department of Children and Adolescents, Oulu University Hospital, 13M. Tarkiainen Introduction: Evaluating disease activity in juvenile idiopathic arthritis (JIA) is mainly done by rating the juvenile arthritis disease activity score in which the physician global assessment (PhGA) of disease activity has an important role. However, it is not known what affects physicians scoring the PhGA. Objectives: To assess the heterogeneity of factors affecting PhGA scoring through a global web-based survey. Methods: An electronic questionnaire regarding factors affecting PhGA was sent to all PRINTO and PR-COIN members. The responders were asked to rate from 0 to 100 the relevance of 17 factors possibly affecting PhGA scoring. These factors were chosen based on consensus of the study panel. Results were analyzed also in groups based on the responders’ level of experience in the field of pediatric rheumatology (<5 years, 5-10 years, or >10 years). Coefficient of variation (CV) was used to measure the heterogeneity in the 17 factors contributing to PhGA soring. Results: 491 (431 from PRINTO and 60 from PR-COIN) responders completed the questionnaire. A large individual variation was observed in the impact of different factors on PhGA assessing JIA (Table). For systemic JIA, the smallest variations (mean/SD/CV) were seen in fever (89.2/16.4/18.4) and serositis (81.0/20.2/25.0), and the largest in the presence of erosions (48.2/33.8/70.1). To the question, “If a patient with oligoarticular non-systemic JIA and a polyarticular patient with non-systemic JIA had the same clinical picture would your VAS be different?” 244 (49,7%) physicians replied “NO” and 244 (49,7%) “YES”. Conclusion: In a global perspective, scoring of PhGA is divergent. Especially the roles of the patient’s clinical history (i.e. oligoarticular or polyarticular disease), presence of extra-articular manifestations, or patient-reported outcome measures should be discussed. To obtain consistent patient assessment in clinical trials and routine practice, a consensus on guidelines for scoring the PhGA is required. Trial registration identifying number: N/AY. Cetin 1 , E. Comak 2 , S. Akman 2 1 Department of Physiotherapy and Rehabilitation, Faculty of Health Sciences, 2 Department of Child Health and Diseases, Faculty of Medicine, Akdeniz University, Antalya, Turkey Correspondence: S. Y. Cetin Introduction: Juvenile idiopathic arthritis (JIA) is the most common childhood rheumatic disease characterized by pain, stiffness and joint swelling. JIA is associated with muscle weakness, impaired mobility, balance, and exercise capacity, resulting in decreased aerobic capacity and functional ability in children. It is stated that exercise programs such as strengthening, stretching and balance are beneficial in terms of increasing functionality and improving the quality of life in these children. Tai Chi is a mind-body Chinese exercise method that consists of a series of gentle movements characterized by balance. As a form of physical exercise, Tai Chi appears to improve cardiovascular fitness, muscle strength, balance, coordination and physical function in rheumatic diseases, and is also associated with improvements in psychological well-being. To the best ourknowledge, there is no study in literature that has investigated the effects of Tai Chi in children with JIA. Consequently, in this study, it was thought that due to the positive effect of Tai Chi in rheumatic diseases, it would also improve balance, functionality and increase the quality of life in children with JIA. Objectives: The aim of this study was to investigate the effect of Tai Chi exercise program on balance, functional mobility, muscle strentgh of lower extremity, exercise capacity, fatigue and quality of life and compare with home exercises group in children with JIA. Methods: 20 children with JIA (11 girls, 9 boys) with an average age of 11.85±3,16 years were included in the study. Children were divided into two groups by block randomization method. Group 1 received 1-hour Tai Chi exercise program twice a week for 10 weeks. Group 2 received a 1-hour strengthening, stretching and balance exercise program twice a week for 10 weeks as a home exercises. Functional Reach Test (FRT) was used to evaluate the balance, Timed Up and Go Test (TUG) for functional mobility, 30-sec Sit to Stand Test for muscle strentgh of lower extremity, 6-Min Walk Test (6MWT) for exercise capacity, PedsQL Multidimensional Fatigue Scale for fatigue, PedsQL Arthritis Modul for quality of life and Childhood Health Assessment Questionnarie (CHAQ) for health assessment. All evaluations were performed at baseline and at the end of the 10th week. Results: When the pre-training values of the groups were compared, there was no statistically significant difference in parameters (p > 0.05). After training, there was a statistically significant difference within groups for the Tai Chi and the home exercise groups for all parameters (p: 0.00-0.04) except for CHAQ scores. When the groups compared after training, FRT, TUG, 6MWT scores and fatigue scores of PedsQL were found to be significantly in favor of the Tai Chi group (p:0.00- 0.04). Conclusion: Tai Chi should be considered for inclusion in rehabilitation programs as a safe mind-body type of exercise to improvein patients with JIA. References 1-Kuntze G, Nesbitt C, Whittaker JL, Nettel-Aguirre A, Toomey C, et al. Exercise Therapy in Juvenile Idiopathic Arthritis: A Systematic Review and Meta-Analysis Arc Phys Med Rehabil 2018;99(1):178-193 2-Wang C. Role of Tai Chi in the treatment of rheumatologic diseases. Curr Rheumatol Rep. 2012;14(6):598-603 3-Cetin SY, Erel S, Bas Aslan U. The effect of Tai Chi on balance and functional mobility in children with congenital sensorineural hearing loss, Dis Rehabil, 2020; 42:12, 1736-1743 Patient Consent: Yes, I received consent Disclosure of Interest : None declared
O28. Sex differences in regulatory T-cells may contribute to autoimmune disease susceptibility O29. FOXP3-specific deletion of CREB generates ST-2 positive regulatory T-cells with shifts towards type 2 immune responses O30. A gene signature for regulatory T cell fitness as a measure of disease activity in juvenile idiopathic arthritis O31. Evidence-based occupational and physical therapy for children and adolescents with inflammatory pediatric rheumatic diseases suffering from fatigue 32. Ultrasound versus MRI in the evaluation of ankle/midfoot joints in juvenile idiopathic arthritis O33. High-dimensional immunophenotyping reveals altered regulatory T cell fitness between inactive and active disease in juvenile idiopathic arthritis O34. New biomarkers from plasma and synovial fluid of oligoarticular juvenile idiopathic arthritis patients 5. Expansion of autoreactive CD27-IGD- double negative b cells in the joints of antinuclear antibody positive JIA patients O36. Traditional laboratory parameters and new biomarkers in Macrophage Activation Syndrome (MAS) and Secondary Hemophagocytic Lymphohistiocytosis (SHLH) G. A. Robinson 1 , J. Peng 1 , H. Peckham 1 , G. Butler 2 , I. Pineda-Torra 3 , C. Ciurtin 1 , E. C. Jury 4 1 Centre for Adolescent Rheumatology Versus Arthritis, 2 Department of Paediatric and Adolescent Endocrinology, 3 Centre for Cardiometabolic and Vascular Science, 4 Centre for Rheumatology, University College London, London, United Kingdom Correspondence: G. A. Robinson Introduction: Men and women have differential immune responses resulting in variation in response to infection, vaccination and autoimmune disease risk. An example is in juvenile-onset systemic lupus erythematosus (JSLE), which predominately affects young women with a common disease onset at puberty. Sexual dimorphisms have been described across both the innate and adaptive immune system which vary depending on age group and pubertal status. We hypothesised that sex-hormones could influence inflammation and autoimmune disease susceptibility following puberty. Objectives: The aim of this study was to investigate the role of sex hormones and chromosomes in driving inflammatory profiles by sex and/or gender using unique cohorts of young individuals. Methods: Flow cytometry was used to measure the frequency of 28 immune-cell subsets from young post-pubertal healthy and JSLE cis-men/women (n=17/22, mean age 18/17.5 and n=12/23, mean age 18/20) recruited from University College London Hospital, UK. Immune phenotype data was analysed by logistic regression and balanced random forest machine learning (BRF-ML). RNA-sequencing was used to assess the phenotype of regulatory T-cells (Tregs) isolated from matched healthy and JSLE individuals (n=5 per group), and from age matched transgender individuals under cross-sex-hormone treatment (trans-men/women, n=5/5, mean age 18.2/18.7, puberty blocked for 12-months followed by testosterone/oestradiol treatment). Differentially expressed gene (DEG) data was analysed by cluster, extended-network, pathway and open-target disease analysis. Suppression assays assessed the anti-inflammatory function of Tregs in vitro . Results: BRF-ML identified increased circulating anti-inflammatory Tregs and decreased pro-inflammatory responder T-cells in healthy young cis-men compared to cis-women (p=0.0097 and p<0.0001, ranked highest in the BRF-ML-model). Tregs from healthy young cis-men were also more suppressive of activated T-cells in vitro . From RNA sequencing analysis, 82 Treg DEGs were identified between healthy young cis-men and cis-women, which could confidently cluster individuals by sex and significantly enriched the PI3K/AKT signalling gene ontology pathway. Importantly, 58.5% of these genes were not located on the X/Y chromosomes, suggesting a role of sex-hormones in Treg function. Despite having no influence on Treg frequency, cross-sex-hormone treatment altered many Treg transcriptomic pathways, including increased cytokine production and decreased immune activation in trans-men and trans-women, respectively, supporting a role of sex-hormones in Treg function. Many of the sex-hormone-induced Treg functional DEGs overlapped with the 82 DEGs identified between cis-men and cis-women and were significantly associated with PI3K/AKT signalling and with SLE by open-target disease analysis (p=0.02). Strikingly, sex differences in Tregs were lost or reversed in young JSLE patients, including Treg frequency, suppressive capacity and gene expression, suggesting that sex-hormone signalling could be dysregulated in autoimmune pathogenesis. Conclusion: Sex-chromosomes and hormones may drive specific changes in circulating Treg frequency and function, respectively. Healthy young cis-men have a more anti-inflammatory Treg profile which could explain autoimmune susceptibilities by sex and inform sex-tailored therapeutic strategies. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
K. Ohl 1 , S. H. Subramanyam 1 , E. Verjans 1 , T. Clarner 2 , S. Böll 3 , I. G. Costa Filho 4 , L. Gan 5 , H. Huehn 6 , T. Bopp 7 , R. Beyaert 8 , B. Lambrecht 8 , M. Scheld 2 , T. Goodarzi 3 , N. Wagner 1 , M. Zenke 1 , K. Tenbrock 3 1 RWTH AACHEN University, Aachen, Germany, 2 Anatomy, 3 Pediatrics, 4 Computational Genomics, 5 IZKF, RWTH AACHEN University, Aachen, 6 HZI, Braunschweig, 7 Institute of Immunology, Mainz, Germany, 8 VIB, Ghent, Belgium Correspondence: K. Tenbrock Introduction: Regulatory T cells (T regs ) are gatekeepers of immune homeostasis and characterized by expression of Foxp3, which maintains T reg identity. The transcriptional activator CREB critically stabilizes Foxp3 expression in vitro. Objectives: To analyze the effect of CREB on regulatory T cells, we generated mice with a deletion of CREB in all hematopietic cells (VAV-CRE) as well as a specific deletion in regulatory T cells (Foxp3-CRE) Methods: Foxp+ CD4 T cells of mice were analyzed virgorously with flow cytometry, whole transcriptome analysis, ATACseq, TSDR methylation in vitro and in animal models of experimental colitis, ovalbumin induced asthma and TLR7 induced lupus in vivo Results: Here we demonstrate that in mice with a Foxp3-specific knockout of CREB, T regs show a reduced Foxp3 expression in vivo , but surprisingly enhanced expression of IL-13, IL-10, ST-2 and CREM. This rendered such T regs highly suppressive in vitro . In vivo , Foxp3-specific knockout of CREB prevents colitis in a T cell mediated transfer colitis model in an IL-10 dependent way however aggravates disease activity in a murine lupus and asthma model. Mechanistically, in cooperation with CREM, CREB expression in T regs alters chromatin accessibility to different loci like the ST-2 region and thereby influences T cell specific immune responses by regulation of IL-10. Conclusion: Our data suggest that CREB expression in T regs is important for the balance between Th1 and Th2 responses by regulating ST-2 and IL-10. Disclosure of Interest : None declared
M. H. Attrill 1 , D. Shinko 1 , T. Martins Viveiros 1 , L. R. Wedderburn 2,3,4 , S. G. CHARMS 2,3,4 , S. G. JIAP 2,3,4 , A. M. Pesenacker 1 1 Infection and Immunity, UCL Institute of Immunity and Transplantation, 2 UCL Great Ormond Street Institute of Child Health, 3 Centre for Adolescent Rheumatology Versus Arthritis at UCL UCLH and GOSH, 4 NIHR Biomedical Research Centre at GOSH, London, United Kingdom Correspondence: A. M. Pesenacker Introduction: Juvenile Idiopathic Arthritis (JIA) is a disease of flare-ups, which do occur without warning or known trigger on or off treatment, yet there is no reliable biomarker to predict disease course. Better predictability of inflammation flare-ups could lead to more personalised disease management with preventative treatment if a flare is likely, or withdrawing medication safely when sustained remission is indicated. Objectives: A regulatory T cell (Treg) defect has previously been suggested in JIA. Using our Treg gene signature as a measure of Treg fitness, we aim to identify the nature of Treg fitness changes in active JIA, which may be used as biomarker to predict disease trajectory and those at more at risk of flare-ups. Here, we focus on peripheral blood (PB) samples from clinically active and inactive JIA as well as synovial fluid (SF) from acutely inflamed joints. Methods: SF mononuclear cells (SFMC, n=30), PBMCs from healthy adults (n=20) and healthy children (n=5) and from active (n= 30) and inactive (n= 17) JIA individuals were thawed, PB/SFMC lysates stored, and remaining cells sorted into CD4+CD25hiCD127low Tregs and CD4+CD25lowCD127hi T-conventional cells (Tconvs) and lysed in RLT buffer. Cell lysates were hybridised to a custom nanoString gene set (48 gene Treg signature plus) and analysed on nanoString nCounter Pro Analysis System. Normalisation by positive control and total sum normalisation, gene expression analysis and biomarker discovery pipeline, via machine learning, were performed using R software. Biomarker discovery analysis used combinations of feature select and classifier-building with logistic or elastic net regression and leave-one-out cross-validation (LOOCV) was used to obtain performance estimates, with the best algorithm chosen. Results: As expected, the 48 gene signature successfully distinguished between Tregs and Tconvs, regardless of activation or disease state. PCA analysis clustered cells from SF and PB separately, with Tregs also distinguished from unfractionated PBMC and SFMC lysates. Genes usually associated with Treg functionality, such as FoxP3, CTLA-4 and TIGIT, were upregulated in SF Tregs. Interestingly, genes connected to the TGF-beta pathway, although not TGF-beta itself, were reduced in SF Tregs compared to blood Tregs. Crucially, using machine learning we could identify differences between blood Tregs from active and inactive JIA samples. Conclusion: Our Treg gene signature serves as a measure of Treg fitness and has biomarker potential for immune-mediated conditions, such as JIA. We identified possible mechanisms behind loss of Treg fitness in the inflamed joint with changes in genes stabilising and responding to TGF-beta signalling. Moreover, our biomarker discovery pipeline could be used to establish an algorithm distinguishing active from inactive JIA blood Treg, indicating biomarker potential that could be ultimately used to track and possibly predict disease flares versus sustained remissionU. Nilsson, I. H. Bolstad, K. Krosby, B. W. Njølstad, S. Hansbø, H. Sanner, K. Risum slo University Hospital, Oslo, Norway Correspondence: U. Nilsson Introduction: Fatigue is common in children and adolescents with inflammatory pediatric rheumatic diseases (PRD) and there is a need to develop an evidence-based guideline for physiotherapy (PT) and occupational therapy (OT) interventions to standardize and improve the quality of care offered to patients with PRD. Objectives: To develop an evidenced-based guideline for PT and OT interventions, including identifying measurement tools to assess fatigue and interventions to treat fatigue. Methods: The guideline is developed according to The Appraisal of Guidelines for Research and Evaluation (AGREE) Instrument, which includes scientific evidence, clinical experience and patient preferences. A systematic literature search of various medical databases was performed, and identified studies were critically evaluated. Occupational therapists and physiotherapists across Norway, working in both hospitals and primary care, contributed with clinical expertise. Patient experiences and preferences were obtained by interviewing representatives from the Norwegian Patient Association for Children with Rheumatic Diseases. Relevant representatives across Norway reviewed the guideline, and then final adjustments were performed. Results: In total, 159 studies were identified and 17 included. In patients with Juvenile Idiopathic Arthritis, the Visual Analogue Scale is the most used unidimensional measurement tool to assess fatigue and the Pediatric Quality of Life Inventory Multi Fatigue Scale is the most used multidimensional questionnaire to assess fatigue. There is insufficient scientific evidence of the efficacy in interventions to reduce fatigue. However, a few studies showed that exercise significantly could reduce fatigue. The therapists often used a biopsychosocial approach in assessment and treatment of fatigue in PRD. They mainly used self-constructed questions to assess how fatigue affects body functions and structures, activities and participation. The therapists often used a time-schedule to get an overview of patient’s activity pattern and any need for adjustments. Since fatigue is a complex symptom, it is necessary to take several factors into consideration, such as sleep, pain and physical activity. The intervention is adapted by individual factors affecting the patient. The patient representatives highlighted a need for focus on fatigue and support by health care professionals to manage their fatigue. Patients experienced that management to reduce pain also could reduce fatigue. The guideline is based on a biopsychosocial understanding of fatigue. It is divided into two parts; assessment and intervention. The assessment part contains patient interview, uni- and multidimensional measurements, activity registration, and assessment of different factors that can influence fatigue. The intervention part consists of patient education, understanding, awareness and regulation of activity level, enhancing self-efficacy, intervention of contributing factors to fatigue, and collaboration with other professionals involved in the care of the patients. A brochure with patient information was developed as a part of the guideline. The guideline will be published and available at the Norwegian Electronic Health Library after final approval by Oslo University Hospital. Conclusion: Scientific evidence is limited for assessment and interventions of fatigue in children and adolescents with PRD. Thus, the evidenced-based guideline is mainly based on clinical experience and patient preferences. Fatigue is a complex symptom and the guideline provides a framework for PT and OT in clinical care of children and adolescents with PRD suffering from fatigue. When applying the guideline, individual adjustments are necessaazzoni 1 , S. Lanni 2 , F. Magnaguagno 3 , M. Valle 3 , A. Pistorio 4 , C. Lavarello 5 , M. Gattorno 5 , A. Ravelli 4 , C. Malattia 5,6 1 Struttura Complessa Pediatria, Ospedale di Lavagna, Lavagna, 2 UOC Pediatria a Media Intensità di Cura Clinica de Marchi, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milano, 3 Radiologia, 4 Direzione Scientifica, 5 Clinica Pediatrica e Reumatologia, IRCCS Istituto G. Gaslini, 6va, Italy Correspondence: M. Mazzoni Introduction: Arthritis of the ankle/midfoot occurs commonly in all subtypes of JIA and might cause considerable functional impairment. Clinical assessment of this region is often challenging due to the multiplicity of joint recesses and surrounding tendons . Objectives: To set-up an ultrasound (US) scoring system to assess synovial inflammation in ankle/midfoot joints and provide preliminary evidence of its validity by comparing US and MRI findings in JIA patients with this region involvement. Methods: JIA patients who underwent contrast-enhanced (CE) MRI and US at the study Centre on the same day were included. The tibio-talar (TT) (anterior recess), the subtalar (ST) (lateral aspect) and the talonavicular (TN) (dorsal aspect) joints were examined by B-mode (synovial hypertrophy and/or joint effusion) and power Doppler (PD) mode. The anterior, lateral and posterior ankle tendons were assessed for tenosynovitis. Synovitis was graded using a specifically devised 0-3 semiquantitative joint-specific scoring system for B-mode and PD signal. MRIs were scored by using the Outcome Measure in Arthritis Clinical Trials Rheumatoid Arthritis MRI Scoring System. The agreement between MRI and US scores in each recess was evaluated by calculating Cohen’s kappa coefficient (k). The interpretation of the k coefficient values was as follows: 0–0.20 poor, 0.20–0.40 fair, 0.40–0.60 moderate, 0.60–0.80 good, and 0.80–1 very good. Results: 67 patients (89.5% F; median age 12.7 y; median disease duration 7.3 y) were included. The agreement between MRI and US B-mode synovitis scores was moderate for the TT (Cohen’s kappa = 0.58; 95% CI = 0.35 - 0.81) and TN joints (Cohen’s kappa = 0.55; 95% CI = 0.31 - 0.79); it was good for the ST joint (Cohen’s kappa = 0.62; 95% CI = 0.39 – 0.85). The agreement between contrast-enhanced MRI and PD score was fair for the TT (k=0.24; 95% CI = 0.07 - 0.41) and ST joints (K= 0.38; 95% CI = 0.19 - 0.57) and moderate for TN joint (K= 0.5; 95% CI = 0.27 - 0.73). Concordance between MRI and MSUS for tenosynovitis was good (k = 0.79; 95% CI = 0.55 -1.00). Conclusion: The joint specific US B-mode scoring system was accurate to assess and quantify synovitis in the ankle/midfoot joints and tendons. The discordances between US and MRI might be explained by MRI ability to visualize the entire joint, while the US can visualize only the superficial joint recesses. US scanning protocol and equipment settings might explain the lower sensitivity of PD mode compared to CE MRI in detecting hypervascularity in JIA synovial tissue that is an expression of active inflammation. Further studies are required to assess the reproducibility and sensitivity to change of the joint specific ankle/midfoot US score. Disclosure of Interest : None declared
M. H. Attrill 1 , D. Shinko 1 , L. R. Wedderburn 2,3,4 , S. G. CharmsM. H. Attrill Introduction: Juvenile Idiopathic Arthritis (JIA) is an autoimmune condition in children characterised by unpredictable, T-cell rich inflammatory flares of the joints. Despite increased number of CD4+FoxP3+ regulatory T cells (Tregs) within the synovial fluid (SF) of active joints, these fail to suppress autoimmunity. Objectives: Here, we take a closer look at the CD4+FoxP3+ Treg populations in JIA. We proposed that through in-depth high-dimensional phenotyping we will be able to identify and distinguish unfit Treg sub-populations present in JIA SF and clinically active peripheral blood (PB) which may be disrupting the immunoregulatory balance leading to ongoing disease. Methods: Using 5-laser full spectrum flow cytometry, we designed and verified a 33-parameter panel to assess differences in the cellular composition and Treg phenotype between JIA SF mononuclear cells (SFMCs, n=18), JIA PBMCs from clinically active (n=29) and inactive disease (n=17), and healthy adult control PBMCs (n=18). The panel was designed to identify monocyte, B, NK and dendritic cell subsets in addition to T cell phenotype. Incorporating markers and receptors associated with Treg functionality, activation, memory, co-stimulation, and co-inhibition, we were able to characterise Tregs in terms of overall ‘fitness’. In addition to traditional gating analysis via Flowjo, unbiased high dimensional analysis was achieved using the R package and computational tool Spectre, allowing raw data integration, clustering through FlowSOM, dimensionality reduction via UMAP and quantitative statistical analysis and visualisation. Results: 12/18 clusters in overall cell composition, with most changes among T lymphocytes, and 8/10 Treg clusters had altered frequencies in SF. We found an emergence of more active subtypes in both CD4- and CD4+ T cells, with increased expression of GITR, HLA-DR, CD71 and CD69. This highly activated phenotype was also mimicked in SF Tregs, which additionally adopted a classically “suppressive” phenotype, with high CTLA-4 and PD-1 levels compared to PB. However, despite a shift to a more active state, SF Tregs were not any more proliferative than blood Tregs, with no change in %Ki67+ in Tregs across SF and PB from JIA or healthy adults. Analysis of co-receptors found the upregulation of the co-stimulatory receptor CD226 on SF Tregs, mostly co-expressed with TIGIT, while blood Tregs rarely co-expressed both co-receptors. Moreover, we identified a sub-population of CD137low ID2intermediate Tregs in PB of inactive individuals, with reversed expression pattern in PB Tregs from active disease, highlighting differences in Treg maintenance with disease activity. Conclusion: High dimensional flow cytometry revealed subsets of Tregs within the inflammatory joint of JIA with a highly active and suppressive phenotype, yet we have identified changes to Treg overall fitness. A shift to a more co-stimulatory receptor presence on SF Tregs could be backing the persistence of inflammation within the joint. Alterations to Treg stability and maintenance in the blood during active disease may also hint towards immunoregulatory disruption in the periphery. These may have biomarker potential for predicting flare-ups and offer new potential mechanistic targets for restoring the immunological balance in JIA. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
S. Pelassa 1 , C. Rossi 1 , F. Raggi 1 , D. Cangelosi 2 , M. Bartolucci 3 , A. Petretto 3 , F. Antonini 3 , P. Bocca 4 , F. Penco 4 , M. Rossano 5 , F. Baldo 5 , G. Filocamo 5 , C. Trincianti 6 , A. Eva 1 , A. Ravelli 4 , A. Consolaro 4 , M. C. Bosco 1 1 Laboratory of Molecular Biology, 2 Clinical Bioinformatic Unit, 3 Core Facilities, 4 Pediatric Rheumatology Clinic, IRCCS Istituto G. Gaslini, Genova, 5 Fondazione IRCCS Ca’ Granda, Ospedale Maggiore Policlinico, Milano, 6 University of Genova, Genova, Italy Correspondence: S. Pelassa Introduction: Extracellular vesicles (EVs) from plasma (PL) and synovial fluid (SF) could represent a valuable source of early predictive biomarkers for Oligoarticular Juvenile Idiopathic Arthritis (OJIA), the most common pediatric rheumatic disease in Western countries Objectives: To identify new low-invasive biomarkers for early diagnosis of OJIA and prediction of disease course through the characterization of the EV miRNome (EVs-miR) and proteome (EV-prot) combined with the study of mononuclear cells in specimens from children with new-onset OJIA Methods: EVs were isolated using a membrane affinity spin column from PL and SF of 50 OJIA patients at disease onset. Patients were followed up for 12 months, and clinical data were collected. PL samples were also obtained from 24 age/gender-matched control children (CTR-PL). EV-miR and EV-Prot expression profiling was carried out using the TaqMan Array RT-PCR and mass spectrometry, respectively. EVs surface protein expression was assessed by the MACSPlex Exosomes Kit. Macrophages and T cells subsets were isolated from the SF aspirates of 14 patients by MACS magnetic beads and characterized by cytofluorimetry Results: EVs-miR expression profiles were compared between OJIA-SF, OJIA-PL, and CTR-PL samples. We identified 46 EV-miR differentially expressed in SF vs both paired and control PL targeting genes involved in processes related to inflammation, cartilage/bone homeostasis, and hypoxia, suggesting EV-miR role in disease pathogenesis and potential as biomarkers of OJIA development. Comparative analysis of the EV-miR Nome in OJIA-PL vs CTR-PL samples identified 57 EV-miRs able to differentiate OJIA patients from CTR children, of which 55 were significantly upregulated and 2 significantly downregulated. 15 EV-miR were consensually overexpressed in OJIA-SF and OJIA-PL compared to CTR samples, potentially representing a disease-specific EV-miR signature at both local and systemic levels. Correlation with clinical data demonstrated that a subset of 3 EV-miR was able to stratify OJIA patients in distinct subgroups, suggesting their potential as predictive biomarkers. Comparative analyses were also carried out among EV-Prot expression profiles of SF- and PL from 30 OJIA patients and 24 CTR children. Significant up- and down-regulation of 268 and 263 EV-Prot were demonstrated in SF vs both paired and CTR PL, respectively, several of which are involved in inflammatory and immunological processes. A subset of EV-Prot able to discriminate subgroups of patients was also identified within the OJIA cohort. We also evaluated the expression of surface proteins on EVs from SF and PL patients by flow cytometry, showing modulation of specific immunologic markers. Finally, we phenotypically characterized macrophages and T cell subsets from OJIA-SF samples. We demonstrated that the ratio between T cells (CD3+):macrophages (CD14+) and between cytotoxic T cells (CD8+):helper T cells (CD4+) were higher in patients who developed polyarticular course respect to patients with oligoarticular course . Conclusion: We provide the first database integrating EV-miR, EV-Prot, and mononuclear cell phenotypic data from PL and SF of new-onset OJIA patients. Results may lead to a better understanding of the molecular mechanisms underlying OJIA development and the identification of new biomarkers for the diseaseStrenzel 1 , J. Dirks 1 , G. Haase 1 , U. Fischer 1 , J. Fischer 1 , A. Holl-Wieden 2 , C. Hofmann 2 , H. Morbach 1 1 Pediatric Immunology, 2 Pediatric Rheumatology and Osteology, University Children’s Hospital, Wuerzburg, Germany Correspondence: C. Strenzel Introduction: Juvenile idiopathic arthritis (JIA) is a chronic inflammatory disease of unknown origin which is assumed to be mediated by an autoimmune response. The appearance of antinuclear antibodies (ANAs) in almost half of the patients suggests B cell dysregulation as a distinct pathomechanism of JIA. Furthermore, ANA positive JIA patients seem to constitute a clinically homogenous group of patients. However, the site of dysregulated activation of autoreactive B cells as well as the B cell subsets involved are still unknown. Objectives: To dissect the distribution of different synovial fluid (SF) B cell subsets in JIA patients and assess their B cell receptor (BCR) specificities on a clonal level. Methods: Distribution of different B cell subsets within SF were assessed by flow cytometry in a large cohort of JIA patients and correlated to disease subgroup (oligo-JIA, poly-JIA) and ANA status. Single cell sorting was used for generation of monoclonal antibodies (mAbs) from individually sorted B cells by expression cloning of the respective immunoglobulin genes. Generated mAbs from single B cells were assessed for antinuclear autoreactivity using HEp-2 based immunofluorescence tests. The phenotype of each B cell corresponding to the HEp-2 reactive mAbs was retraced by an index sorting algorithm. Results: SF B cells in JIA patients mainly displayed a phenotype of activated CD21lo/-CD11c+ B cells. We used single B-cell based cloning technology to isolate and express immunoglobulin genes from SF B cells of 5 active JIA patients. By this, we generated 140 mAbs from SF CD21lo/-CD11c+ B cells from 3 ANA positive and 2 ANA negative JIA patients. Of these mABs 17-33% per patient were HEp-2 reactive and thus could be considered as autoreactive. The binding pattern of these mAbs in HEp-2 immunofluorescence tests revealed reactivity against cytoplasmic as well as nuclear antigens. Further flow cytometric analysis of SF B cells in a larger cohort of JIA patients (n=46) revealed a significant expansion of CD21 lo/- CD11c + CD27-IgD- “double-negative” (DN) B cells in ANA positive JIA patients. Retracing by index sort analysis showed that these DN B cells in ANA positive JIA patients were also enriched in autoreactive clones. Interestingly, HEp-2 reactive (“autoreactive”) B cell clones within the DN B cell and CD27+IgD- switched memory B cell compartment differed significantly in the frequency of somatic hypermutation (SHM) events with lower SHM frequencies in the first subset. Conclusion: Accumulation of autoreactive B cell clones in the inflamed joints is a common phenomenon in JIA patients. However, CD21 lo/- CD11c + DN B cells are particularly expanded in the SF of ANA+ JIA patients and enriched in autoreactive B cell clones. The specific pattern of SHM within autoreactive DN B cell clones particularly expanded in ANA positive JIA patients suggests that different activation pathways may drive this B cell subset. Disclosure of Interest : None declared
A. De Matteis 1 , D. Pires Marafon 1 , I. Caiello 1 , M. Pardeo 1 , G. Marucci 1 , E. Sacco 1 , F. Minoia 2 , F. Licciardi 3 , A. Miniaci 4 , I. Maccora 5 , M. C. Maggio 6 , G. Prencipe 7 , F. De Benedetti 8 , C. Bracaglia 8 1 Division of Rheumatology, IRCCS Ospedale Pediatrico Bambino Gesù, Roma, Italy, Roma, 2 Pediatric Rheumatology, Fondazione IRCCS Ca’ Grande Ospedale Maggiore Policlinico, Milano, 3 Department of Pediatrics and Infectious Diseases, School of Medicine, University of Turin, Regina Margherita Children’s Hospital, Torino, 4 Department of Pediatrics, University of Bologna, S. Orsola-Malpighi Hospital, Bologna, 5 Pediatric Rheumatology Unit, Meyer Children’s University Hospital, Firenze, 6 University Department Pro.Sa.M.I. “G. D’Alessandro”, University of Palermo, Palermo, 7 Division of Rheumatology, ERN-RITA Cent, IRCCS Ospedale Pediatrico Bambino Gesù, 8 Division of Rheumatology, ERN-RITA center, IRCCS Ospedale Pediatrico Bambino Gesù, Roma, Italy Correspondence: C. Bracaglia Introduction: MAS and sHLH are hyperinflammatory conditions, in which IFNγ plays a pivotal role. Early recognition and treatment improve the outcome and the mortality rate. Objectives: This is a retrospective multicenter study. We correlate traditional laboratory parameters of hyperinflammation with IL-18 and IFNγ related biomarkers. We have also evaluated the diagnostic and prognostic role of IL-18, CXCL9, CXCL10 and neopterin in patients with MAS and sHLH. Methods: One hundred-six patients from 6 Italian centers were enrolled: 41 with sHLH, 41 with MAS in the context of sJIA, and 24 with sJIA without MAS. The samples were collected at three different time points: active disease (T0), 7-10 days from starting therapy (T1) and in clinical inactive disease on medication (from 1 to 3 months from onset) (T2). Results: A total of 378 samples were collected. Laboratory features at T0 are detailed in table 1. Using the 2016 classification criteria for MAS, we can confirm that platelet count is a specific parameter, whilw ferritin is a sensitive parameter. Lactate dehydrogenase (LDH) values were statistically higher in MAS and sHLH compared to sJIA. ROC curve of LDH values in MAS showed a statistically significant area under the curve (AUC= 078.2%, p-value <0.0001). A cut-off of 683 U/L had a sensitivity of 73.6% and a specificity of 70.3%. IL-18, CXCL9, CXCL10 and neopterin levels in T0 were significantly higher in MAS and sHLH. In MAS, IL-18 levels were significantly higher compared to sHLH (p<0.0001). The ROC curves performed for each biomarker showed a statistically significant AUCs (p<0.01), except for IL-18 in sHLH. We have identified a cut off value for each biomarker in MAS (CXCL9 900 pg/ml, CXCL10 260 pg/ml, neopterin 5.0 ng/ml, IL-18 82996 pg/ml) and sHLH (CXCL9 2145 pg/ml, CXCL10 270 pg/ml, neopterin 7.1 ng/ml). In T0 neopterin correlates significantly with IL-18, CXCL9 and CXCL10 in MAS group but not in sHLH. We found also strong correlation between CXCL9 and CXCL10 only in MAS group. Conclusion: Platelet count and ferritin have high specificity and sensitivity, respectively, to diagnose MAS in the context of sJIA. Even if LDH is not included in 2016 classification criteria for MAS in sJIA, we have found that this parameter could help to discriminate MAS in sJIA, in addition to the others. Our results confirm that IL-18 and the IFN-γ related biomarkers are significantly higher in patients with MAS and sHLH and might be useful to diagnose MAS/sHLH in addition to the traditional laboratory parameters. Moreover, IL-18 could help to distinguish sHLH from MAS and MAS from active sJIA. Patient Consent: Yes, I received consent Disclosure of Interest : A. De Matteis: None declared, D. Pires Marafon: None declared, I. Caiello: None declared, M. Pardeo: None declared, G. Marucci: None declared, E. Sacco: None declared, F. Minoia: None declared, F. Licciardi: None declared, A. Miniaci: None declared, I. Maccora: None declared, M. C. Maggio: None declared, G. Prencipe: None declared, F. De Benedetti Consultant with: Abbvie, SOBI, Novimmune, Novartis, Roche, Pfizer, Employee with: SOBI, C. Bracaglia Consultant with: SOBI and Novartis
P001. A retrospective study of subcutaneous golimumab in Juvenile Idiopathic Arthritis (JIA), an alternative biologic in refractory disease P002. The potential of CD161+ T cells as a surrogate measure of IL-17A expressing T cells in the synovial fluid of JIA patients P003. Inflammatory arthritis as the presentation finding of autoimmune polyendocrinopathy with candidiasis and ectodermal dystrophy syndrome P004. The predictors of etanercept efficacy and treatment outcomes in non-systemic juvenile idiopathic arthritis: the data of 375 patients P005. Feasibility study of taking minimally invasive, ultrasound-guided tissue biopsies of synovial tissue in children with juvenile idiopathic arthritis for research P006. IL-4 and S100A9 can differentiate acute lymphoblastic leukemia from juvenile idiopathic arthritis better than existing laboratory values P007. Economic impact of juvenile idiopathic arthritis in Mexico P008. Predictors of 2 and 5 years sustained disease remission among children and young adults with extended oligoarticular, enthesitis-related, or psoriatic juvenile idiopathic arthritis: results from clipper studies P009. Understanding the role of ultrasound in detecting temporomandibular joint involvement in juvenile idiopathic arthritis: a pilot study P010. Study of diagnostic delay in patients with juvenile idiopathic arthritis P011. Increasing incidence of juvenile idiopathic arthritis? a trend over 31 years in Southern Sweden 12. Interferon signature as possible new marker for the stratification of patients with JIA 3. The factors affecting the duration of remission of polyarticular JIA P014. Improving sleep reduces pain in childhood arthritis; a crossover randomized controlled trial P015. Acceptability of preliminary Arabic PGALS in patients with juvenile idiopathic arthritis P016. Quantifying cost impact of withdrawing biologic dmards in children with JIA P017. Juvenile psoriatic arthritis - clinical characterization and differences from adult-onset disease P018. Validation of a lateral flow rapid test for adalimumab drug monitoring in patients with juvenile idiopathic arthritis P019. Cutaneous disorders and systemic manifestations, the importance of the multidisciplinary approach P020. Biomarkers related to myeloid derived cells obtained at baseline and at 18-year follow-up in juvenile idiopathic arthritis. relation to remission status and inactive disease P021. Clinical patterns in psoriatic juvenile idiopathic arthritis in a pediatric rheumatology unit from a tertiary hospital P022. The effect of nutritional status on disease activity and disease course in juvenile idiopathic arthritis and use of malnutrition screening tests P023. A10-year inception cohort of mexican-mestizo patients with juvenile idiopathic arthritis (JIA) from 3 tertiary centers in Mexico City. A Real-World Evidence (RWE), based on real world data from routine clinical assessment P024. Validation of the parent global assessment as a quality of life measure in juvenile idiopathic arthritis: results from reacch-out P025. Juvenile psoriatic arthritis: a case report P026. Evaluation of comorbidities in patients with juvenile idiopathic arthritis: a cross-sectional single center study P027. Neutrophils extracellular traps formation may serve as a biomarker for disease activity in oligoarticular juvenile idiopathic arthritis P028. Functional state of the right ventricle of the heart in adolescents with juvenile idiopathic arthritis P029. Α2-fraction and haptoglobin as biomarkers for disease activity in oligo- and polyarticular juvenile idiopathic arthritis P030. Liver stiffness in low-dose methotrexate use in JIA patients 31. A clinical case of a patient with down syndrome and juvenile idiopathic arthritis: difficult to diagnose, difficult to treat, difficult to manage severe comorbid problems P032. Ana status is not associated with altered b cell subset distribution in the peripheral blood and synovial fluid of JIA patients P033. Juvenile psoriatic arthritis: data from the pera psoriasis study group P034. Investigation of physical activity levels of patients with juvenil idiopatic arthritis era COVID 19 P035. Serum calprotectin (S100A8/A9): a promising biomarker of activity and erosive changes in different subtypes of juvenile arthritis P036. Disease activity assessment for juvenile idiopathic artritis in transitional care P037. Patterns of biological switching among children with non-systemic juvenile idiopathic arthritis P038. Normal neonatal TREC and KREC levels in early onset juvenile idiopathic arthritis 9. Temporomandibular Joint (TMJ) involvement in Juvenile Idiopathic Arthritis (JIA) at disease onset: a rheumatologist’s, orthodontist’s and radiologist’s joint venture P040. Factors increasing the risk of flare in JIA with inactive disease under MTX monotherapy P041. Charcot’s neuropathic arthropathy – when no pain is not pleasure! P042. Predicting drug-free inactive disease two years after diagnosis of non-systemic juvenile idiopathic arthritis P043. Short term outcomes following tolerated disease activity level for individuals with juvenile idiopathic arthritis in the childhood arthritis and rheumatology research alliance registry P044. Impact of weekdays versus weekend days on accelerometer measured physical behavior in adolescents with Juvenile Idiopathic Arthritis (JIA): results from the ActiMON study P045. Parents´ experiences of a one-year support program when their child was diagnosed with juvenile idiopathic arthritis – a qualitative interview study 46. The parent/child juvenile idiopathic arthritis disease activity score: responsiveness to change and factor analysis P047. A decade of progress in juvenile idiopathic arthritis treatments and outcomes in Canada: results from reacch-out and the CAPRI registry P048. Tofacitinib in the therapy of different kind of pediatric rheumatic deseases: real clinical practice experience of a single center P049. Drug survival of abatacept therapy in children with rheumatic diseases: 10 years experience in a single center P050. Assessment of liver stiffness and steatosis in juvenile idiopathic arthritis patients treated with methotrexate P051. Cognitive impairment associated with higher C-reactive protein values in patients with juvenile idiopathic arthritis: a prospective cohort study 52. Cluster analysis of patients with oligoarticular, RF negative polyarticular and undifferentiated juvenile idiopathic arthritis P053. How to diagnose juvenile psoriatic arthritis? Multicenter prospective observational study in children with suspected diagnosis P054. Early cervical involvement in children with juvenile idiopathic arthritis and its variants: a frequently neglected domain in paediatric pathology P055. Juvenile psoriatic arthritis: a case series P056. The current state of global research on juvenile idiopathic arthritis 7. The adulthood transition of patients with polyarticular juvenile idiopathic arthritis in the era of biologic agents 8. Calcium intake and bone mineral density in juvenile idiopathic arthritis P059. Defining KLRB1/CD161 expression in synovial fluid immune cells in patients with juvenile idiopathic arthritis using single-cell RNA sequencing P060. Juvenile arthritis or recessive multiple epiphyseal dysplasia – differential diagnosis 61. Phenotypes of presentation of psoriatic arthritis in pediatric patient P063. Efficacy of secukinumab in enthesitis-related arthritis and juvenile psoriatic arthritis: results from a phase 3 study (JUNIPERA) P064. Exploratory methods identify novel autoantigens in juvenile idiopathic arthritis P065. Polyarticular JIA has a distinct co-inhibitor receptor profile among other JIA subtypes 6. Evaluation of comorbidities in patients with juvenile idiopathic arthritis P067. Conventional dendritic cells type 1 (CDC1) are strongly enriched, quiescent and relatively tolerogenic in local inflammatory arthritis P068. Effect of drug therapy for juvenile idiopathic arthritis on the level of cystatin c as a marker of renal dysfunction P069. A key role of IL-6/STAT signaling and cell-cell interactions in monocyte function in oligoarticular juvenile idiopathic arthritis P070. Rare association of juvenile polyarthritis and steroid-resistant nephrotic syndrom – case report P071. Analysis the survival of genetically engineered biological therapy in children with juvenile idiopathic arthritis P072. Clusters of JIA at methotrexate initiation identified using topological data analysis P073. What are the outcomes of persistent oligoarthritis? A systematic review plan 74. Circulating mirnas as non-invasive biomarkers for juvenile idiopathic arthritis disease activity monitoring P075. Cardiovascular risk assessment in children with juvenile idiopathic arthritis based on aerobic capacity, echocardiographic and laboratory parameters P076. The relationship between pain, kinesiophobia and proprioception in children with juvenile idiopathic arthritis P077. Adalimumab and anti-drug antibodies in a cohort of children with juvenile idiopathic arthritis: a single-center experience P078. Intra-Articular Glucocorticoid Injections (IAGI) in Juvenile Idiopathic Arthritis (JIA): a single center experience straddling COVID-19 pandemic P079. Children with extended oligoarticular and polyarticular juvenile idiopathic arthritis have a cytokine profile in peripheral blood sustaining b cell activation P080. Do we all score the physician global assessment in the same way? Results from a large international survey with 17 case scenarios 81. Natural antibodies levels depend on the activity of juvenile idiopathic oligoarthritis 2. Body mass index is related to disease variables in young adults with juvenile idiopathic arthritis P083. Prevalence of autoimmune diseases in parents of children with juvenile idiopathic arthritis: results from the international pharmachild register 4. How ready are adolescents with JIA for transfer to adult care?- parental perspective P085. Adaptation and validation of the methotrexate intolerance scale (Miss - Methotrexate Intolerance Severity Score) in children with juvenile idiopathic arthritis S. Zhukov, V. A. Malievsky Pediactric, BSMU, Ufa, Russian Federation Correspondence: S. Zhukov Introduction: Juvenile idiopathic arthritis (JIA) is the most common rheumatic disease in children. According to modern criteria, JIA is arthritis of unknown etiology, which begins before the age of 16 years, with a duration of more than 6 weeks. International and domestic protocols for the management of children with this pathology provide for the early prescription of basic antirheumatic drugs, mainly methotrexate (MT). Methotrexate is widely used for the treatment of JIA as a starting, basic drug with proven efficacy, but at the same time, the possibility of long-term therapy with methotrexate may be limited by a number of undesirable effects, the risk of which increases in proportion to the duration of the drug. A group of Dutch scientists developed the Methotrexate Intolerance Inventory (MISS). which have proven to be highly effective in clinical practice. In Russia, in the Republic of Bashkortostan, the necessary questionnaire was validated, which made it possible to successfully introduce the methotrexate intolerance questionnaire into the work of the department. Objectives: Adaptation and validation of the Russian version of the MISS questionnaire (assessment of the severity of methotrexate intolerance). Methods: The text of the questionnaire was translated and tested at the departments of hospital pediatrics (head of the department - Doctor of Medical Sciences, Professor Malievsky V.A.) and foreign languages with a Latin language course of the Bashkir State Medical University (head of the department - candidate of philological sciences, associate professor Mayorova O.A.). The validation procedure for the MISS questionnaire included language and cultural adaptation, which, according to international recommendations for the validation of questionnaires, took place in several stages. Under our supervision there were 100 children whose age at the onset of the disease ranged from 3 to 17 years inclusive (32 boys, 32.0% and 68 girls, 68.0%). The most common variant of JIA was polyarthritis (58 children, 58.0%). 36 children (36.0%) had persistent oligoarthritis. 3 children (3.0%) had juvenile arthritis with a systemic onset. Spreading oligoarthritis was diagnosed in 3 children (3.0%). Results: The value of the ɑ-Kronbach coefficient according to the scales of the MISS questionnaire All scales of the questionnaire have a ɑ-coefficient >0.7, which corresponds to the optimal level of internal constancy of the questionnaire adopted for group studies. In addition, these data are comparable with the results obtained by the authors of the original questionnaire. Conclusion: The MISS questionnaire has been adapted and validated in accordance with generally accepted requirements. An analysis of the reliability of the Russian version of the questionnaire based on the calculation of the Cronbach’s alpha coefficient indicates its sufficient reliability and the possibility of use in clinicalR. Al Mheiri, O. Killeen Rheumatology, National Centre for Paediatric Rheumatology, CHI@Crumlin, Ireland, Dublin, Ireland Correspondence: A. R. Al Mheiri Introduction: The introduction of biologics at the turn of this century has greatly improved the clinical outcome of JIA. Golimumab, a monoclonal tumour necrosis factor (TNF)-antagonist antibody was approved for paediatric patients with Polyarticular JIA in 2020. Objectives: The purpose of this study was to assess the effectiveness of Golimumab and to document our experience of this biologic agent to date in our National Centre. Methods: This retrospective, observational chart review included all patients followed in our service with JIA, who were treated with golimumab, between November 1, 2017, and November 30, 2020. Data were collected for a minimum of twelve weeks after the start date, or until discontinuation of anti-TNF therapy, whichever occurred last. The primary outcomes were clinical effectiveness at 4-6 months, defined as 1. Active clinical response (decrease in total number of active joints), 2. Clinical inactive disease (no active arthritis; no fever, no rash, serositis, splenomegaly, or generalized lymph- adenopathy attributable to JIA; no active uveitis; normal erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) levels), 3. Clinical no response (same total active disease or worsening of active joint cunt). A total of 17 patients met the inclusion criteria and included in the final analysis. Results: Five patients (29.4%) experienced a reduction in the total number of joints with active disease, six patients (35.3%) were in clinical inactive disease, four patients (23.5%) had same number of joints with active disease and two patients (11.8%) were worse at follow-up. No significant change was noticed in the inflammatory markers or CHAQ score. There were no reported significant side effects or any flare of uveitis or psoriasis in any of the patients. Golimumab therapy was discontinued in eight patients (47%) for lack of efficacy or loss of effectiveness. Conclusion: Data from this analysis demonstrate that Golimumab is an effective therapy in treating patients with refractory JIA. It was well tolerated with no major adverse reactions or flare of extra-articular co morbidities of psoriasis or uveitis. Disclosure of Interest : None declared
V. Alexiou 1,2 , B. Jebson 1,2 , E. Ralph 1,2,3 , M. Kartawinata 1,2 , E. Vigorito 4 , L. R. Wedderburn 1,2,3 on behalf of the CLUSTER Consortium 1 Centre for Adolescent Rheumatology Versus Arthritis at UCL University College London Hospital (UCLH) and Great Ormond Street Hospital (GOSH), 2 Infection, Immunity and Inflammation Programme, UCL Great Ormond Street Institute of Child Health, 3 National Institute for Health Research (NIHR) GOSH Biomedical Research Centre, London, 4 MRC Biostatistics Unit, University of Cambridge, Cambridge, United Kingdom Correspondence: V. Alexiou Introduction: CD161+ T cells are highly enriched in the synovial fluid (SF) of juvenile idiopathic arthritis (JIA) patients. The group has previously shown that CD161 (a C-type lectin-like receptor encoded by KLRB1 ) is expressed on Th17 cells undergoing transition to an intermediate Th17/Th1 fate in response to inflammation 1 . Therapeutic agents targeting the Th17 cytokine, IL-17A, such as Ixekizumab and Secukinumab are currently used primarily for psoriatic arthritis and ankylosing spondylitis. Their use in JIA could be informed by assessing the levels of IL-17A production in JIA patients. Identifying a reliable surrogate marker for IL-17A which can be easily clinically tested, such as cell surface marker CD161, could be valuable for patient stratification for the administration of IL-17A targeting agents. Objectives: To explore the relationship between the CD161+ CD4 T cell population and the pro-inflammatory environment of the joint in JIA, and to test the hypothesis that the CD161 molecule is a surrogate marker of IL-17A-expressing T cells in the joints of JIA patients. Methods: Multi-parameter flow cytometry analysis was performed on paired (collected on the same day or timepoint) samples of peripheral blood mononuclear cells (PBMC) and synovial fluid mononuclear cells (SFMC) of JIA patients (n=47, male:16, female:31). The JIA patients were grouped by ILAR subtype: oligo-articular JIA (n=21), poly-articular JIA (n=15), systemic JIA (n=6), enthesitis-related JIA (n=3) and psoriatic JIA (n=2). T cell populations were analysed based on the expression of CD3, CD4, CD8 and CD161. Following PMA/Ionomycin stimulation in the presence of Brefeldin A, intracellular production of GM-CSF, IFNγ, IL-17A. TNFα and IL-10 was analysed. Results: Analysis confirmed that the proportion of CD161+ CD4 T cells was increased in SFMC relative to peripheral blood PBMC of JIA patients (p <0.0001). Correlation analysis showed that CD161+ CD4 T cells strongly, positively correlate with IL-17A-expressing CD4 T cells in both PB (r =0.935, P<0.0001) and SF (r =0.823, P=0.0003). The analysis also showed that CD161+ positively correlated with CD4 T cells expressing other pro-inflammatory cytokines, TNFα (r = 0.626, P=0.0165) and GM-CSF (r =0.563, P=0.045), but not those expressing IFNg (r = −0.100, P=0.733). This concurs well with previous studies which have shown that IFNγ is also expressed by the CD161− CD4 T cell population in the joint 1,2 . Additionally, IL-17A expressing CD4 T cells correlate between PB and SF of JIA patients (r = 0.414, P=0.013). Conclusion: These data show that the proportions of IL-17A+ CD4 T cells correlate between the PB and SF of JIA patients and that the CD161+ CD4 T cell population may serve as a surrogate marker of IL-17A expressing CD4 T cells in both compartments. CD161 may represent a reliable indicator of the Th17/Th1 type of inflammation in the joint and extent of inflammation. This could have translational potential, as the detection of a cell surface marker provides a readily measurable biomarker and could be valuable for the provision of treatments targeting IL-17A in JIA. References: 1. Nistala K, Adams S, Cambrook H, et al. Th17 plasticity in human autoimmune arthritis is driven by the inflammatory environment. Proc Natl Acad Sci U S A . 2010;107(33):14751-14756. doi:10.1073/pnas.1003852107 2. Piper C, Pesenacker AM, Bending D, et al. T cell expression of granulocyte-macrophage colony-stimulating factor in juvenile arthritis is contingent upon Th17 plasticity. Arthritis Rheumatol . 2014;66(7):1955-1960. doi:10.1002/art.38647 Patient Consent: Yes, I received consent Disclosure of Interest : V. Alexiou Grant / Research Support with: NIHR BRC at GOSH, B. Jebson Grant / Research Support with: NIHR BRC at GOSH, E. Ralph Grant / Research Support with: NIHR BRC at GOSH, M. Kartawinata Grant / Research Support with: NIHR BRC at GOSH, E. Vigorito : None declared, L. Wedderburn Grant / Research Support with: NIHR BRC at GOSHR declares in kind contributions to CLUSTER by AbbVie, GSK, UCB, Sobi and Pfizer inc and non-renumerated collaborations with Lilly and Novartis.
G. Aytac 1 , B. Guven 2 , I. Aydin 2 , E. Topyildiz 3 , A. Aykut 4 , G. Aksu 1 , N. E. K. Edeer Karaca 3 , N. Kutukculer 1 1 Pediatric rheumatology, Ege University, 2 Pediatrik romatoloji, Ege Üniversitesi, 3 Pediatrik immunolgy, 4 Ege University, Izmir, Turkey Correspondence: G. Aytac Introduction: Introduction: Autoimmune polyendocrinopathy with candidiasis and ectodermal dystrophy (APECED) is an autosomal recessive disorder of immune regulation caused by mutations in the autoimmune regulatory ( AIRE ) gene. AIRE is a protein involved in the development of central immunological tolerance. APACED is characterized by multiorgan autoimmunity leading to chronic mucocutaneous candidiasis (CMC), hypoparathyroidism and adrenocortical failure. Objectives: Case: A 3-year-old boy was admitted with complaints of fever, pain and swelling on ankles and nail dystrophy on thumbs for 2 months. He was born to third-degree consanguineous healthy parents. On physical examination, the patient had splenomegaly, onychomycosis on both thumbs with oral candidiasis. Musculoskeletal examination demonstrated joint effusion with a decreased range of motion of wrist and ankles bilaterally and swelling of the knee. Laboratory investigations showed elevated acute phase reactants, hypergammaglobulinemia, and antinuclear antibody positivity. Biochemical parameters, including calcium, were normal. Rheumatic factor, antineutrophil cytoplasmic antibody, and antithyroid antibodies were negative. The patient responded well to nonsteroid antiinflammatory medications. Due to the coexistence of onychomycosis, arthritis, splenomegaly, consanguinity, and hypergammaglobulinemia, targeted next-generation sequencing of a Primary Immune Deficiency Research Panel was performed and a homozygous mutation (c.769C>T, p. Arg257Ter) in the AIRE gene was detected. Methods: There is no method section as it is a case report. Results: None Conclusion: Conclusion: The classical triad of APACED syndrome is CMC, hypoparathyroidism and adrenal failure. Inflammatory arthritis is rarely described in association with APECED. The clinical presentation of our patient reinforces the idea that non-classical manifestations can occur before classic symptoms develop. It is, therefore, useful to consider the diagnosis in patients with CMC and arthritis beyond the first year of life. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
K. Belozerov 1,1 , A. Lobacheva 1 , E. Gaidar 1 , A. Yakovlev 1 , N. Gordeeva 2 , A. Khabirova 1 , A. Kupreeva 1 , V. Masalova 1 , T. Kornishina 1 , E. Isupova 1 , L. Snegireva 1 , O. Kalashnikova 1 , L. Sorokina 1 , M. Kaneva 1 , I. Chikova 1 , T. Likhacheva 1 , M. Dubko 1 , V. Chasnyk 1 , M. Kostik 1 1 Department of Hospital Pediatrics, Federal State budgetary Educational Institution of Higher Education «St. Petersburg State Pediatric Medical University» of the Ministry of Healthcare of the Russian Federation, 2 Cardiorheumatology, Children’s City Hospital #2 of Holy Mary Magdalene, Saint-Petersburg, Russian Federation Correspondence: M. Kostik Introduction: TNF-⍺ is a one of the predominating cytokines in the pathogenesis of juvenile idiopathic arthritis (JIA). There are more than 20 years experience of using TNF-ibhinitors for treatment of JIA. Objectives: to report about our 10 years experience of treatment JIA with etanercept (ETN). Methods: We retrospectively studied cases of 375 patients, who were diagnosed as JIA with ILAR 2001 criteria and who used ETN. The treatment compliance, remission achievement, flare, as well as the switching to next biologic and the appearance of uveitis (de-novo) were taken into account. Results: according to our data, 90.4% of children were receiving DMARDs (mostly methotrexate) at the time of ETN initiation. Other biological agents were used before ETN in 6.9% of cases. Initial JIA remission was observed in 254/322 (78.9%) cases after 6 months (0,3-1,6 years) after starting therapy. Patients who hadn’t previously received biological therapy achieved remission faster (LogRank test, p=0.001), as well as those who used therapy regularly (LogRank test, p=0.004). Cumulative likelihood of flare was higher in ANA-positive patients (LogRank test, p=0.060) and RF-positive polyarthritis. The presence of concomitant non-biological treatment didn’t affect the likelihood of flare. The switch from etanercept to another biological drag occurred in 47/270 (17.4%) children, which in a quarter of cases is associated with the development of de-novo uveitis. Conclusion: ETN is effective, especially in distinct JIA categoriC. Bolton 1 , C. G. Smith 2 , A. McNeece 1 , S. Sultan 3 , V. Alexiou 1 , A. Hackland 2 , J. Crook 4 , H. D. Nguyen 1 , C. C. - 5 , M. Thyagarajan 3 , Z. Shiekh 6 , C. Cotter 3 , P. Reis Nisa 2 , E. Al-Abadi 3 , S. Chippington 7 , S. Compeyrot-Lacassagne 7 , A. Filer 3 , L. Wedderburn 1 , A. Croft 2 1 UCL, London, 2 University of Birmingham, 3 Birmingham Women’s and Children’s NHS Foundation Trust, Birmingham, 4 Guy’s and St Thomas’ Hospital, London, 5 CLUSTER Consortium, UK wide, 6 Birmingham Women’s and Children’s NHS Foundation Trust, 7 Great Ormond Street Hospital, London, United Kingdom Correspondence: H D Nguyen Introduction: When investigating disease mechanisms, site-specific differences in immune cell phenotype and function have highlighted the need to analyse cellular and molecular mechanisms at the tissue site directly 1 . In adults, the ability to obtain synovial tissue biopsies using ultrasound-guided techniques, combined with advanced tissue analytics, has revolutionised our understanding of the cellular ecosystem that operates within the joint and how it contributes to disease 2 . However, a similar approach in paediatric disease is lacking. Objectives: 1) To describe the protocol for undertaking minimally-invasive ultrasound-guided synovial tissue biopsies in children and young people with arthritis, for the purpose of research, alongside routine clinical care. 2) To investigate whether high-quality synovial tissue can be obtained that is suitable for downstream applications including single cell profiling technologies, histology and digital spatial profiling. Methods: Following ethical approval, treatment-naïve children with a diagnosis of Juvenile Idiopathic Arthritis were recruited from two large UK Paediatric Rheumatology centres. Participating families completed questionnaires prior to and following synovial biopsy. We established a workflow pipeline for performing synovial tissue biopsies in child and young people with arthritis, using standardised procedures for biopsy and sample processing. Procedures were performed by experienced paediatric interventional radiologists with experience of joint biopsy for diagnostic purposes. In brief, children who were referred for aspiration and corticosteroid joint injection were recruited. Following a general anaesthesic, required as part of routine clinical care and the establishment of sterility, synovial fluid was aspirated. Needle-biopsies were undertaken from the same needle insertion site and subsequently corticosteroid was injected into the joint. Thickened synovium was graded via ultrasonography. Results: 10 participants were recruited to the study over a nine month period, with a median age of 7 years (range 1-16 years); 90% were female. Samples obtained included core synovial biopsies, paired synovial fluid and peripheral blood. Synovial tissue fragments were processed for histology by formalin fixation and cryopreserved for downstream applications, including RNA sequencing and cell culture. Quality control indices included histological analysis to ensure the biopsied material was characteristically synovium and to grade the severity of inflammation. No significant complications were reported, however one child had a mild hemarthrosis controlled with cold saline wash out and cold compresses. Conclusion: Obtaining biopsies of synovial tissue in children with Juvenile Idiopathic Arthritis for the purpose of research, alongside clinical care is feasible. Analysis of tissue direct from the site of inflammation with single-cell RNA sequencing in children is achievable. 1 Veale DJ, Fearon U. Next-generation analysis of synovial tissue architecture. Nat. Rev. Rheumatol. 2020; 16 : 67–68. 2 Croft AP, Campos J, Jansen K, Turner JD, Marshall J, Attar M et al. Distinct fibroblast subsets drive inflammation and damage in arthritis. Nature 2019; 570 : 246–251. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
N. Brix 1,2 , M. Glerup 1 , D. Foell 3 , C. Kessel 3 , H. Wittkowski 3 , L. Berntson 4 , A. Fasth 5 , S. Nielsen 6 , E. Nordal 7 , M. Rygg 8,9 , H. Hasle 1 , S. Hagstrøm 2 , T. Herlin 1 on behalf of the Nordic Study Group of Pediatric Rheumatology (NoSPeR) group 1 Department of Pediatrics and Adolescent Medicine, Aarhus University Hospital, Aarhus, 2 Department of Pediatrics and Adolescent Medicine, Aalborg University Hospital , Aalborg , Denmark, 3 Department of Pediatric Rheumatology and Immunology, Medical Center (UKM), Muenster, Germany, 4 Department of Women’s and Children’s Health, Uppsala University, Uppsala, 56 Department of Pediatrics, Rigshospitalet, Copenhagen University Hospital, Copenhagen , Denmark, 7 Department of Pediatrics, University Hospital of North Norway, and Department of Clinical Medicine, UiT The Arctic University of Norway, Tromsø, 8 Department of Clinical and Molecular Medicine, NTNU - Norwegian University of Science and Technology, 9 Department of Pediatrics, St. Olavs Hospital, Trondheim, Norway Correspondence: N. Brix Introduction: Acute lymphoblastic leukemia (ALL) may be misdiagnosed as juvenile idiopathic arthritis (JIA) with the risk of mistreatment and prolonged diagnostic interval. In demand are sensitive and specific biomarkers in order to optimize the diagnostic process. Objectives: To evaluate the predictive value of biomarkers of inflammation like phagocyte-related S100 proteins and a panel of cytokines in order to differentiate the child with ALL and arthropathy from the child with JIA. Methods: In this cross-sectional study, we measured S100A9, S100A12 and 14 cytokines in serum from children with ALL (n=150, including 27 with arthropathy) and JIA (n=238) by multiplexed bead array assay on a MAGPIX instrument using Luminex software. We constructed predictive models using logistic regression, including ‘ten-fold cross-validation and recalibration. Thereby computing the area under the curve (AUC) as well as predicted probabilities in order to differentiate ALL from JIA. Results: The proinflammatory S100A12 was three-fold higher and the anti-inflammatory IL-10 two-fold lower in the patients with ALL and arthropathy (n = 27) compared to the ALL patients without any arthropathy (p = 0.002 and p = 0.03, respectively). The median levels of IL-4 was 12 pg/mL (IQR 10-14) in ALL with arthropathy versus 89 pg/mL (IQR 55-133) in the JIA patient, p < 0.001. The median levels of S100A9 was 47 pg/mL (IQR 30-113) in ALL with arthropathy versus 511 pg/mL (IQR 315-1281) in JIA, p < 0.001. In predictive models, we found IL-4 and S100A9 to be valuable markers to separate the child with ALL from JIA, with AUCs of 98% (95% CI: 98-100%) and 95% (95% CI: 91-97%), respectively, exceeding both hemoglobin, CRP, ESR and for IL-4 also platelets. Conclusion: The IL-4 and S100A9 serum concentration may be used as biological serum markers to distinguish ALL with arthropathy from JIA. Disclosure of Interest : None declared
R. C. Calderón Zamora 1 , A. K. Leos-Leija 1 , N. Rubio-Perez 1 , A. V. Villarreal-Treviño 1 , F. García-Rodríguez 1 , I. Peláez-Ballestas 2 , E. Faugier-Fuentes 3 1 Department of Pediatrics, Universidad Autónoma de Nuevo León, Hospital Universitario “Dr. José E. González”, Monterrey, 2 Rheumatology Unit, Hospital General de México “Dr. Eduardo Liceaga”, 3 Rheumatology Department, Hospital Infantil de México “Federico Gómez” , Cd de México, Mexico Correspondence: R. C. Calderón Zamora. Due to the multidisciplinary treatment and the wide range of therapeutic options, this disease is considered to have a high economic cost (1), however, it depends on factors as family income, coverage for medical services, the cost of medications, the frequency of exams and interventions, and disease activity and progression (2, 3). There are few reports about economic cost of JIA in the world (4), especially in those low and middle-low-income countries. Objectives: The aim of this study is to estimate the economic costs for the family of patients with Juvenile Idiopathic Arthritis in Mexico. Methods: Observational and descriptive study including caregivers of pediatric patients with JIA (according to the ILAR Classification) from January to May 2022. Data related to health care, medication, complementary exams, indirect costs, and total family income were collected by the adaptation of the questionnaire “Determination of the Economic Impact on Rheumatic Diseases, Section B. Interview regarding demographic and socioeconomic aspects”. A descriptive analysis of the data was carried out, reporting measures of central tendency and proportions. Results: Twenty-nine caregivers of patients with JIA were included, with an average age of 37 years (IQR 23-53), 25 were female, 15/29 caregivers had 12 or more years of formal education, and 48% had a remunerated job. The patients had a mean age of 10 years (IQR 5-19), 69% female, the predominant subtype is polyarticular arthritis (18, 62%), followed by oligoarticular and systemic arthritis (4, 13.7% each), no patient had psoriatic or undifferentiated JIA. One third patients had history of hospitalization due to disease activity, only 3 patients reported disability. The most prescribed treatment is DMARDs, 75% synthetics and 31% biologicals. The proportion of total family income used for JIA related costs was 34%, 14.5% associated with treatment and 19.5% from other direct and indirect health care (Table). Half of the caregivers responded that after the diagnosis of JIA in their patient, their economic situation worsened a little, and a third of the relatives considered that the situation worsened significantly. Conclusion: The results of this study demonstrate the impact of JIA on families. The direct costs of the disease are influenced by the partial or total coverage of public health institutions, however, indirect costs reflect the difficulties to specialized health care for the patients. The impact that JIA causes in the economy of the patient and his family, can significantly affect adherence to treatment and multidisciplinary care, worsen their prognosis. Trial registration identifying number: 1. Angelis A, BURQOL-RD Research Network, Kanavos P, López-Bastida J, Linertová R, Serrano-Aguilar P. Socioeconomic costs and health-related quality of life in juvenile idiopathic arthritis: a cost-of-illness study in the United Kingdom. BMC Musculoskelet Disord [Internet]. 2016;17(1). 2. Lapsley HM, March LM, Tribe KL, Cross MJ, Courtenay BG, Brooks PM, et al. Living with rheumatoid arthritis: expenditures, health status, and social impact on patients. Ann Rheum Dis [Internet]. 2002;61(9):818–21. 3. Mould-Quevedo J, Peláez-Ballestas I, Vázquez-Mellado J, et al. El costo de las principales enfermedades reumáticas inflamatorias desde la perspectiva del paciente en México. Gac Med Mex. 2008;144(3):225-231. 4. García-Rodríguez F, Gamboa-Alonso A, Jiménez-Hernández S, Ochoa-Alderete L, Barrientos-Martínez VA, Alvarez-Villalobos NA, et al. Economic impact of Juvenile Idiopathic Arthritis: a systematic review. Pediatr Rheumatol Online J [Internet]. 2021;19(1) Patient Consent: Yes, I received consent Disclosure of Interest : None declared
V. Chasnyk 1 , I. Nikishina 2 , P. Dolezalova 3 , I. Rumba-Rozenfelde 4 , N. Wulffraat 5 , R. Burgos-Vargas 6 , J. Chaitow 7 , A. Martini 8 , V. Tsekouras 9 , D. Graham 9 , C. Borlenghi 9 , B. Vlahos 9 , C. Zang 9 , N. Ruperto 10 on behalf of Paediatric Rheumatology International Trials Organisation (PRINTO) 1 Saint-Petersburg State Pediatric Medical University, Saint-Petersburg, 2 Pediatric Department, V.A. Nasonova Research Institute of Rheumatology, Moscow, Russian Federation, 3 General University Hospital, Faculty of Medicine, Charles University in Prague, Prague, Czech Republic, 4 University Children Hospital, Riga, Latvia, 56 Department of Rheumatology, Hospital General de Mexico, Mexico City, Mexico, 7 Sydney Adventist Hospital, Wahroonga, NSW, Australia, 8 Università degli Studi di Genova, Genoa, Italy, 9 Pfizer, New York, NY, United States, 10 Istituto Gianina Gaslini, Genoa, Italy Correspondence: N. Ruperto Introduction: CLIPPER2 (NCT01421069) was an 8-year, open-label extension of the phase 3b, 2-year CLIPPER study (NCT00962741) investigating the long-term safety/efficacy of etanercept (ETN) in patients with 3 subtypes of juvenile idiopathic arthritis (JIA): extended oligoarticular JIA (eoJIA), enthesitis-related arthritis (ERA), or psoriatic arthritis (PsA). Objectives: To identify predictors of sustained clinical remission over 2 and 5 years. Methods: Patients with eoJIA (2–17 years), ERA, or PsA (each 12–17 years) who received ≥1 ETN dose in CLIPPER could enter CLIPPER2. Inactive disease was defined according to the American College of Rheumatology (ACR)-approved Juvenile Arthritis Disease Activity Score (JADAS, Consolaro et al. 2009) and Wallace cut-off criteria (Wallace et al. 2004). Sustained clinical remission was defined as consecutive ≥2 years or ≥5 years of inactive disease using either set of criteria. Predictors of sustained disease remission were identified using a stepwise logistic regression model. Results: Of 127 patients enrolled in CLIPPER, 109 enrolled into CLIPPER2. 84 patients completed CLIPPER2; 27 still received active treatment. 26 patients were in clinical remission ≥2 years after starting CLIPPER according to JADAS criteria and 13 according to Wallace criteria. 6 patients were in JADAS remission and 2 in Wallace remission after ≥5 years. Predictors of response are shown in the Table. Conclusion: JADAS low disease activity (LDA) at 3 months and lower prorated number of active joints were significant predictors of 2-year sustained JADAS remission; Wallace remission at 3 months, higher Childhood Health Assessment Questionnaire score at baseline, JADAS LDA at 3 months, and baseline methotrexate use were significant predictors of 2-year Wallace remission. The low number of patients remaining in remission over 5 years limited the predictive power of these data. Trial registration identifying number: NCT01421069, NCT00962741 Patient Consent: Not applicable (there are no patient data) Disclosure of Interest : V. Chasnyk Consultant with: Amgen, Bristol Myers Squibb, Eli Lilly, Pfizer, GlaxoSmithKline, UCB, Novartis, I. Nikishina Consultant with: Pfizer, Novartis, MSD, Roche, Sobi, UCB, Eli Lilly, Ipsen, P. Dolezalova Consultant with: Sobi, Novartis, Pfizer, I. Rumba-Rozenfelde Consultant with: Pfizer, N. Wulffraat Consultant with: Sobi, Novartis, UBC, R. Burgos-Vargas: None declared, J. Chaitow: None declared, A. Martini Consultant with: Aurinia, Bristol Myers Squibb, Eli Lilly, EMD Serono, Janssen, Pfizer, Roche, V. Tsekouras Employee with: Pfizer, D. Graham Employee with: Pfizer, C. Borlenghi Employee with: Pfizer, B. Vlahos Employee with: Pfizer, C. Zang Employee with: Pfizer, N. Ruperto Consultant with: Ablynx, Amgen, AstraZeneca-MedImmune, Aurinia, Bayer, Cambridge Healthcare Research, Celgene, Domain Therapeutic, EMD Serono, GlaxoSmithKline, Idorsia, Janssen, UCB, Bristol Myers Squibb, Eli Lilly, Novartis, Pfizer, Sobi, F. Hoffmann-La Roche.
C. Eboli 1 , E. A. Conti 1 , F. Chironi 1 , S. Testa 1 , F. Lucioni 1 , G. B. Beretta 1 , A. Marino 2 , P. Cressoni 3 , S. Lanni 1 , M. Rossano 1 , G. Filocamo 1 1 Clinica Pediatrica De Marchi, IRCCS Ca’ Granda Ospedale Maggiore Policlinico, 2 ASST Gaetano Pini-CTO, 3 IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy Correspondence: A. Conti Introduction: Involvement of the temporomandibular joint (TMJ) is frequent in juvenile idiopathic arthritis (JIA). Early recognition and treatment of TMJ arthritis may potentially reduce long-term damage, such as facial asymmetry, micro and retro-gnathia, dental malocclusion, and joint ankylosis. A TMJ clinical examination protocol has been recently developed to screen TMJ involvement in JIA, and showed acceptable construct validity and reliability [1]. Ultrasound (US) has been suggested as a useful tool to evaluate inflammation and damage of TMJ in JIA [2]. Objectives: The aim of the study was to establish the validity of US in detecting signs of TMJ arthritis in JIA irrespective of the presence of clinical signs of involvement. Methods: A sample of consecutive JIA patients, either in remission or with active disease, seen at our Centre underwent clinical evaluation of TMJs. Patients with TMJ disorders (dental problems, pre-existing cranio-maxillofacial disorder, history of facial trauma) unrelated to JIA were excluded. History of TMJ pain or dysfunction was assessed by a TMJ screening questionnaire for parents together with the Italian version of the Juvenile Arthritis Multidimensional Assessment Report (JAMAR). Clinical assessment was performed according the published TMJ screening protocol. Clinical and US examinations were carried out on the same consultation and blindly each other. TMJ was considered clinically involved in case of presence of either signs of active TMJ arthritis or evidence of TMJ deformity. Clinical data were then compared to US assessment. Results: : A sample of 23 consecutive patients were recruited in the study. Overall clinical and US examinations resulted well accepted by patients and parents. The table shows the preliminary results on clinical and US assessment. Conclusion: The results observed in this preliminary cohort of patients do not allow to evidence a strong agreement between US and clinical data in detecting signs of previous or ongoing involvement of TMJ in JIA. Further studies with a larger cohorts of patients and involving the use of MRI will help to clarify the role of US in the assessment of TMJ arthritis in JIA. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
M. Corbeto, E. M. Ruzafa, L. M. Mitjana Universitari Vall d’Hebron, Barcelona, Spain Correspondence: M. Corbeto Introduction: Juvenile idiopathic arthritis (JIA) is the most common cause of inflammatory arthritis in children under 16 years of age. The only epidemiological project carried out to date in southern Europe estimated a prevalence rate of 39.2/100,000 children. However, we do not have studies evaluating the patient’s journey towards accurate diagnosis and care in our area. The time to diagnosis (TD) is a fundamental quality measure since increases in this can lead to a higher comorbidity of patients with JIA. Objectives: To describe and analyze the time to diagnosis from the onset of symptoms in patients with JIA and to identify factors associated with the patient related to a longer time to diagnosis. Methods: We conducted a retrospective cohort study by reviewing the medical records of patients diagnosed with JIA according to the ILAR criteria and treated at the Pediatric Rheumatology Unit (PRU) of the Vall d’Hebron University Hospital from 2009 to the present. Epidemiological variables were collected. Time to diagnosis (TD) was defined as the time elapsed between the onset of symptoms and the diagnosis established in the PRU. Delay in TD has been categorized as times greater than two months. Time to referral (TTR), defined as the time elapsed between the onset of symptoms and the date of referral to the PRU, was also evaluated. Qualitative variables are expressed in absolute values and percentages. Quantitative variables are expressed as medians and IQR as they present a non-normal distribution. In the bivariate analysis, the qualitative variables have been compared with the χ2 test and the quantitative variables with logistic regression. The level of statistical significance has been set at 0.05. Statistical analyzes have been performed with STATA/IC 15.1 Results: 60 patients have been included, 40 girls (66.67%) with a measured age of 8.01 years (SD 4.43). All patients come from an urban environment. The most frequent JIA subtype is oligoarticular with 28 patients (46.67%). 98% of the patients presented joint symptoms at debut followed by systemic manifestations in 21.67% of the patients. 61.67% of the patients were referred by primary care pediatrics. The TD was a median of 3.68 months (IQR 2.34 – 10.10), and the TTR was a median of 2.76 months (IQR 1.25 – 8.32). 11 patients (18.33%) did not present a diagnostic delay compared to 49 (81.66%) who were diagnosed with a delay of more than 2 months. The JIA subtype with the highest TD was polyarticular RF negative 9.26 months, but without finding significant differences between the different subtypes (p=0.523). No significant associations were observed between the delay in diagnosis and the epidemiological, clinical or analytical variables. Conclusion: The TD in patients with JIA in our cohort is a median of 3.68 months, a time similar to other cohorts previously described at a European level, with RF polyarticular JIA being the one with the highest TD, probably due to the wide differential diagnosis that characterizes this subtype. No significant differences have been found that relate the diagnostic delay and the referral professionals or previous consultations to the emergency room or previous admissions. The retrospective design of the study and the non-inclusion of socioeconomic variables or rural population constitute important limitations of the study to be considered in future researchE. Berthold 1,2,3 , A. Dahlberg 2,4,5 , H. Tydén 1,3 , B. Månsson 1,3 , R. Kahn 1,2,5 1 Skåne University Hospital, Lund and Malmö, 2 Wallenberg Center for Molecular Medicine, 3 Department of Rheumatology, Clinical Sciences Lund, Lund University, Lund, 4 Helsingborg Hospital, Helsingborg, 5 Department of Pediatrics, Clinical Sciences Lund, Lund University, Lund, Sweden Correspondence: A. Dahlberg Introduction: The incidences of chronic immune diseases are proposedly increasing(1), but whether this is applicable to juvenile idiopathic arthritis (JIA) is unestablished. Previous studies present conflicting results and reported incidence differs profoundly based on different study design and geographic regions(1-5). Objectives: To investigate the incidence of JIA over a period of 31 years, using two population-based JIA cohorts. Methods: Incident cases of juvenile arthritis 1980-2001 in the Swedish region Skåne (population 1 023 479 by 1980) were identified with registered International Classification of Diseases (ICD)-code of juvenile arthritis from both in- and outpatient care, through an ICD-code search at the local database at the regional center for pediatric rheumatology, Lund, and at the diagnosis register at the National Board for Health and Welfare (NBHW). Included ICD-codes were: 696.00, 712, 713.10-19, and 714.93 (ICD-8); 696A, 713B and 714 (ICD-9); and M08-M09 (ICD-10). To validate the diagnoses, medical records were reviewed. In total, 400 cases were confirmed 1980-2001, and to compare the incidences of JIA over time with rate ratios (RR), this new cohort of 400 cases was combined with the previously published population-based cohort of 251 validated JIA cases in Skåne 2002-10(6). Results: The annual incidence of juvenile arthritis 1980-2010 was 9.9 (95% CI 9.2-10.7) per 100 000 children <16 years, increasing from 5.9 (4.5-7.5) in 1980-84 to 14.0 (12.1-16.2) in 2005-10 with a significant RR of 2.4 (95% CI 1.8-3.2) (Table 1). When comparing the rates from the 90’s to the rates after the millennium, they were significantly higher when comparing the rate in 2005-10 to the rate in 1990-94 and 1995-99 (RR 1.6 (1.2-2.1) and 1.4 (1.1-1.7) respectively). The incidence of JIA in the cohort of 2002-2010 was increasing from 12.1 in 2002-05 to 13.9 in 2006-10, however this increase was not significant (RR 1.2 (0.9-1.5)). Conclusion: We show a significantly increasing incidence of JIA over a period of 31 years in two population-based cohorts, supporting the theory of increasing occurrence of chronic immune diseases. Although possibly missing a few mild outpatient cases in the 80’s and 90’s due to non-compulsory outpatient registration, most cases of JIA in the region are believed to be included since most cases during this period required inpatient care for treatment. To evaluate if an increasing incidence of JIA is a continuous trend, further studies are necessary. References: 1. Sevelsted A, et al. Eur J Epidemiol. 2021;36(11):1179-85. 2. Krause ML, et al. Arthritis Rheumatol. 2016;68(1):247-54. 3. Shiff NJ, et al. Arthritis Care Res (Hoboken). 2019;71(3):413-8 4. Cardoso I, et al. Int J Environ Res Public Health. 2021;18(16). 5. Danner S, et al. J Rheumatol. 2006;33(7):1377-81. 6. Berthold E, et al. Arthritis Res Ther. 2019;21(1):218De Nardi 1 , F. Rispoli 1 , S. Pastore 2 , A. Taddio 1,2 , A. Tommasini 1,2 1 University of Trieste, 2 Pediatrics, IRCCS “Burlo Garofolo” - Trieste, Trieste, Italy Correspondence: L. De Nardi Introduction: Interferon signature (IS) is a valuable instrument to quantify the expression of interferon-stimulated genes in the bloodstream, providing an indirect estimate of cells’ exposition to type I Interferon-mediated inflammation. Apart from interferonopathies, where the pivotal role of type I interferon (IFN) is recognized, dysregulation in IFN-I production and function has been observed also in some autoimmune diseases. An over-reactive IFN-I signaling pathway has been first described in Systemic Lupus Erythematosus (SLE) and Juvenile Dermatomyositis (JDM), leading to high levels of IS in such conditions. Unfortunately, there is no agreement in literature on the role of IS in other polygenic autoimmune diseases such as Juvenile Idiopathic Arthritis (JIA), whose inflammation can be dominated by other types of cytokines. Furthermore, the growing evidence of JIA heterogeneity encourages physicians to research new markers for integrating clinical and biological data, to identify subgroups of patients which could benefit from a specific therapeutical approach. Objectives: This study aims to explore the clinical significance of IS among a cohort of patients with JIA, especially concerning its association with disease stratification and prognosis. Methods: This is a consecutive case series conducted at the Institute for Maternal and Child Health “Burlo Garofolo” of Trieste, Italy. Pediatric patients between 6 months and 18 years old who accessed the Rheumatology Department of the Institute between May 2017 and December 2021 with a diagnosis of JIA (according to 2001 ILAR criteria) were recruited; patients with systemic JIA were excluded. For each patient the following variables were collected: age, sex, age at onset, family history, arthritis type, involved joints, JADAS-27 score, presence of enthesitis, tenosynovitis, uveitis, IS, erythrocyte sedimentation rate (ESR), C-reactive protein, immunoglobulins, anti-nucleus antibody and rheumatoid factor levels. Also information about previous and ongoing therapies were collected. Results: A total number of 44 patients were recruited (35 F, 9 M). Subtypes of arthritis were distributed as follows: 19 polyarticular, 19 oligoarticular (of which 6 extended), 5 psoriatic (2 oligo- and 3 polyarticular). Twenty-seven patients had a disease which required 2 nd line therapy with any biological drug, while 17 showed disease remission on 1 st line medication. Out of 44 patients, 15 had a positive IS (considered if ≥ 3). This subgroup of patients did not show statistically significant higher frequency of tenosynovitis (p=0.31), enthesitis (p=0.52), uveitis (p=0.43) or cervical spine involvement (p=0.57). IS positivity was not associated with higher levels of JADAS score (p=0.17). However, an association between IS positivity and the number of involved joints was found (p=0.019). Notably, a correlation between IS positivity and polyarticular type was detected (including extended oligoarticular and polyarticular psoriatic type, p=0.013), along with a correlation between ESR values and IS frequency (p=0.010). Patients who required biological drugs, did not have significantly higher IS levels (p= 0.33). Finally, no correlation between IS and age at onset was found. Conclusion: This study shows an association between the presence of IS positivity and the polyarticular involvement, along with a correlation between IS frequency and ESR levels. Even though these evidences are too limited to consider IS a novel biological marker for the stratification of JIA, we could speculate on the possible role of JAK inhibitors therapy for some refractory cases of JIA with elevated IS levels, in which they could be beneficial and safer. Further studies are needed to explore such clinical associations in a larger cohort of patientE. Esen 1 , A. P. Kısaarslan 1 , S. Ö. Çiçek 2 , M. H. Poyrazoğlu 1 1 Pediatric Rheumatology, Erciyes University Faculty of Medicine, 2 Pediatric Rheumatology, Kayseri City Hospital, Kayseri, Turkey Correspondence: A. P. Kısaarslan Introduction: Polyarticular Juvenile idiopathic arthritis(polyJIA) is divided into seronegative (RF-) and seropositive (RF+) polyarticular JIA. Objectives: In this study, we aimed to determine the factors affecting the duration of remission with or without treatment in polyarticular JIA patients. Methods: The data of 88 patients diagnosed with polyarticular JIA according to the ILAR diagnostic criteria in the Erciyes University pediatric rheumatology were evaluated retrospectively. The factors affecting the duration of remission were analysed by using univariate cox regression analysis. Results: Seventy(79.5%) patients were female, median age was 175.5(28-270) months,and median follow-up time was 45(4-238) months. Median joint involvement was 10(5-36). Rheumatoid factor positivity was in 11(12.5%), and anti-CCP positivity in 10(11.4%) patients. In our study, 82 (93.2%) patients had at least one large joint involvement with small joints, 8 (9.1%) patients had temporamandibular joint, and 11 (12.5%) patients had cervical vertebral involvements. 65 (73.9%) patients had acute phase reactant elevations at their first admission. Fifteen (17%) patients had accompanying diseases. Ten(11.4%) patients had uveitis. Sixty-three (71.6%) patients received systemic steroids, 22 (25%) patients received intra-articular steroid injection. All of our patientsterated with the disease-modifying drug, which was methotrexate. Sixty-five of them (73.9%) treated with at least one of the biological agents. At the last visit, 68 patients were in remission. JADI-A median score was 0(0-22) and JADI-E median score was 0(0-2). Fifty-one(58%) patients did not have articular sequelae,68 (77.3%) patients did not have extra-articular sequelae. The median time to remission with DMARD was 2(0-47) months. The median time to remission with biological agents was 14(0-50) months, and the median time to remission without medication was 0(0-96) months. The median total remission time (with treatment and without treatment) was 18 (0-114) months. The factors affecting the duration of remission analysed by using cox regression analysis are shown in table 1. Conclusion: Our results show that steroid dose and duration did not affect the duration of remission periods. Because the total remission time without treatment was very short, we think that especially biological therapy should not be interrupted. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
S. Dover 1 , H. Clairman 1 , D. Beebe 2 , B. Cameron 1 , R. Laxer 1,3 , D. Levy 1,3 , I. Narang 1,3 , R. Schneider 1,3 , L. Spiegel 1 , S. Stephens 1 , J. Stinson 1,3 , G. Tomlinson 3 , S. M. Tse 1,3 , S. Weiss 1,3 , K. Whitney 1 , B. M. Feldman 1,3 1 The Hospital for Sick Children, Toronto, Canada, 2 Cincinnati Children’s Hospital, Cincinnati, United States, 3 University of Toronto, Toronto, Canada Correspondence: B. M. Feldman Introduction: Childhood arthritis is a chronic childhood disease that is very prevalent and negatively impacts quality of life. It can be a source of discomfort and in refractory cases, despite modern biologic therapies, can lead to persistent pain and stiffness. Adolescents have poor sleep practice, and their poor sleep has been associated with negative outcomes, including pain. Poor sleep, combined with childhood arthritis, may lead to even worse health outcomes. Previous studies have shown a strong link between sleep duration and pain in children with childhood arthritis. Objectives: The aim of the present study was to determine if longer sleep duration leads to reductions in self-reported pain when compared to restricted sleep duration in adolescents with childhood arthritis. Methods: Adolescents between 12-18 years of age, with a minimum pain score of 1 on a 10cm visual analog scale, completed a 3-week sleep manipulation protocol involving a baseline week, followed by a restricted sleep (RS) condition (6.5 hrs in bed per night) and a health sleep (HS) condition (9.5 hrs in bed per night). We used a randomized crossover experimental design. Participants’ sleep was monitored at home via self-report and actigraphy. Pain was assessed up to 3 times daily using the iCanCope with Pain app. At baseline and the end of each sleep condition, participants completed validated questionnaires about pain interference, pain behaviour, daytime fatigue, and had their disease activity assessed by a trained clinician. We used Bayesian hierarchical models to estimate the effect of sleep duration on pain, as measured by iCanCope, as well as the effects on pain interference, pain behaviour, and disease activity. Results: Participants (n=31) had a mean age of 15.0 ± 1.8 years (see Table 1 for basic demographic information). Participants averaged 1.4 (95% credible interval, CrI 1.2-1.6) more hours of sleep per night during the HS condition relative to the RS condition. Compared to the RS condition, participants were 40% (odds ratio 0.61, 95% CrI 0.39-0.95) less likely to report pain in the HS condition. There were no differences between the RS and HS conditions on pain interference, pain behaviour, daytime fatigue, or disease activity. Conclusion: Adolescents with childhood arthritis improved their sleep duration, and this longer sleep duration resulted in a moderate reduction in pain. These findings complement prior correlational studies and confirm a causal relationship between reduced sleep duration and increased pain. Trial registration identifying number: Clinicaltrials.gov registration: NCT04133662 Disclosure of Interest : None declared
H. Ferjani 1 , E. Rabhi 1 , S. al mayouf 2 , D. Hadef 3 , L. Loucif 3 , W. Hamdi 1 , H. Foster 4 1 Rheumatology, Kassab orthopedics institute, Ksar Said, Tunisia, 2 pediatrics, King Faisal specialist hospital and research center, Riyadh, Saudi Arabia, 3 pediatrics, Batna 2 University, Batna, Algeria, 4 pediatrics, Population Health Sciences Institute, Newcastle upon Tyne, United Kingdom Correspondence: H. Ferjani Introduction: The paediatric Gait, Arms, Legs, and Spine (pGALS) is a quick, easy tool for evaluating musculo-skeletal problems in school-age children. It facilitates early recognition of joint problems and prompts referral to specialist center to optimize clinical outcomes. pGALS hasbeen shown to be practical and useful, with excellent acceptability. Its use was limited in Arabic-speaking countries because of the lack of an Arabic version. Objectives: Under the umbrella of the musculoskeletal matter, we aimed to translate the English pGALS into Arabic form using the Delphi approach and to evaluate the acceptability of the preliminary version in children and their parents. Methods: The original pGALS was translated by three native-speaker translators. The different propositions were mixed in a consensual way by a children’s musculoskeletal specialist. The version was validated according to the Delphi method. Delphi method is the consensus-building method, providing the consensual opinion of the experts. For each translated item of the pGALS, the experts assessed the relevance using a scale ranging from 1 to 9 (not relevant-completely relevant). Then the median was calculated giving for each item the position of the group: disagree (if the median < 3), equivocal (median between 4-6) and agreement (median >7). The degree of the convergence with the group was assessed to clarify this result: the group’s opinion is consensual if 70% of the responses were within the range of the median. For the non consensual and no relevant item, the experts propose a comment to reformulate the sentence. The acceptability of the first form was evaluated using the visual analog scale in patients with juvenile idiopathic arthritis (JIA). Results: Six experts from different Arabic-speaking countries (pediatricians and rheumatologists) were interviewed during 3 rounds by electronic survey individually and anonymously to validate the Arabic form. After each round: the median, consensus, and comments of every item are collected and a meeting with experts was held to analyze the results. During the first meeting, we were consensual and we had an agreement on 83 % of the items using the Arabic version (30 items were validated, and 6 items were reformulated. Then the forms were reformulated using the results of the preliminary rounds: opinions of the experts and their proposals during the last meeting). We were in agreement and we validated the remaining item during the second meeting. In the last round, we obtained a consensual preliminary version of the Arabic pGALS. After translation–back-translation, the Arabic pGALS was used in five pairs (child and parent) with JIA. The acceptability varied between 8 to 9, and all the questions and maneuvers were understood by the pairs. Conclusion: After the consensual translation of the Arabic pGALS and evaluation of its acceptability in the small sample, the second step is to validate this form in a large paediatric population and to assess its sensibility and specificity of this form in screening musculoskeletal disorders in Arabic countries. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
A. A. Florax 1 , M. J. Doeleman 2,3 , S. de Roock 2,3 , N. van der Linden 1 , E. Schatorjé 4,5 , G. Currie 6 , D. A. Marshall 6 , M. J. IJzerman 1 , R. S. Yeung 7 , S. M. Benseler 6 , S. J. Vastert 2,3 , N. Wulffraat 2,3 , J. F. Swart 2,3 , M. M. Kip 1,2 on behalf of UCAN CAN-DU and UCAN CURE consortia 1 University of Twente, Enschede, 2 University Medical Center Utrecht, 3 Utrecht University, Utrecht, 4 St. Maartenskliniek, 5 Radboud University Medical Center, Nijmegen, Netherlands, 6 University of Calgary, Calgary, 7 University of Toronto, Toronto, Canada Correspondence: M. M. Kip Introduction: The cost impact of withdrawing biologic DMARDs (bDMARDs) in JIA patients in clinically inactive disease is currently unknown. Objectives: To quantify the difference in costs of hospital-associated care before and after withdrawing bDMARDs (discontinuing or tapering) in JIA patients <18 years old, after they achieved clinically inactive disease on bDMARDs. Methods: Retrospective analysis of prospective data from electronic medical records of JIA patients at the Wilhelmina Children’s Hospital (Utrecht, the Netherlands), aged <18 years between 8 April 2011 and 8 April 2022, and treated with TNF-α bDMARDs, which were discontinued or tapered during this period. The hospital-associated resource use and associated costs during clinically inactive disease (i.e. pre-withdrawal) were compared to the costs within the first year after initiating withdrawal (i.e. post-withdrawal), grouped by discontinuing and tapering. The paired t-test was used to evaluate the significance of the cost difference between the pre- and post-withdrawal period. Unit prices were obtained from Dutch reimbursement lists, pharmaceutical price lists, and hospital price lists. Results: Of the 55 JIA patients, 25 discontinued and 30 tapered bDMARDs. The mean annual costs of hospital-associated care per patient were €9,906 in the pre-withdrawal period (mean follow-up of 448 days) and decreased significantly to €5,633 in the post-withdrawal period (fixed follow-up of 365 days, p<0.05). The mean absolute difference in the entire study population is €-4,273 per patient per year (i.e. -43%), Table 1. The medication costs represented 80% and 60% of total mean annual costs in the pre- and post-withdrawal period, respectively. When distinguishing between withdrawal strategies, mean annual costs per patient reduced significantly from €9,670 to €4,338 in the discontinuation group (i.e. -55%, p<0.05), and from €10,103 to €6,712 in the taper group (i.e. -34%, p<0.05). Conclusion: The mean annual costs of bDMARDs within the first year post-withdrawal period are significantly lower than the annual costs pre-withdrawal. In addition, abruptly discontinuing bDMARDs results in greater cost reductions, although there is substantial patient-level variation. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
V. Fraga, A. Ana Catarina, S. Sousa, M. J. Santos Garcia de Orta, Almada, Portugal Correspondence: V. Fraga Introduction: Juvenile psoriatic arthritis (jPsA) is a category of juvenile idiopathic arthritis (JIA), clinically heterogenous. Objectives: Characterize clinical features of jPsA and compare with adult-onset psoriatic arthritis (aPsA). Methods: Children fulfilling the ILAR criteria for jPsA and adults fulfilling CASPAR criteria for PsA were included. Demographic and clinical data was retrieved from the Portuguese registry Reuma.pt. A cross-sectional descriptive analysis of jPsA followed by a comparison with aPsA was performed. Results: A total of 11 jPsA and 132 aPsA were included. jPsA represented 8.9% of JIA patients: 8 girls (72.7%), mean age at disease onset 10.4 ± 3.7 years and at diagnosis 11.9 ± 3.9 years, all Caucasian. Five (45.5%) developed psoriasis before arthritis and in 2 patients, psoriasis followed the onset of arthritis. Family history of psoriasis in 1 st degree relatives was positive in 63.6%. The most frequent articular pattern was oligoarthritis (63.6%, n=7) and the most common musculoskeletal features were dactylitis (27.3%, n=3) and enthesitis (27.3%, n=3). Axial involvement was present in only one case. All jPsA patients were rheumatoid factor negative, six (54.5%) were ANA positive and none was HLA-B27 positive. Seven were treated with methotrexate, in combination with a biologic in four of them. The mean age of aPsA onset was 40.5 ± 18.9 years, 51.5% (n=68) were males, and the most common joint pattern was polyarthritis (59.1%, n=78). Presence of psoriasis at diagnosis was significantly more frequent in aPsA than in jPsA (84,8% vs 45.5%; p-value= 0.01). A total of 77 patients were treated with a biologic, most frequently adalimumab (n=27), etanercept (n=21) and infliximab (n=15). Seventy-two patients were treated with csDMARDs alone or in combination with a biologic. Conclusion: At disease onset, less than half of the jPsA had psoriasis, and the most common joint pattern was oligoarthritis. The presence of dactylitis, enthesitis, and a positive family history of psoriasis are helpful in establishing the diagnosis. Conversely, aPsA patients were more often male, with psoriasis at the time of diagnosis and with polyarticular disease. No differences were found concerning extra-articular manifestaS. Fuehner 1 , A. Mureseanu 1 , S. de Roock 2 , J. F. Swart 2 , G. Simonini 3 , E. Marrani 3 , P. Rovero 4 , F. Real-Fernandez 4 , J. Weber 5 , R. Cotti 5 , K. Minden 6 , D. Foell 1 , H. Wittkowski 1 1 Department of Paediatric Rheumatology and Immunology, University Hospital Münster, Münster, Germany, 2 Department of Paediatric Immunology and Rheumatology, University Medical Centre Utrecht, Utrecht, Netherlands, 3 Rheumatology Unit, Meyer Children Hospital, University of Florence, Florence, 4 Interdepartmental Laboratory of Peptide and Protein Chemistry and Biology, Department of NeuroFarBa, University of Florence, Sesto Fiorentino, Italy, 5 BÜHLMANN Laboratories AG, Schönenbuch, Switzerland, 6 Charité University Medicine and Epidemiology Unit, German Rheumatism Research Centre, Berlin, Germany Correspondence: H. Wittkowski Introduction: Therapeutic drug monitoring in patients with juvenile idiopathic arthritis (JIA) could guide dose adjustments and treatment intervals and thereby further improve patient outcomes during treatment with biologic agents. Point-of-care tests become increasingly available for routine clinical care and provide the possibility of immediate therapy decisions at the outpatient clinic. Objectives: We have tested Lateral Flow Immunoassays (LFIA) with a Point-Of-Care-Device (POC, Quantum Blue® Reader) to investigate whether they can be helpful in the context of therapy decisions and drug monitoring in patients with JIA. Methods: We included 91 JIA patients from the biobank of the German multicenter inception cohort (ICON), 71 of whom received the anti-TNF biologic adalimumab and a control group of 20 patients treated with etanercept. From these patients, 148 serum samples (stored at -80°C) were identified to measure adalimumab levels with the BÜHLMANN Quantum Blue® Adalimumab rapid test (measuring range 1.3 - 35.0μg/ml) in at least one sample per patient. Anti-drug antibodies (ADAs) were determined with the BÜHLMANN Quantum Blue® Anti-Adalimumab rapid test (measuring range 0.2 - 12μg eq /ml). Since this assay is described as drug sensitive, only samples with adalimumab levels below the detection limit (n = 41) were analyzed. For external validation 10 selected samples were analyzed in a routine laboratory with an enzyme immunoassay (EIA). An additional cross-check was performed with 21 samples from two external cohorts. In these, adalimumab was measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay and assayed for ADAs by radioimmunoassay (RIA) or ELISA (LisaTracker). Results: In our cohort, adalimumab levels above the detection limit were measured in 87 of 128 samples, from 57 out of 71 JIA patients treated with adalimumab. In 14 patients, although adalimumab treatment was documented, no adalimumab could be measured at any time point. The samples selected for external validation were measured in a routine laboratory with a correlation (Spearman r) of 0.93 ( p = 0.0012 ), thus confirming the rapid test as highly reliable. Furthermore, the comparison of rapid tests with LC-MS/MS measured adalimumab in the external partners’ samples showed a correlation of 0.86 ( p = 0.0278 ). In patients treated with Etanercept no interference occurred and no false positive result was measured using the LFIA. All samples with adalimumab levels below the detection limit were then tested for ADAs and in only one of these samples, antibodies could be detected with the used rapid test. The external laboratory was able to measure ADAs positively in two out of 10 selected samples. This may indicate that the rapid test is less sensitive than the EIA method. This trend was also observed in the cross-check measurements using the samples of the external partners. Here, the BÜHLMANN ADA test showed no false-positive result (positive predictive value of 100%) compared to the results measured with RIA and ELISA, but a negative predictive value of only 40% and 85%, respectively. Conclusion: Adalimumab LFIA rapid test with a POC-Device can be a useful tool for drug monitoring in patients with JIA. The handling is simple and tests are time saving and yet reliable. In patients with undetectable adalimumab levels, a rapid ADA test may be helpful in interpreting the absence of adalimumab. None-detectable ADA levels according to POC-Device should be validated and/or confirmed with conventional laboratory testing. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
L. Gago 1,2 , M. H. Lourenço 1,2 , R. Pinheiro Torres 1,2 , M. Guedes 3 , C. Amaro 4 , M. Costa 1 , J. C. Branco 1,2 , A. F. Mourão 1,2 1 Rheumatology, Hospital Egas Moniz, 2 Centro de Estudos de Doenças Crónicas (CEDOC), Universidade Nova de Lisboa, 3 Ophthalmology, 4 Dermatology, Hospital Egas Moniz, Lisboa, Portugal Correspondence: L. Gago Introduction: Wooly hair nevus is a hair change, where there is a curly, hypopigmented patch of hair in a restricted area of the scalp. It may be associated with ophthalmologic, auditory, renal, cutaneos, cardiac and skeletal manifestations. Objectives: The authors describe a case of a 4-year-old girl with woolly hair nevus and psoriatic arthritis associated with chronic anterior uveitis. Methods: A 4-year-old girl presented to the dermathology department with confluent erythematous papules on plaques on her thighs, elbows, and knees, and a curly patch of hair that contrasted with her straight hair. No relevant family history. In addition, the patient had a history of frequent falls. She denied arthralgias, myalgias, dactylitis. She also denied visual acuity changes. After careful objective examination she was diagnosed with psoriasis and wooly hair nevus. An ophthalmological screening was performed which detected the presence of bilateral chronic anterior uveitis, and treatment with topical corticotherapy was initiated. Due to her history of frequent falls and bilateral uveitis she was referred to our department. On physical examination she presented with arthritis of the right knee, with no associated pain, no enthesitis, dactylitis or other swollen or painful joints. She also had no strength deficits. Blood tests showed no elevation of inflammatory parameters. She tested positive for the antinuclear-antibodies, and negative for extractable nuclear antigens. She also tested positive for the human leukocyte antigen B27 (HLA-B27). An ultrasound of the right knee was performed with evidence of synovitis of this joint. An echocardiogram was also performed and did not show any alterations. Results: Considering the presence of right knee arthritis, bilateral uveitis and psoriasis associated with positive HLA-B27, the diagnosis of Psoriatic Juvenile Idiopathic Arthritis was made and the patient was medicated with Methotrexate 7.5 mg/week and prednisolone 7.5 mg, with significant improvement of symptoms. Three weeks after starting therapy the patient had no arthritis or skin lesions, however, bilateral uveitis persisted and for this reason topical ocular corticosteroid therapy was maintained. Conclusion: Woolly hair nevus may be associated with ophthalmological, auditory, renal, cutaneous, and musculoskeletal manifestations, which makes referral to different specialties extremely important. The ophthalmologic examination is always indicated in these children. Psoriatic arthritis is rare in children , representing the adult form of spondyloarthritis, but axial involvement is uncommon. Joint involvement may be mono- or polyarticular, symmetrical or asymmetrical. About 30% of patients are HLA-B27 positive. In this particular case, the ophthalmologic involvement may be related to both wooly hair nevus and psoriatic arthritis, since both conditions are associated with ocular pathology. Thus, early ophthalmologic screening is of utmost importance, for diagnosis and treatment at early stages of the disease, in order to avoid irreversible changes. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
M. Glerup 1 , C. Kessel 2 , D. Foell 2 , M. Høllsberg 1 , L. Berntson 3 , A. Fasth 4 , S. Nielsen 5 , E. Nordal 6 , V. Rypdal 6 , M. Rygg 7 , E. Arnstad 8 , S. Peltoniemi 9 , K. Aalto 10 , T. Herlin 1 on behalf of the Nordic Study Group of Pediatric Rheumatology (NoSPeR) 1 Department of Paediatrics and Adolescent Medicine, Aarhus University Hospital, Aarhus , Denmark, 2 Department of Pediatric Rheumatology and Immunology, University Hospital Münster, Münster, Germany, 3 Department of Women’s and Children’s Health, Uppsala University, Uppsala, Sweden, 4 Department 5 Department of Pediatrics, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark, 6 Department of Pediatrics, University Hospital of North Norway, Department of Clinical Medicine, UiT The Arctic University of Norway, Tromsø, Norway, 7St. Olavs Hospital, Trondheim, NorwayDepartment of Pediatrics, Levanger Hospital, Nord-Trøndelag Hospital Trust, Levanger, Norway, 9 Clinic of Rheumatology, Helsinki University Hospital, Helsinki, Finland, 10 Department of Pediatrics, New Children’s Hospital, Helsinki University Hospital, Pediatric Research Center, University of Helsinki, Helsinki, Finland Correspondence: M. Glerup Introduction: Several blood biomarkers have been suggested to track with disease activity in juvenile idiopathic arthritis (JIA) and facilitate prediction of clinical outcome. Objectives: To evaluate whether levels of biomarkers of inflammation in serum from patients with JIA close to disease onset was related to disease activity at baseline and at long-term follow-up (FU). Methods: Patients from the population-based Nordic JIA cohort study were recruited at disease onset from defined regions of Denmark, Sweden, Norway, and Finland between 1997-2000. Serum was obtained at baseline (within 6 months from disease onset) and at 18-yr FU. S100 proteins, and 14 other inflammatory markers (cytokines, chemokines) were determined by multiplexed bead array assay. Data acquisition and analysis were performed on a MAGPIX instrument using xPONENT v4.2 software (Luminex). The analyzing laboratory in Muenster was blinded for the patients’ clinical data. Results: Of the 510 patients from the Nordic cohort, serum samples from 236 patients at baseline and 284 patients at 18-yr FU were analyzed. Of these, 150 patients had paired samples taken at both time-points. Median age at onset was 6.0 yrs (IQR 2.9-10.4) and 23.6 yrs (IQR 20.5-27.6) at last FU.4% of the patients. At baseline levels of IL-1b, IL-6, IL-10, IL-17, IL-18, and sCD25 ranged 1.4-5.5 times higher than at 18-Y FU (all p<0.001) whereas baseline levels for IL-12 and MPO was half the values obtained at FU (both p<0.001). Sampled at baseline IL-6, IL-12p70, GM-CSF and S100A12 correlated significantly with baseline JADAS71 (p<0.01), but IL-1b, IL-4, IL-13 and MMP-3 correlated only weakly (0.01 Patients with active disease at 18-yr FU had significantly higher levels of S100A9, S100A12, IL-1β, IL-6, IL-12p70, IL-13, MMP-3, and GM-CSF at baseline than patients with inactive disease at FU, but levels of TNFα, IL-4, IL-10, IL-17, IL-18, CCL-2, sCD25, and MPO were not significantly different. Patients who achieved remission off medication at 18-y FU had significantly lower levels of IL-1β, IL-12p70, IL-13, MMP-3 measured at baseline compared to the rest of the cohortmay even contribute to future prediction models of long-term outcome. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
I. Gonzalez Fernandez 1 , B. Lopez Montesinos 2 , M. Marti Masanet 1 , L. Lacruz Perez 2 , I. Burgos Berjillos 2 , I. Calvo Penades 2 1 Pediatric Rheumatology Unit, Medical Research Institute Hospital La Fe, 2 Pediatric Rheumatology Unit, Hospital Universitario y Politécnico La Fe, Valencia, Spain Correspondence: M. I. Gonzalez Fernandez Introduction: Psoriatic Juvenile Idiopathic Arthritis (PsJIA) is reported to account for around 5% of Juvenile Idiopathic Arthritis (JIA). It is considered a heterogeneous entity, with observation of a bimodal distribution of age at the disease onset (as in JIA globally) and description of an early-onset and a late-onset form. Objectives: To describe a cohort of patients with PsJIA from a paediatric rheumatology unit in a tertiary hospital. Methods: Patients seen at our paediatric rheumatology unit between January 2014 and April 2022 who met PsJIA ILAR criteria and with at least 18 months of follow-up were included. Medical records were reviewed to collect demographic, clinical, laboratory, treatment and evolution data. Results: 39 patients met Vancouver criteria for probable or definite Juvenile Psoriatic Arthritis, of which 34 (87%) fulfilled ILAR criteria for PsJIA. The other 5 patients were all female, ANA-positive, early-onset JIA patients with dactylitis who met probable Vancouver criteria (four of them with psoriasis in a second-degree relative and one with nail pitting). Median (IQR) age at JIA onset was 2,78 (1,58-6,81) years. 71% were female. Median (IQR) follow-up was 11,45 (8,01-13,74) years. As expected, distribution of the age at JIA onset was not consistent with normality, resembling a bimodal distribution. Patients were divided into 2 subgroups according to the age at disease onset, group of early onset (<=6 years) with 25 patients and group of late onset (>7 years) with 9 patients. Female gender predominance was found in both groups: 18/25 (72%) in early onset and 6/9 (67%) in late onset. With respect to arthritis, 12/34 (35%) patients had oligoarticular involvement at the beginning of the disease. 26/34 (76%) had polyarticular course: 21/25 (84%) in the early-onset group and 5/9 (56%) in the late-onset group. Only one patient presented sacroilitis, in the late-onset group. 19/34 (56%) had dactylitis and 5/34 (15%) enthesitis. ANA were positive in 20/34 (59%) patients (68% early-onset group vs 33% late-onset group). HLA-B27 was positive in 5/34 (15%) patients (8% early-onset group vs 33% late-onset group). 9/34 (26%) patients had anterior uveitis, all in the early-onset group. Nail pitting was observed in 15/34 (44%) patients. Psoriasis appeared in 23/34 (68%) patients over the course of follow-up: 16/25 (64%) in the early-onset group and 7/9 (78%) in the late-onset group. Psoriasis was diagnosed after the arthritis in all patients in the early-onset group and in 3/7 (43%) patients in the late-onset group (p<0,05 Fisher test). Median (IQR) time from JIA diagnose to psoriasis diagnose 6,04 (3,27-9,16) years. Regarding treatment, all patients received MTX and 24/34 (71%) biological DMARDS. 12/34 (35%) patients switched to a second biologic drug and 2/34 (6%) patients switched to a third biologic agent (early-onset patients). Median (IQR) time until clinical remission with treatment was 1,62 (1,32-2,46) years. At last visit, 23/34 (68%) patients where in clinical remission (5/34 (15%) off therapy). Conclusion: In our cohort of PsJIA, the form of early onset is more represented, with higher tendency to ANA positivity and chronic anterior uveitis. Female gender predominance was observed in both groups. High percentage of patients were treated with biological agents, in relation with uveitis and high frequency of polyarticular course. Psoriasis diagnosis was made a median (IQR) of 6,95 (4,24-9,46) years after arthritis diagnosis in early-onset patients. Disclosure of Interest : M. I. Gonzalez Fernandez Consultant with: Novartis, B. Lopez Montesinos: None declared, M. Marti Masanet: None declared, L. Lacruz Perez: None declared, I. Burgos Berjillos: None declared, I. Calvo Penades Consultant with: Novartis, Speaker Bureau with: Novartis, Sobi, Abbvie
A. Günay 1 , Ö. F. Beşer 2 , A. Adroviç Yıldız 3 , S. Şahin 3 , M. Yıldız 3 , F. Haşlak 3 , A. Günalp 3 , Ö. Kasapçopur 3 , K. Barut 3 1 Department of Pediatrics, 2 Department of Pediatric Gastroenterology, Hepatology & Nutrition, 3 Department of Pediatric Rheumatology, Istanbul University, Cerrahpaşa - Cerrahpaşa Medical Faculty, Istanbul, Turkey Correspondence: K. Barut Introduction: Juvenile idiopathic arthritis is the most common chronic, systemic, autoimmune connective tissue disease of unknown etiology in children. Malnutrition can be seen frequently during the course of chronic diseases. Depending on the severity of malnutrition, the course of chronic diseases may be adversely affected. For these reasons, it is emphasized that malnutrition screening tests should be routinely applied in chronic patients in order to detect nutritional status disorders that may occur during the course of chronic diseases. During the course of JIA, nutritional status disorders can often be encountered as a result of various complications or related to the severity of the disease and this may adversely affect the course of the disease. There are screening tests used to determine the risk of malnutrition. Pediatric Yorkhill Malnutrition Score (PYMS) and Screening Tool for Risk of Impaired Nutritional Status and Growth (STRONGKids) are two accepted tests. Objectives: We aimed to determine the frequency of malnutrition in JIA and the possible malnutrition status that may develop in JIA patients with screening tests. Also we aimed to reveal the factors in patients with existing malnutrition and/or patients who will develop malnutrition. Methods: JIA (n=150) and FMF (n=156) patients were included in the study. All physical examinations of the patients were performed, height and weight measurements were recorded with the scale and meter in the outpatient clinic, BMI was calculated, laboratory values at the time of admission were examined, disease activities of JIA patients were determined according to JADAS and Wallace criteria. All patients’ malnutrition risk scores were determined by applying PYMS and STRONGKids malnutrition screening tests. Afterwards, the files of all patients were analyzed retrospectively. All patients were made an appointment again at least 3 months after the examination. Anthropometric measurements of each patient were repeated in the outpatient clinic, and their BMIs were calculated. Physical examinations of all JIA patients were performed, laboratory parameters were requested and the results were recorded, and the activity level of the disease was determined again. Results: According to PYMS values, malnutrition risk is seen in 11.3% (n=17) of JIA group cases. According to STRONGKids values, malnutrition risk is observed in 9.3% (n=14) of JIA group cases. According to the two screening tests, there is no statistically significant difference in the subtypes, ages, age at onset of disease, diagnosis age, drugs used, duration of drugs used and total doses of prednisolone used according to malnutrition risk. Although it is not statistically significant, it is noteworthy that the probability of malnutrition is higher in polyarticular JIA and enthesitis-related arthritis subgroups compared to other subgroups. According to PYMS and STRONGKids; JADAS applied at the time of admission and at least 3 months later and Wallace applied at the time of admission, the rate of being active was found to be statistically significantly higher than those without the risk of malnutrition. Conclusion: Our study indicates the importance of determining the risk of malnutrition in this patient group by showing that the frequency of disease activity increases in patients at risk for malnutrition in JIA cases. It is emphasized that patients in polyarticular and enthesitis-related arthritis subgroups, whose prognosis is worse than other subgroups, should be followed more closely in this regard. Disclosure of Interest : None declared
R. Gutiérrez Suárez Pediatric Rheumatology, Shriners Hospital of Mexico City, Mexico, Mexico Correspondence: R. Gutiérrez Suárez Introduction: Cohort studies can provide useful access to RWE which reflects the reality of daily clinical practice. RWE is becoming highly valuable and complementary to randomized clinical trials (RCT) in the generation of clinical evidence due to their high external validity, better generalisability, time and resource-efficiency and the possibility of long term surveillance. Objectives: To evaluate propectively several disease outcome measures, in a 10-year period inception cohort of Mexican-Mestizo patients JIA, with the aim of generating real world diagnostic, therapeutic and prognostic evidence of this patients. Methods: A10-year inception cohort was designed and conducted in three tertiary referal centers in Mexico City. Incident and prevalent JIA Mexican-Mestizo patients according to ILAR criteria were included in the cohort since its inception on January, 2012; and evaluated clinically and with laboratories at 12, 26 and 54-weeks periods and thereafter at year: 2, 3, 5, 7.5 and 10. Different demographical (referal time, time to adequate diagnosis and treatment, family income, parents education and social security); clinical (MD-centered measures: MD-VAS for disease activity and joint count; and parent/patient-centered measures: VAS of well-being and pain; disease activity (JADAS-71), functional capacity (CHAQ), articular and extra-articular damage (JADI-A and JADI-E); criteria for minimal disease activity (MDA), remission with (RWT) and without treatment (RWOT)); laboratory (RF, ESR CRP, antinuclear antibodies and HLA-B27) and therapeutic (NSAID’s, Steroids, Non biological and biological drugs: time and availability) variables were collected. Descrptive and some inferential statistics were performed for the 7-JIA subtypes according ILAR Results: A total of 265 (60 % female) Mexican-Mestizo JIA patients according with ILAR criteria were included in the inception cohort. Age at onset (median ± IR); 6.5 (3-16) years; referal time (years): 2.5 (0.1-7.2); time to adequate diagnosis (years): 2.8 (0.1-7.5) time to adequate treatment; (years): 2.8 (0.2-7); family income per month; 300 USD (50-1100 USD). There were several differences between different subtypes in terms of disease activity (JADAS-71) functional capacity (CHAQ), articular and extra-articular damage (JADI-A and JADI-E). Also a high frequency of oligoarhritis patients complete criteria for minimal disease activity (MDA) and remission with (RWT) and without treatment (RWOT)): Patients with enytehsitis related arthritis and polyarthritis RF (+) were unable in a high proportion of patients to complete MDA or RWT and without treatment RWOT. Biological availabilty for treatment (4% of patientes in the inception cohort) Principally TNF inhibitors were used.. Time for Biological treatment (years): 1.5 (0.3-10). a large proportion of patients in RF (+) polyarthritis required combination or 2 or 3 non biological drugs related to the biological avaliability. Conclusion: RWE reflects the reality of daily clinical practice and is a complement for RCT in the generation of clinical evidence. This studies enables research into the general quality of pediatric Rheumatogy care and provide insights into a broader optimization of pediatric Rheumatogy care, refined therapeutic strategies for patient subgroups as well as avenues for further research in pediatric RheumatologyK. Oen 1 , K. Toupin-April 2 , B. M. Feldman 3 , R. A. Berard 4 , C. M. Duffy 5 , L. B. Tucker 6 , J. Tian 7 , D. G. Rumsey 8 , J. Guzman 6 on behalf of ReACCh-Out Investigators 1 Department of Pediatrics and Child Health, University of Manitoba, Winnipeg, 2 School of Rehabilitation Sciences and Department of Pediatrics, University of Ottawa, Ottawa, 3 Pediatrics and Medicine, University of Toronto, Toronto, 4 Pediatrics, London Health Sciences Centre, London, 5 Pediatrics, Children’s Hospital of Eastern Ontario and University of Ottawa, Ottawa, 6 Pediatrics, BC Children’s Hospital and UBC, Vancouver, 7 Statistics and Actuarial Science, Simon Fraser University, Burnaby, 8 Pediatrics, Stollery Children’s Hospital and University of Alberta, Edmonton, Canada Correspondence: J. Guzman Introduction: The Juvenile Idiopathic Arthritis (JIA) parent global assessment (parent global) is a visual analogue scale anchored by the words 0 very well and 10 very poor, and headed by the instruction: “Considering all the ways that arthritis affects your child, rate how your child is doing by placing a mark on the line.” Despite its extensive use there has been no formal conceptualization or validation of the parent global, resulting in uncertainty as to what it measures. We hypothesized that the parent global is an assessment of a child’s health as affected by arthritis, and should be considered a disease-specific health-related quality of life (HRQoL) measure. Objectives: To 1) validate the parent global as a HRQoL measure, 2) evaluate measurement properties of accepted HRQoL measures relative to those of the parent global, and 3) assess causal pathways determining parent global scores. Methods: Data from the Research in Arthritis in Canadian Children emphasizing outcomes (ReACCh-Out) cohort were used. Measurement properties were assessed in 344 patients at enrolment and 6 months later. Causal pathways were tested by structural equation modelling to understand root causes and mediators leading to parent global scores. Results: Construct validity was supported by moderate to high Spearman correlations (0.53-0.70) of the parent global with the Juvenile Arthritis Quality of Life Questionnaire (JAQQ), Quality of My Life health scale (HRQoML), Pediatric Quality of Life Inventory (PedsQL)-Parent, and Child Health Questionnaire (CHQ)-Physical; and lower correlations (0.14-0.49) with disease activity measures (physician global assessment of disease activity (PGADA), active joint count, erythrocyte sedimentation rate(ESR)). Responsiveness of the parent global to improvement according to parent ratings (0.51) was acceptable and within the range (0.32-0.71) of that of other measures. Reliability estimates and measurement errors for all measures were unsatisfactory, likely due to the prolonged time between assessments. Causal pathways for the parent global matched those previously reported for HRQoML [1]. Conclusion: Our results support the parent global as a valid measure of HRQoL. If confirmed, the findings of previous studies using this measure and its role in JIA core sets and composite measures should be re-interpreted in this light. [1] K Oen et al, Causal Pathways to Health-Related Quality of Life in Children with Juvenile Idiopathic Arthritis: Results from the ReACCh-Out Cohort. Rheumatology (Oxford) 2021;60(10):4691-4702 Disclosure of Interest : None declared
A. Harbi, N. Balhoudi, B. Ibrahim, H. Mejaouel Medical School, Kairawan , Tunisia Harbi Introduction: Psoriatic arthritis is a heterogeneous entity defined by the association of psoriasis and arthritis with two forms: one more frequent in girls with an age of onset of about 6 years, close to oligoarthritis with a risk of uveitis, the other later, around 11-12 years, more frequent in boys, close to spondylarthropathies. It is an autoimmune inflammatory disease with hyperproduction of pro-inflammatory cytokines, particularly TNF alpha. Objectives: The objective of our case study is to describe a rare and interesting case of Juvenile psoriatic arthritis . Methods: We describe a clinical case of a child aged 13 years, complete vaccination according to age with a history of myositis post viral infection 5 years ago. Currently he presents walking disorder for 3 weeks, spinal pain, diffuse polyarthralgia with functional repercussions and interruption of his education. The muscular testing globally at 3+ but it is generated by the pain, X-ray of the painful joints: without abnormalities with an ENMG having shown no myogenic attack nor motor or sensory deficit with a cerebral-medullary MRI without abnormalities. Ultrasound of the joints: discrete effusion of the knees bilaterally .Biology: antinuclear antibodies, antiAMAM2 rheumatoid factor, anti LKM, anti SLA LC1: negative, thyroid check-up correct with the fundus showing no uveitis. On skin examination, he has erythematous scaly patches on 2 knees with post-inflammatory hypopigmentation on the elbows, nails punctuated with thimbles, distal interphalangeal attack, oral mucosa: no abnormalities: it is psoriasis, hence the need for local treatment. It is a case of juvenile psoriatic arthritis and is treated with NSAIDs, with a good outcome. Results: Juvenile idiopathic arthritis is considered an autoimmune disease, which may result from an abnormal immunological response triggered by environmental factors such as infection or trauma in a genetically susceptible individual. Psoriatic arthritis is less than 10% of JIA. In more than 60% of cases, arthritis precedes the skin manifestations of psoriasis, sometimes by several years, and usually presents as an asymmetric oligoarthritis. Monoarthritis is relatively common initially, with isolated involvement of the knee and small joints of the hands and feet. The Edmonton diagnostic criteria are used. Psoriatic arthritis is characterized by the presence of arthritis and psoriasis, or failing that, by arthritis accompanied by at least two of the following signs: - Dactylitis - Nail staining or onycholysis - Family history of psoriasis in a first degree relative. Management should be done in a specialized setting, in the context of a pediatric rheumatology consultation. As with other JIAs, treatment is based on non-steroidal anti-inflammatory drugs (NSAIDs). NSAIDs are indicated as the first-line treatment; they are prescribed in sufficient and continuous doses. They may be combined with intra-articular injections of delayed corticoids. Background treatment involves methotrexate and biotherapies. Conclusion: Psoriatic arthritis is characterised by the presence of arthritis associated with psoriasis. Non-steroidal anti-inflammatory drugs have traditionally been the main treatment for all forms of juvenile idiopathic arthritis (JIA) and other paediatric rheumatic diseases. Trial registration identifying number: . Patient Consent: Yes, I received consent Disclosure of Interest : None declared
F. Haslak, V. Guliyeva, B. Hotaman, C. Duman, M. Yildiz, A. Gunalp, A. Aliyeva, A. Adrovic, S. SahinThe most common subtype is the oligoarticular one. While autoimmunity is generally responsible for the pathogenesis of subtypes other than systemic, evidence of autoinflammation is usually pointed out in the pathogenesis of the systemic subtype. Although there are previous studies evaluating JIA patients in terms of other accompanying autoimmune diseases, psychiatric diseases, and cardiovascular diseases, there is a lack of data in the literature to evaluate all comorbidities together. Objectives: We aimed to evaluate all the comorbidities of our JIA patients and compare the results between those with systemic JIA and those with other subtypes. Methods: Among the patients with JIA under 21, who were admitted for the routine control between September 2020 and November 2020 were included in the study. Those with additional rheumatic diseases other than JIA and those with less than six months of follow-up duration were excluded from the study. Data were obtained from face-to-face interviews and their medical records cross-sectionally during these three months. Results: The study included 459 participants (Female: 62.1%) (Systemic JIA group: 57, Non-systemic JIA group: 402). The median age was 12.87 (1.53-20.95) years, and 36.8% (n=169) of the patients were under biologic therapy. About one-third of the patients (n=155) had at least one non-rheumatic disease. The most common comorbidities were allergic rhinitis (n=38, 8.27%), attention deficit-hyperactivity disorder (n=35, 7.62%), atopic dermatitis (n=28, 6.1%), allergic asthma (n=14, 3.05%) and migraine (n=10, 2.17%). While atopic diseases were seen in 16.5% (n=76) of the patients, autoimmune disease frequency was 3.26% (n=15). Although the incidence of autoimmune disease and atopic disease was not significantly different in systemic and non-systemic JIA groups, no autoimmune disease was detected in any patient in the systemic JIA group. Conclusion: The increased comorbidity burden in JIA patients brings problems of care, follow-up, treatment adherence, and decreased quality of life. This study showed that JIA patients need to be followed up with a multidisciplinary approach. Although autoimmunity was held responsible for the pathogenesis of most subtypes of JIA, it was remarkable that the most common comorbidities were atopic diseases, not autoimmune. On the other hand, none of the detected autoimmune diseases were in the systemic JIA group, and this finding was compatible with the idea of autoinflammation rather than autoimmunity is responsible for the pathogenesis of systemic JIAS. Baron 2,3 , G. Schulert 4 , A. Shusterman 3 , R. Shukrun 2,3 , Y. Binenbaum 2,3 , R. Elhasid 2,3,5 1 Pediatric Rheumatology, Tel Aviv Medical Center, 2 Sackler School of Medicine, Tel Aviv University, 3 Pediatric Hemato-Oncology Research Laboratory, Tel Aviv Medical Center, Tel Aviv, Israel, 4 Division of Rheumatology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, United States, 5 Department of Pediatric Hemato-Oncology, Tel Aviv Medical Center, Tel Aviv, Israel Correspondence: M. Heshin-Bekensteinren, causing significant morbidity. Despite the dramatic improvement in treatment, many patients do not achieve complete remission, and biomarkers for subclinical disease, flares and response to treatment are lacking. Neutrophils and neutrophil extracellular traps (NETs) were revealed to partake in pathogenesis of autoimmune and inflammatory conditions, but their role in JIA pathogenesis and as biomarkers for JIA disease activity are largely unexplored. Objectives: In this study, we aimed to characterize the neutrophil enzymatic activity and NETs in oligoarticular and polyarticular JIA and explored its association with disease activity. Methods: Neutrophils from 7 patients with oligoarticular and RF negative polyarticular JIA were isolated at time of diagnosis and after glucocorticoid intraarticular injection with or without DMARD treatment. Enzymatic activity of neutrophil granular enzymes were monitored by colorimentry, while NET formation was assessed using confocal fluorescence microscope. Results: In oligoarticular JIA patients (n=4), all disease activity parameters and neutrophil function-related parameters decreased following intraarticular steroid injection. However, only three parameters exhibited statistically significant decline, including Physician Global Assessement (PhGA), Juvenile Activity Disease Activity Score-10 (JADAS-10) and clinical JADAS-10 (cJADAS-10). Importantly, NET formation was strongly positivily correlated with the cJADAS-10 at all time points (R=0.93, p=0.007). In polyarticular JIA patients (n=3), neither clinical disease activity parameters, the CRP, or the neutrophil function-related parameters showed consistent and significant decreases after steroid injections. Similarly, there was no significant correlation was found between NETs formation and cJADAS-10 in polyarticular JIA patients. Conclusion: This is the first study exploring the link between NETs formation and JIA activity. In this pilot study we demonstrated a statistically significant linear correlation between cJADAS-10 and NETs formation in oligoarticular but not in polyarticular JIA patients. Hence, we suggest that NETs might play a role in JIA activity and may serve as a putative biomarker for treatment response. Further work is needed to validate these initial results and determine dynamics of NETosis in JIA. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
T. Holovko 1,2 , N. S. Shevchenko 1,2 , L. BogThe relevance of cardiovascular complications in patients with rheumatoid arthritis remains. This is due to the high frequency of vascular accidents (myocardial infarction and stroke) against the background of atherosclerotic vascular lesions, as well as the development of cardiopathy with the formation of heart failure. The development of chronic heart failure is manifested by a violation of the left ventricle of the heart and, above all, a decrease in its systolic function. The state of the right ventricle is not characterized. However, it is known that the functioning of the left and right ventricles of the heart is closely interconnected due to their common blood supply, the anatomical structure of muscle fibers, the common interventricular septum, pericardium, and innervation. Objectives: To study the systolic and diastolic function of the right ventricle (RV) of the heart in adolescents with juvenile idiopathic arthritis (JIA). Methods: 53 patients with polyarticular JIA, mostly girls (69.82%), mean age 13.36±0.39 years, disease duration 68.29±5.67 months were examined. All patients were on basic therapy with methotrexate at a dose of 10-15 mg/m2 of body surface, the average dose was 11.74 ± 0.37 mg/m2, the duration of treatment was 44.64 ± 5.17 months. The obtained results were compared with the indicators of healthy peers comparable in sex and age (54 adolescents, mean age 14.63 ± 0.29 years), who were in the control group. The study program included an ultrasound study of the morpho-functional state of the heart with Doppler echocardiography using a LOGIO V2 General Electric device (USA), a 3Sc-RS sensor. The systolic function of the right ventricle was characterized by indicators: ejection fraction (EFF), stroke volume (SV), right ventricular minute volume (RV). To assess the diastolic function of the right ventricle, the maximum flow rate in the early diastolic filling phase (E), the flow rate in the late diastolic flow phase (A), their ratio (E/A), the flow rate deceleration time in the early diastolic filling phase (DT) and isovolumetric relaxation time (IVRT). Results: Conclusion: The study of RV systolic function in patients with JIA showed a significant decrease in RVF compared with adolescents in the control group, and RV stroke volume and RV minute volume were significantly higher than in healthy children. Thus, in adolescents with JIA, there is a decrease in the systolic function of the right ventricle, which is accompanied by an increase in the strength of its contraction. There is also a significant increase in the rate of blood flow during the period of late diastolic flow, which indicates the inclusion of cardiac compensatory mechanisms to ensure proper intracardiac hemodynamics. Disclosure of Interest : None declared
L. Zeller 1 , P. Tyrrell 2 , S. Wang 2 , N. Fischer 1 , J.-P. Haas 1 , B. Hügle 1 1 German Center for Pediatric and Adolescent Rheumatology, Garmisch-Partenkirchen, Germany, 2 Department of Statistical Sciences, Institute of Medical Science, Toronto, Canada Correspondence: B. Hügle Introduction: Unlike in adult rheumatology, in most forms of juvenile idiopathic arthritis (JIA) no reliable biomarkers currently exist to assess joint and disease activity. However, electrophoresis is frequently changed in active juvenile arthritis. Objectives: The objective of this study was to evaluate the α2-fraction of serum electrophoresis and its main components as biomarkers for JIA, categories extended/persistent oligoarthritis and seronegative polyarthritis, in comparison with the conventionally used erythrocyte sedimentation rate and C-reactive protein. Methods: Serum samples and clinical data from 181 patients with JIA were collected. Serum electrophoresis and α2-fraction and its components were determined using standard methods. Relationship between calculated α2-fraction of serum electrophoresis (CA2F) and its components, acute-phase parameters and cJADAS27 was assessed using Pearson’s correlation coefficient and linear regression modelling, adjusting for confounding effects. Results were confirmed in a second cohort with 223 serum samples from 37 patients, using a mixed model to account for repeated measures. Results: Compared to ESR and CRP, CA2F showed higher correlation to cJADAS27, in particular for persistent oligoarthritis. Of the three components of the α2-fraction, haptoglobin showed the highest correlation to cJADAS27. Regression analysis demonstrated higher ability to predict cJADAS27 for CA2F, and especially for haptoglobin as a component thereof, than for CRP and ESR. Conclusion: Compared to conventional methods, α2-fraction of serum electrophoresis and specifically, haptoglobin show higher correlations with disease activity in common subtypes of JIA, representing excellent candidates as biomarkers for disease activity. Further studies are necessary to determine diagnostic value and correlations in other subtypes. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
V. Iacomi, N. Revenco, R. Eremciuc SUMPh “Nicolae Testemitanu”, Chisinau, Moldova, Republic of Correspondence: V. Iacomi Introduction: Evaluation of methotrexate treatment results in JIA patients is challenging in the of absence of validated predicting models or screening methods. The disease activity score is the main reference value in monitoring the therapeutic effects. Low-dose treatment may involve the liver damage that expresses the outcomes. Objectives: Assessment of the interdependence of JIA disease activity score and the degree of liver fibrosis in children that use low-dose methotrexate. Methods: Children with JIA who received low-dose methotrexate at least 6 months underwent evaluation by transient unidimensional liver elastography with the M probe. The data results were adjusted according to the EFSUMB guidelines. Results: Out of 68 JIA who were examined in our clinic, only 50 eligible patients have performed liver elastography. A sample of 38 (76%) were confirmed with the liver stiffness median greater than 4,8 kPa after 24 weeks of methotrexate use (p < 0,005). We noted a substantial decrease in DAS28, up to 1,09 compared to 2,87 (p <0,0005), in children with high liver stiffness. We also noted a decrease in liver stiffness after 144 weeks of methotrexate administration and a remaining low disease activity score for this sample. Conclusion: Low-dose methotrexate treatment has an impact on the liver stiffness in JIA. This is the toxicity outcome. The use of liver elastography as a method of its evaluation, allows to monitor it in compliance with the disease activZarina, V. Matkava, N. Irina, B. Leonid, A. SvetlanaN. Irina Introduction: There is a lot of difficulties in the medical management of patient with Down syndrome and joints involvement on the stage of diagnosis, choice of therapy and resolving numerous problems associated with comorbid conditions, including COVID19 infection. Objectives: To describe the difficulties of caring for a patient with Down’s syndrome and multiple comorbidities who has had a COVID-19 infection. Methods: Case report : Girl N., 9 years old was admitted to our observation in November, 2021. Diagnosis: Down syndrome; congenital heart defect, high pulmonary hypertension. Grade 2A circulatory insufficiency. Dislocation of left patella, subluxation of right patella Since 2018 she had pain in the wrist, knee, ankle joints and the diagnosis of Down’s arthropathy was suspected. Juvenile idiopathic arthritis (JIA) was diagnosed in March 2020, methotrexate (MTX) was started with insufficient effect. Persistent tendency to leukopenia limited the regular therapy. Results: The patient had swelling and hypermobility of all joints, inadequate assessment of the patient’s subjective pain perception. There were no laboratory activity, ANA, RF were negative. A Whole-body MRI (WB-MRI) scan detected polyarthritis with synovitis and areas of osteitis, which confirmed the inflammatory origin of the disease. Due to inefficacy of MTX therapy, leukopenia, severe multimorbidity, planned orthopedic surgery and a psychological barrier for use of injections, oral JAK inhibitor Tofacitinib (TOFA) 5 mg BID was chosen. Good results were achieved rapidly. Open reduction of the left patella was performed in January 2022. The early post-operative period was uneventful. Before discharge from the surgical hospital on 1 February a PCR revealed COVID-19 infection, up to 15% lung involvement according to CT, persistent hypercoagulability (D-dimer >10000). Girl was hospitalized in infectious department. She was treated by monoclonal antibodies, Heparin with further switched to Warfarin 5mg/d under INR control (target 2-3). This choice was apparently made due to a previously performed cardiac surgery. Warfarin was continued but adequate INR monitoring was not performed. The spokes were removed at the outpatient orthopedic clinic on 30 March as was planned. The surgeon was not informed about the warfarin treatment. Bleeding from the post-operative wound and haemarthrosis of the left knee were developed a day later. The girl admitted to the ICU in a severe condition with haemorrhagic syndrome, great anaemia, no coagulation. Warfarin an TOFA were withdrawn, blood components transfusion and aspiration of blood from the left knee were done. The condition was improved. In May 2022 the girl was admitted to our center with the clinical picture of JIA exacerbation. Control WB-MRI showed inflammatory lesions with polyarthritis, increased areas of osteitis. Due to the positive effect of earlier therapy with TOFA and the negative dynamics of interrupted treatment, the decision was made to continue treatment with TOFA. In addition, for the preventing of infectious complications intravenous immunoglobulin at the dose of 0.2 mg/kg was added to the treatment. Conclusion: In this case we met a lot of problems and obstacles regarding to diagnosis verification and choice of medicine. WB-MRI is a useful tool for identifying the inflammatory origin of arthritis in a child with Down syndrome. Peroral TOFA is preferred for patients with multimorbidity and intolerance to injections. It extremally important to keep good coordination between doctors and parents, especially in case of life-threatening condition during COVID-19 pandemic. Because of the high risk of hospital-acquired COVID-19, it is recommended to postpone any planned surgery, as possible. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
B. Jebson 1,2 , N. de Gruijter 1,3 , M. Kartawinata 1,2 , V. Alexiou 1,2 , L. Wedderburn 1,2,4,5 , E. Rosser 1,3 1 Centre for Adolescent Rheumatology Versus Arthritis at UCL, UCL, UCLH, GOSH, 2 UCL GOS Institute of Child Health, 3 Centre for Rheumatology, Division of Medicine, UCL, 4 Great Ormond Street Hospital for Children NHS Trust, NHS, 5 NIHR Biomedical Research Centre at GOSH, NIHR, London, United Kingdom Correspondence: B. Jebson Introduction: In Juvenile Idiopathic Arthritis (JIA), many patients are positive for autoantibodies known as anti-nuclear antibodies (ANA), which are secreted by B cells. Production of ANA is a hallmark of a breakdown in B cell tolerance and cellular dysregulation. Despite this, B cell biology remains understudied in JIA and the mechanisms controlling ANA production, and their contribution to pathogenicity, remain unclear. Objectives: To assess whether B cell phenotype is altered in thecompared to PBMC from age-matched healthy children and whether altered B cell subset distribution is associated with ANA status. Methods: B cell phenotype was analysed based on CD19, CD24 and CD38 expression using multiparameter flow cytometry analysis and was performed on PBMC from JIA patients (n=185), age-matched healthy controls (n=37) as well as SFMC from JIA patients (n=52, n=47 of which were paired with blood samples from the same patient). JIA patients were further stratified by ANA titre with levels of ≥1:160 being positive and <1:80 negative tested at Great Ormond Street Hospital (PBMC n=117 ANA+, n=56 ANA-), (SFMC n=24 ANA+, n=16 ANA-). Where ANA status was not available, patients were excluded from later analysis. Results: In our large cohort, the frequency of total B cells (CD19+) was significantly higher within PBMC of JIA patients compared to healthy control samples (p=0.0004). There were also higher levels of immature B cells (CD19 pos CD24 hi CD38 hi , p=0.0037) and lower levels of memory B cells (CD19 pos CD24 hi CD38 lo , p=0.0104) within PBMC of JIA patients compared to healthy controls. In agreement with previously published data 1 , when comparing B cell phenotype between JIA PBMC and SFMC, we observed that there were significantly higher levels of ‘atypical’ (CD19 pos CD24 lo CD38 lo , p=<0.0001) memory B cells, memory (CD19 pos CD24 hi CD38 lo , p=<0.0001) B cells and plasmablast (CD19 pos CD24 lo CD38 hi , p=<0.0001) within JIA SFMC. Interestingly, despite a recently published study describing B cell subset differences in the SFMC of ANA+ JIA patients compared to ANA- patients 2 , using our B cell phenotyping strategy we found no differences in the frequency or phenotype of PBMC and SFMC B cells in this cohort of ANA+ and ANA- JIA patients. Conclusion: In this JIA cohort, we found that there were B cell subset abnormalities in patients versus controls, but that these differences were independent of ANA. These data suggest that mechanisms other than altered B cell subset distribution are driving a breakdown of B cell tolerance and ANA production in JIA. Uncovering these mechanisms will form the basis of our future work. REFERENCES 1. Corcione, A. et al. Phenotypic and functional characterization of switch memory B cells from patients with oligoarticular juvenile idiopathic arthritis. Arthritis Research & Therapy 11 , R150 (2009). 2. Dirks, J. et al. CD21lo/−CD27−IgM− Double-Negative B Cells Accumulate in the Joints of Patients With Antinuclear Antibody-Positive Juvenile Idiopathic Arthritis. Frontiers in Pediatrics 9 , 143 (2021). Patient Consent: Yes, I received consent Disclosure of Interest : B. Jebson: None declared, N. de Gruijter: None declared, M. Kartawinata: None declared, V. Alexiou: None declared, L. Wedderburn Grant / Research Support with: MRC, Versus Arthritis, GOSCC, AbbVie, GSK, UCB, Sobi, Pfizer, Novartis, Lilly, Sobi, E. Rosser Grant / Research Support with: NIHR BRC
S. G. Karadağ 1 , T. Coşkuner 2 , F. G. Demirkan 3 , H. E. Sönmez 4 , S. Özdel 5 , M. Çakan 2 , G. Otar Yener 6 , K. Öztürk 7 , F. Demir 8 , B. Sözeri 2 , N. Aktay Ayaz 3 1 Pediatric Rheumatology, University of Health Sciences, Basaksehir Cam and Sakura City Hospital, 2 Pediatric Rheumatology, University of Health Sciences, Ümraniye Research and Training Hospital, 3 Pediatric Rheumatology, Istanbul University, Faculty of Medicine, İstanbul, 4 Pediatric Rheumatology, Kocaeli University, Faculty of Medicine, Kocaeli, 5 Pediatric Rheumatology, University of Health Sciences, Sami Ulus Maternity and Children’s Diseases Training and Research Hospital, Ankara, 6 Pediatric Rheumatology, Şanlıurfa Training and Research Hospital, Şanlıurfa, 7 Pediatric Rheumatology, Istanbul Medeniyet University, Göztepe Prof. Dr. Süleyman Yalçın City Hospital, 8 Pediatric Rheumatology, Acıbadem Healthcare Group, İstanbul, Turkey Correspondence: S. G. Karadağ Introduction: Juvenile psoriatic arthritis (JPsA) is one of the 7 subtypes of juvenile idiopathic arthritis (JIA) according to the International League of Associations for Rheumatology (ILAR) classification criteria. JPsA is the rarest subgroup of JIA, so the corresponding literature describing the clinical characteristics, long-term outcomes, or treatment status of JPsA is limited Objectives: To describe demographic and clinical features of children with JPsA and to compare distinct patterns of the disease between early onset and late onset groups. Methods: Patients classified as JPsA according to ILAR criteria in 7 different pediatric rheumatology centers and followed regularly for at least 6 months between 2010 and 2020 were included in the study. The files of the patients were reviewed retrospectively, and their demographic, clinical and treatment characteristics were evaluated. Results: A total of 87 (46 male/41 female) patients were included in the study. The mean age at diagnosis of JPsA was 11.9 ± 4.5. While 57 (65.5%) of the patients had psoriasis at the time of diagnosis, arthritis preceded psoriasis in 10 (11.5%) patients. There were 32 (36.8%) patients with a history of psoriasis in the first-degree relatives. Thirty (34.5%) patients had dactylitis, 28 (32.2%) had nail pitting, 36 (41.4%) had small joint involvement, 20 (23%) had enthesitis, and 14 (16.1%) had axial involvement. Sacroiliitis was detected in 11 (12.6%) patients on magnetic resonance imaging. Uveitis developed in 4 (4.5%) patients during the follow-up period. Anti- nuclear antibodies (ANA) were positive in 35 (40.2%) patients. Twelve children were in the early-onset (<5 years) group. Uveitis and ANA positivity were more common in the early early-onset group. Conclusion: About one-third of patients with JPsA do not have psoriasis at the time of diagnosis. In some patients, no skin lesion is seen during the course of the disease. Children with psoriatic arthritis seem to displaying two different phenotypes. Younger children tend to have female predominance, ANA positivity and uveitis, while older children have more axial involvement . Patient Consent: Yes, I received consent Disclosure of Interest : None declared
E. P. Kısa 1 , E. Tarakcı 2 , G. Leblebici 3 , Ö. Kasapçopur 4 1 Department of Physiotheraphy and Rehabilitation,Health Science Faculty, Biruni University, 2 Physiotheraphy and Rehabilitation,Health Science Faculty, İstanbul University- Cerrahpasa, 3 Department of Physiotheraphy and Rehabilitation,Health Science Faculty, Medeniyet University, 4 Department of Pediatric Rheumatology, Cerrahpaşa Faculty of Medicine, İstanbul University- Cerrahpasa, İstanbul, Turkey Correspondence: E. P. Kısa Introduction: Childhood rheumatic diseases are a group of diseases that can affect many organs and systems with problems such as pain, joint stiffness, atrophy and weakness. The presence of physical inactivity is one of the most frequently reported conditions. Exercise and physical activity are recognized as an important part of the treatment of children with rheumatic disease. Physical inactivity and the accompanying sedentary lifestyle aggravate problems common in pediatric rheumatic diseases such as weakness, atrophy and muscle dysfunction, chronic pain, fatigue, bone loss, insulin resistance, and decreased health-related quality of life. Conversely, some of these symptoms create a fear of the thought that physical activity may cause an exacerbation of the disease and may be significant barriers to increasing physical activity. In addition to all these problems we have already encountered in the course of the disease, the increase in the time spent at home due to the Covid-19 pandemic has made these problems even more evident. Objectives: The aim of the study was to examine the physical activity levels of children with Juvenile Idiopathic Arthritis in the COVID 19 era and to compare them with their healthy peers. Methods: Fifty-five patients with a diagnosis of oligoarticular JIA, aged 7-18 years, who applied to the Istanbul University-Cerrahpaşa Pediatric rheumatology clinic, and 52 healthy controls in the same age group were included. Participants were invited to the study by making an announcement. All participants were informed about the purpose and procedure of the study before the evaluation. The participant statement was read to the families of all participants included in the study. The sociodemographic characteristics of the participants (age, gender, height, body weight, disease duration, joint involvement, exercise habits) were evaluated with a sociodemographic questionnaire. Pain status and general well-being level in activity status were questioned with the Visual Analogue Scale (VAS). Physical activity levels and energy expenditure levels were evaluated with the 1-day physical activity scale (1-day activity diary), and functional levels were evaluated with the Childhood Health Assessment Questionnaire (CHAQ). Statistical analysis was performed using SPSS for Windows (Statistical Package ort he Social Sciences-24.0, Inc., Chicago, IL). P≤0.05 was considered statistically significant in all analyzes. Results: Fifty-five patients and 52 controls with a mean age of 12.43±6.33 and 13.24±3.17 years, respectively, were included. The mean disease duration was 4.5 years. Compared to the control group, the JIA group had significantly less time in physical activity (p=0.001), decreased energy expenditure (p=0.042), and higher CHAQ scores (p=0.001) (Table 1). Conclusion: This study demonstrated that children with JIA had significantly lower levels of physical activity, energy expenditure, and functional ability during the COVID-19 pandemic than healthy controls. It has been revealed that patients with JIA who have low physical activity levels due to pain, fatigue and fear of avoiding movement are more inactive duege to inevitable reasons during the pandemic process. This result shows us that children need correct guidance to use the time they stay at home more efficiently in order to increase their physical activity levelA. Kozhevnikov 1,2 , N. Pozdeeva 1 , S. Vissarionov 1 , S. Bogdanova 1 , G. Novik 2 1 H.Turner National Medical Research Center for Сhildren’s Orthopedics and Trauma Surgery, 2 Saint-Petersburg State Pediatric Medical University, Saint-Petersburg, Russian Federation Correspondence: A. Kozhevnikov Introduction: Juvenile arthritis (JA) is an autoimmune inflammatory joint disease. Some laboratory tests neither rule in nor rule out juvenile arthritis. An elevated ANF titer is not a diagnostic criteria for JA. C-reactive peptide and erythrocyte sedimentation rate can be normal despite marked involvement of arthritis. Calprotectin is a heterodimeric complex of S100A8/9 (MRP8/14) has been proposed as serum biomarker that reflects disease activity (J.Hurnakova et al., 2015; T. Marushko et al., 2019; Y. Boyko et al., 2020; M. Romano et al., 2021). Objectives: The aim of this study was to test the hypothesis that calprotectin is associated with structural joint damage and reflects disease activity in children with juvenile arthritis. Methods: We evaluated the level of serum calprotectin in 70 children with JIA and 20 adolescents with non-rheumatic joint pain. All children fulfilled the ILAR criteria for JIA. 50 children had active JIA, 17 of them had oligoarticular disease subtype (oJA, mean age 7,2±2,2 years), 15 – polyarthritis (pJA, mean age 9±2,3 years), 18 – enthesitis-related arthritis (erJA, mean age 13±2,7 years). 20 children were with inactive disease by DMARDs. Quantitative indicators distribution is given as a median [5th; 95th percentile]. Clinical data, radiology and laboratory results (serum Calprotectin level/sCal, Vimentin, serum IL6/sIL6, serum TNF-alpha/sTNF) were collected and evaluated in all 90 children. Results: Level of sCal in the active oJA was 2,61 μg/ml [1,015; 3,935], inactive oJA was 1,258 μg/ml [0,772; 2,254], active pJA was 5,845 μg/ml [3,408; 8,005], inactive pJA was 1,36 μg/ml [0,678; 2342], active erJA was 2,98 μg/ml [0,897; 6,876], inactive erJA was 0,94 μg/ml [0,429; 1,92]; sCal level in children with non-rheumatic joint pain was 1,288 μg/ml [0,513; 2,364]. All children with active JIA were divided into three groups depending on their treatment and aggressive disease. 1 st gr – consist of 12 children with non-erosive erJA (JADAS10-ESR 3.2 – 4.6), 2 nd gr – 16 children with erosive JA which start to therapy of methotrexate (4 erJA /10 oJA /2 pJA, JADAS10-ESR 4.8 – 8.2), 3 rd gr - 22 children with erosive JA which switch to anti-TNF drugs (2 erJA /7 oJA /13 pJA, JADAS10-ESR > 13). In the 1 st group sCal level were 1,0175 μg/ml [0,45; 2,378], sIL6 - 2,94 pg/ml [1,549; 5,617], Vimentin - 9,872 U/ml [3,87; 18,81], sTNF - 1,144 pg/ml [0,397; 3,757]. In the 2 nd sCal level were 3,81 μg/ml [2,48; 5,992], sIL6 16,15 pg/ml [1,769; 48,85], Vimentin - 13,632 U/ml [2,319; 44,492], TNF - 1,18 pg/ml [0,204; 3,54]. In the 3 rd sCal level were 4,828 μg/ml [2,93; 7,954], sIL6 - 11,048 pg/ml [1,5; 33,7], Vimentin - 17,22 U/ml [4,212; 52,1], TNF - 10,5 pg/ml [0,5; 50,43]. Statistic analysis were revealed a correlation between sCalc and active erosive JA (R 2 = 0.4159, T = 4.336, OR erosive JA = 3.3193, 95%CI 1,7006-6.4789, p=0.0079). Serum Level of vimentin, IL6 and TNF-alpha were not correlated with active stage JA and erosive joint damage. The ROC analysis of the sCalc showed that a cut-off point more of 2,9 μg/ml may be high prognostic factor for related erosive JA (AUC 0,837±0,0553, 95%CI 0,711-0.923). Conclusion: The serum levels of calprotectin are significantly associated with oligo and polyarticular JA disease activity. These results suggest that calprotectin might be superior to serum IL6 and TNF-alpha for erosive joint damage in children with JA. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
F. La Torre 1 , F. Cardinale 1 , M. G. Anelli 2 , F. Cacciapaglia 2 , G. Lopalco 2 , F. Iannone 2 1 Department of Pediatrics, Pediatric Rheumatology Center, Giovanni XXIII Pediatric Hospital, University of Bari, 2 Department of Emergency and Organs Transplantation (DETO), Rheumatology Unit, University of Bari, Bari, Italy Correspondence: F. La Torre Introduction: Although Juvenile Idiopathic Arthritis and Rheumatoid Arthritis have well-defined disease activity scores, there is no validated score during transition. In clinical practice a patient with JIA who transfer to the adult rheumatologists is evaluated with the disease activity scores validated for RA (DAS28, SDAI and CDAI) and not for JIA. Objectives: Given the differences between the “scores” used in adulthood and pediatric age, the primary objective of our study was to identify which of those scores was the best one in assessing disease activity for JIA during transitional care. Methods: Data from all cases of Juvenile Idiopathic Arthritis (JIA) with almost one year of transition follow-up, older than 16 years at time of baseline visit, were collected. In each visit (at baseline and after 12 months of follow-up) we calculated the disease activity scores used in childhood (JADAS and cJADAS) and in adulthood (DAS28, SDAI and CDAI). Because JADAS10 and JADAS27 may have a joint count lower than the active joints in a subject with polyarticular JIA, and since the need to identify a single score to correlate to the adult ones, JADAS71 was chosen. The linear regression model has been applied to continuous variables. For the discrete variables, instead, the Kendall’s tau test was used. For both methods the concordance is poor between 0-0.4; it’s fair between 0.4-0.6; it’s good between 0.6-0.8 and finally if it assumes values between 0.8-1 the agreement is excellent. Results: We recruited 26 patients with JIA, 11 patients were Polyarticular (42.3%) and 15 patients were Oligoarticular (53.1%). No patient was lost during the follow-up. All the disease activity scores were calculated at baseline and 12-month evaluation. The linear regression analysis was used to correlate continuous variables (applied to the values expressed in mean ± SD) and the results were expressed as R-square. The R-square value was 0.690 for the correlation between JADAS71 and DAS28; 0.865 for the correlation between JADAS71 and SDAI; 0.809 for the correlation between JADAS71and CDAI (Table). To correlate the discrete variables (expressed as a percentage) the Kendall’s tau test was used; the cutoffs for correlation are the same seen for R-square in the linear regression analysis. The Kendall’s tau value was 0.797 for the correlation of JADAS71 with DAS28 and 0.788 between cJADAS71 and DAS28; 0.831 for the correlation with SDAI both for JADAS71 and cJADAS71; and 0.831 for the correlation of JADAS71 with CDAI and 0,813 between cJADAS71 and CDAI (Table). Table shows the correlation between JADAS71 and cJADAS71 to the other scores used in adulthood (DAS28, SDAI, CDAI). Conclusion: Our study confirms that the Simplified disease activity score(SDAI)is the adult score that correlates better with JADAS71. This best agreement is expressed by excellent correlation evaluated by linear regression with R-square value for continue variables (0.865) and with of k (Kendall’s) value for discrete variables (0.831). New prospective studies for disease activity score in transitional care with a large number of patients will bdeclared
M. Lindegaard Pedersen 1 , A. Neve-Græsbøll 2 , M. Bonde Glerup 2 , T. Herlin 2 1 MP and AG contributed equally to the study and shares the first authorship. Department of Paediatrics and Adolescent Medicin, 2 Department of Paediatrics and Adolescent Medicin, Aarhus University Hospital, Aarhus N, Denmark Correspondence: M. Lindegaard Pedersen Introduction: With the advent of biological agents (BA) for the treatment of patients with juvenile idiopathic arthritis (JIA) achievement of remission has become a realistic goal. However, clinical remission is unattainable in some patients despite BA therapy and switching to another BA is required. The best choice of second-line BA remains unclear. Objectives: This retrospective observational study aims to describe the pattern, timing, frequency, and reasons for BA switching among children diagnosed with non-systemic JIA treated at Aarhus University Hospital, Denmark. Methods: Patients were identified by combining unique personal identification numbers, the International Code of Diagnosis (ICD10) for JIA and biologic therapy: Etanercept, adalimumab, golimumab, infliximab, anakinra, canakinumab, tocilizumab, abatacept, rituximab, tofacitinib and baricitinib. Clinical characteristics were collected retrospectively from the electronic medical records. 200 children diagnosed with non-systemic JIA who started their first biologic drug between January 1st, 2012, and March 1st, 2021, were included in the study. We compared characteristics of non-switchers vs switchers and early switchers (≤6 months) vs late switchers (>6 months). Results: We found that 37% switched to a different BA after median 6.3 (3.8-9.4) years after diagnosis and 17.5% of patients switched at least twice. In total, 6% of patients switched three or more times. The most common reason for switching was inefficacy (57%) followed by injection/infusion reactions (15%) and uveitis (13%). 77% were late switchers, and switched primarily due to inefficacy, and 23% were early switchers who switched more often due to other reasons (61%). All patients started a tumor necrosis factor inhibitor (TNFi) as initial BA (etanercept (ETN): 49.5%, other TNFi: 50.5%). The patients who started ETN as first-line BA were more likely to be switchers compared to those who started another TNFi. Conclusion: During a 6-year observation period biologic switching was observed in more than one third, primarily due to inefficacy. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
J. A. Gudmundsdóttir 1 , S. Thorgeirsdóttir 2 , V. Lundbäck 3 , C. Göngrich 3 , J. Lingman Framme 2 , K. Rydenman 2 , E. Kindgren 4 , B. R. Ludviksson 5 , S. Nilsson 6 , R. Zetterström 3 , O. Ekwall 7 , S. Lindgren 2,7 1 Children’s Medical Cente, Landspitali, Reykjavik, Iceland, 2 Department of Pediatrics, Institute of Clinical sciences, Göteborg, 3 Centre for Inherited Metabolic Diseases, Karolinska University Hospital, Stockholm, 4 Division of Pediatrics, Biomedical and Clinical Sciences, Linköping, Sweden, 5 Department of Immunology, Landspital, Reykjavik, Iceland, 6 Department of Mathematical Sciences, Chalmers University of Technology, 7 Department of Rheumatology and inflammation research, Institute of Medicin, Göteborg, Sweden Correspondence: S. Lindgren Introduction: Disturbed central tolerance mechanisms predispose to autoimmune diseases. Reduced thymic output as well as compromised central B cell tolerance checkpoints have been proposed in the pathogenesis in juvenile idiopathic arthritis (JIA). Objectives: The aim of this study was to investigate neonatal levels of T cell receptor excision circles (TRECs) and kappa-deleting element excision circles (KRECs), as markers of T- and B-cell output at birth, in patients with early onset JIA. Methods: TRECs and KRECs levels were quantitated by multiplex qPCR from dried blood spots (DBS), collected 2-5 days after birth, in 156 children with early onset JIA and 312 matched controls. Results: When analysed from neonatal dried blood spots, the median TREC level was 78 (IQR 55-113) in JIA cases and 88 (IQR 57-117) copies/well in controls. The median KREC level was 51 (IQR 35-69) and 53 (IQR 35-74) copies/well, in JIA cases and controls, respectively. Stratification by gender and age at onset of disease did not reveal any difference in the levels of TRECs and KRECs. Conclusion: T- and B-cell output at birth, as measured by TRECs and KRECs levels in neonatal dried blood spots, does not differ in children with early onset JIA compared to controlGiancaspro 1,2 , G. Tarantino 3 , P. Festa 2,4 , G. Vallogini 2 , L. M. Gregori 5 , D. Pires Marafon 3 , A. Aquilani 3 , R. Nicolai 3 , E. Marasco 3 , F. De Benedetti 3 , V. Quinzi 1 , A. Galeotti 2 , S. Magni-Manzoni 3 1 Dep.of Health, Life and Environmental Science, University of L’Aquila, L’Aquila, 2 Dentistry Unit, IRCCS Bambino Gesù Children’s Hospital, 3 Rheumatology, IRCCS Bambino Gesù Children’s Hospital, Rome, 4 AORN Santobono-Pausilipon, Naples, 5 Imaging Dep., IRCCS Bambino Gesù Children’s Hospital, Rome, Italy Correspondence: S. Magni-Manzoni Introduction: TMJ arthritis in JIA often occurs asymptomatic in the initial stages. The frequency of TMJ involvement at JIA onset has been scarcely investigated. Objectives: To assess in a multidisciplinary approach the frequency of TMJ involvement in a prospective cohort of patients at JIA onset; to describe demographic and clinical features of JIA patients with TMJ involvement at JIA onset and follow up. Methods: Consecutive patients at JIA onset in a single tertiary care centre, who provided consent, underwent rheumatologic and orthodontic assessments. Demographic, clinical features and joint counts were registered by rheumatologists. Diagnostic Criteria for Temporomandibular Disorders (DC/TMD) Axis I and Axis II assessment protocol was runned in each patient in separate orthodontic examination. The following features were registered by consensus of at least two orthodontists: presence and type of myalgia of the masticatory muscles, myofascial pain, TMJ arthralgia, TMJ associated headache, disc displacement, intermitted locking, limited opening, clinically detectable osseous changes, subluxation. The overall orthodontic assessment required 50-60 minutes. Results of the rheumatologic and orthodontic evaluations in each patient were regularly discussed in periodic multidisciplinary meetings for the definition of the subsequent diagnostic and therapeutic program. Only patients with time frame of less than 8 weeks between rheumatologic and orthodontic assessment were included in the analysis. Descriptive statistics was used. Results: One hundred and fifteen patients at JIA onset seen at the study centre from 2018 to 2021 were invited to participate. Twenty-three (22%, 78% female) performed complete rheumatologic and orthodontic assessment maximum 2 months apart; 66% and 30% were respectively affected by oligoarticular and polyarticular RF-negative JIA subtype, and presented a median age at disease onset of 5.2 years (IQR 2.8-8.5). Five patients (22%) showed at least one pathologic feature at the DC/TMD assessment; 3 of them presented any TMJ sign/symptom also at the rheumatologic examination. TMJ MRI investigation was required in one patient (female, oligo JIA, of 9 years of age), with confirmation of TMJ active involvement. MRI follow up after 11 months, with meaningwhile subcutaneous methotrexate and TNF-inhibitor treatment, showed structural improvement and no disease progression. At the last follow up, after a median of 24 months (IQR 7-27), only 2 of the 5 JIA patients with baseline pathological signs/symptoms still presented clinically detectable TMJ involvement, with no progression; 1 patient negative for TMJ involvement at JIA onset (6%) reported mild TMJ symptoms at the last follow up visit, with no indication to imaging exam or medical treatment. Conclusion: Though the relatively low rate of combined rheumatologist’s and orthodontist’s assessment in the study cohort, TMJ involvement was detected in a relatively high proportion of patients at JIA onset. Joined efforts are required for increasing the performance of the clinical and imaging tools for quick identification and treatment of this potential invalidating feature of JIA. Improvement in the feasibility of the diagnostic investigations are also urgently required. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
R. Isguder, Z. Kizildag, R. Torun, T. Aydın, B. Makay, S. E. Unsal okuz Eylül University Faculty of Medicine, IZMIR, Turkey Correspondence: R. Isguder Introduction: Juvenile idiopathic arthritis (JIA) is a chronic and heterogeneous disease causing structural changes in the joints. Treatment aims to stop inflammation, relieve pain, maintain joint function, prevent damage, and suppress disease activity. As treatment options increase, maintenance of drug-free inactive disease (ID) has gained more importance. Methotrexate (MTX) is the most commonly used agent among synthetic DMARDs in JIA. Nevertheless, there is no consensus on how and when to stop MTX to maintain drug-free remission after ID. Factors that increase the risk of flare, apart from the drug withdrawal strategy are also unclear. Objectives: To determine the factors that increase the risk of disease flare in patients with JIA who stopped MTX monotherapy following inactive disease. Methods: Files of all JIA cases between 1992-2022 were examined retrospectively. Patients who stopped MTX monotherapy following inactive disease were evaluated. Patients with disease flare and ongoing ID were compared. Demographic data, JIA subgroup, age of disease onset, autoantibodies, acute phase reactants, MTX dose and administration route, MTX discontinuation method, and time interval were recorded. Results: Among the files of 1,036 patients with JIA, a total of 333 patients were identified as MTX monotherapy, and ID was achieved in 138 (41.5%). Among 138 patients, 105 (76%) were female and median age was 5.8 years. Oligoarticular JIA was the most frequent type. Other subtypes included polyarticular JIA, systemic-onset JIA (SoJIA), and juvenile psoriatic arthritis (JpsA) (Table-1). Patients reached ID in median 7.9 months after starting MTX, and MTX treatment was discontinued median 1.02 year after ID. The disease flare developed in 67 (48.6%) of the cases. The ID continued in 71 patients (51.4%) (Table-1). The flare developed median 1.6 years after ID and median 8.8 months after discontinuation of MTX. The follow-up period of cases with persistent inactive disease was 3.06 years. The age at disease onset and at diagnosis were lower in the group with flare, and SoJIA cases were less frequent in this group. The anti-nuclear antibody (ANA) positivity was more frequent in cases with flare. At MTX initiation visit, the median C-reactive protein (CRP) value in the group with the flare was found to be higher compared to the patients with ongoing ID (Table-1). The risk of flare increased in cases with a CRP value above 6.7 mg/L at the onset of MTX, age at disease onset below 5.1 years, and age at diagnosis below 5.9 years. There was no difference between the two groups in terms of MTX dose and administration route, the duration to reach inactive disease, MTX discontinuation time, and method. Conclusion: In this study, risk of flare was associated with early disease onset, ANA positivity and high CRP values at the beginning, rather than the administration and discontinuation method of MTXN. Maldar, A. Khan, A. PrabhudesaiN. Maldar Introduction: Charcot’s neuropathic osteoarthropathy (CNA) is a rare eponymous condition associated with autonomic sensory neuropathy, first described in 1868. Warm, painless boggy joints and instability, erosive arthropathies, chronic osteomyelitis and deformities are the predominant manifestations. Differentials are congenital insensitivity to pain and anhidrosis (CIPA), familial dysautonomia(Riley-Day syndrome), hereditary sensory motor neuropathy (Charcot-Marie-Tooth disease), traumatic nerve injuries, syringomyelia and meningomyelocele. CIPA is caused by mutations in NTRK1 gene and is classified as type IV hereditary sensory and autonomic neuropathy according to the Dyck’s classification. Objectives: We present a case series of 3 CNA misdiagnosed as inflammatory/infective conditions. Methods: Case 1 : 18 months-old girl born of third-degree consanguinity, presented with low grade fever, tender hard swelling around the right shoulder extending to the distal forearm with palmoplantar skin rash. Suspecting hereditary autoinflammatory bone disease with chronic non-bacterial osteomyelitis as a feature, a clinical exome was performed. On reverse phenotype corelation there was history of self-mutilation, absence of pain during immunization with anhidrosis. Case 2 : An 8-year-old boy was referred for fever and right ankle swelling, misdiagnosed as JIA, was on steroids and disease modifying drugs. There were hyperkeratotic lesions on both heels and large warm, boggy left ankle swelling albeit with a full and painless range of motion. Neurological evaluation suggested gait disturbance, normal muscle power, intact temperature and deep tendon reflexes with insensitivity to pain. Suspecting a syndromic arthropathy, a whole exome was sent. Case 3 : 7-year-old boy had trauma induced fracture in distal shaft of left tibia. In a few months, this was followed by painful swelling that progressed into chronic osteomyelitis of tibia. He had a limp, boggy left ankle, high steppage gait with foot drop and reduced sensations in the affected foot. He was treated by debridement with saucerization of left distal tibia with common peroneal nerve release. Results: Conclusion: While traditional teaching includes Blau syndrome and pigmented villonodular synovitis as differentials of a large boggy warm joint, addition of the adjective painless, should bring to mind CNA. This entity may remain undiagnosed for long and be mistaken for commoner rheumatological entities. Management consists of counselling regarding the disease, need for orthotics and prevention of accidental or thermal injuries. Simple questions pertaining to pain perception, sweating, local hair loss and bedside neurological evaluation easily provide the diagnosiM. Mannion 1 , C. Chen 2 , O. Halyabar 3 , S. Paetkau 4 , T. Qui 2 , B. Huang 2 on behalf of for the PR-COIN Investigators 1 University of Alabama at Birmingham, Birmingham, 2 Division of Biostatistics, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, 3 Boston Children’s Hospital, Boston, MA, United States, 4 parent, Hospital for Sick Children, Toronto, Canada Correspondence: M. Mannion Introduction: The goal of treatment for juvenile idiopathic arthritis (JIA) is inactive disease (ID). The best treatment for each patient to maximize ID and minimize overtreatment is unknown. Objectives: The objective of this study was to assess if clinical measures could predict the onset of ID or drug-free inactive disease (DFID) within 2 years of diagnosis of non-systemic JIA. Methods: Using an inception cohort from a large pediatric rheumatology clinic in the US from 2009 to 2020, we identified patients with non-systemic JIA in the electronic health record (EHR) with ≥ 2 clinical visits and ≥ 2 years of follow up following diagnosis. JIA category at baseline is reported; oligoarticular (oligo), rheumatoid factor (RF) - polyarticular (poly), RF+ poly, and all other non-systemic. Medications were classified into systemic corticosteroids, non-biologic (nb) disease modifying anti-rheumatic drug (DMARD), biologic (b) DMARD, and intra-articular corticosteroid injections. ID was defined as no active joint count (AJC), no enthesitis, no active uveitis, and physician global (PGA) <1. Descriptive statistics and Kaplan-Meier curves for time to ID and DFID were calculated based on baseline characteristics. Cox Proportional hazard (CoxPH) modeling was used to evaluate the effect of baseline characteristics and 1 st year medication use on onset of ID and DFID. Results: 605 patients with JIA were included (Table). By 1(2) years post diagnosis, 52(73)% and 28(42)% achieved ID and DFID respectively. Time to 1 st ID or DFID is significantly different (Log-rank test P=0.001 for ID; P<.0001 for DFID) by category; median (95% CI) time to ID and DFID in oligo 0.76 years (0.63, 0.90), 1.65 (1.08, 2.24), RF- poly 1.23 (1.01, 1.49), 5.05 (3.21, 7.83), RF+ poly 1.36 (0.63, 2.79), >2.22, and other types 0.90 (0.73, 1.18), 2.87 (1.99, 4.67). Of 30 RF+ poly patients only 10 achieved DFID, the median time to DFID could not be estimated. JIA category was not significant in multivariable CoxPH analyses, but lower clinical juvenile disease activity score (cJADAS), shorter time from symptoms to diagnosis, and private insurance were significantly associated with sooner time to ID and DFID. Less DMARD use (b and nb) was associated with sooner DFID, but more bDMARD use was associated with sooner ID. Conclusion: In the two years after diagnosis, 73% of JIA patients achieved ID and 42% achieved DFID. There are disease characteristics associated with sooner time to ID and DFID, but further research is needed to predict medication needs for patients with JIA. Patient Consent: Not applicable (there are no patient data) Disclosure of Interest : M. Mannion Grant / Research Support with: Rheumatology Research Foundation Norman B Gaylis, MD Clinical Investigator Award, C. Chen: None declared, O. Halyabar: None declared, S. Paetkau: None declared, T. Qui: None declared, B. Huang: None declared
M. L. Mannion, F. Xie, T. Beukelman, J. R. Curtis on behalf of for the CARRA Registry Investigators University of Alabama at Birmingham, Birmingham, United States Correspondence: M. L. Mannion Introduction: Current recommendations suggest treatment escalation for juvenile idiopathic arthritis (JIA) until the disease activity target is reached. Objectives: We compared patient reported outcomes (PRO) 6 months after maximally tolerated disease activity level. Methods: We included all individuals enrolled with non-systemic JIA in the Childhood Arthritis and Rheumatology Research Alliance (CARRA) Registry. We defined the maximally tolerated disease activity at the registry visits with no medication change for >180 days prior. Individuals could contribute more than one observation but were excluded for a medication change between the index and 6 month follow up visit. Individuals were considered to have polyarticular (poly) course if total joint count was >4 and oligoarticular (oligo) course if total joint count was ≤ 4. Disease activity was classified based on clinical Juvenile Arthritis Disease Activity Score (cJADAS) inactive disease (ID; oligo ≤1.1, poly ≤2.5), cJADAS low disease activity (LDA; oligo ≤2, poly ≤3.8), American College of Rheumatology (ACR) provisional criteria for clinical inactive disease (ACRCID) and ACR preliminary criteria for clinical inactive disease (Wallace). Change in outcome variables (Patient Reported Outcomes Measurement Information System [PROMIS] pain interference, mobility, and pediatric global health) from index to 6 month visit was stratified by disease activity status at the index visit and presented as relative change determined by measure specific minimal clinical important difference. The association with tolerated disease activity state was compared by descriptive statistics. Results: 6,235 individuals with JIA and 10,653 observations were included. The tolerated disease activity state was cJADAS ID in 36%, cJADAS LDA in 41%, ACRCID in 32%, and Wallace criteria in 33%. At the index visit the median (interquartile range; IQR) pain interference was 49 (40.6, 56.6, n=6359), mobility was 56 (43, 58.5, n=6670), and global health was 42.1 (37.9, 45.7, n=8445). cJADAS ID, cJADAS LDA, ACRCID, and Wallace were all associated with differences in relative change of mobility, but only cJADAS ID and cJADAS LDA were associated with relative change in pain interference or global health. Conclusion: Individuals with JIA often have higher than ID or LDA at the time of no medication change and have a lower global health than population norms. There were similar frequency of worsening for all measures and tolerated disease activity states; these results do not suggest preference of one criteria set over anotherF. Xie: None declared, T. Beukelman Consultant with: Novartis, UCB, J. Curtis: None declared
F. Milatz 1 , R. Trauzeddel 2 , T. Kallinich 3,4 , M. Klaas 5 , H. Girschick 5 , S. Hansmann 6 , G. Horneff 7,8 , D. Windschall 9,10 , J.-P. Haas 11 , N. Baumeister 12 , M. Niewerth 1 , K. Minden 1,13 1 Epidemiology and Health Services Research, German Rheumatism Research Centre, 2 Department of Paediatrics, Helios Klinik Berlin-Buch, 3 Pathophysiology of Rheumatic Inflammation, German Rheumatism Research Centre , 4 Department of Pediatric Respiratory Medicine, Immunology and Critical Care Medicine, University Medicine Charité Berlin, 5 Children’s Hospital, Vivantes Hospital im Friedrichshain, Berlin, 6 Center for Pediatric Rheumatology, autoinflammation reference centre Tuebingen (arcT), University Children’s Hospital Tuebingen, Tuebingen, 7 Department of Paediatrics, Asklepios Clinic Sankt Augustin, Sankt Augustin, 8 Department of Pediatric and Adolescents Medicine, University of Cologne, Cologne, 9 Clinic of Paediatric and Adolescent Rheumatology, Northwest German Center for Rheumatology, St. Josef-Stift Sendenhorst, Sendenhorst, 10 Medical Faculty, Martin Luther University of Halle-Wittenberg, Halle, 11 Center for Pain Treatment in Young People, German Center for Paediatric and Adolescent Rheumatology, Garmisch-Partenkirchen, 12 Department of Sport and Health Sciences, Technical University of Munich, Munich, 13 Department of Rheumatology and Clinical Immunology, University Medicine Charité Berlin, Berlin, Germany Correspondence: F. Milatz Introduction: JIA have been linked to reduced physical activity (PA) and increased time spent sedentary [1]. While structured activities in which young patients participate (e.g. at school) are consistent and limited in scope, after-school or weekend activities, in contrast, encompass a broader range of behaviors. Rrecording a wide range of intensity under daily life conditions is an important prerequisite for deriving measures to promote PA. Objectives: Since it is assumed that adolescents’ PA is lower on weekends compared to weekdays or school days, this study aimed i) to objectively assess PA in detailed intensity range over the course of a week and ii) to determine the proportion of patients achieving the WHO recommended minimum level of at least 60 minutes moderate-to-vigorous physical activity (MVPA) daily. Methods: Within the framework of the ActiMON study as part of the TARISMA research network, data were collected at several German paediatric rheumatology centres. In the period from June 2021 to April 2022, measurements were collected exclusively outside lockdowns and during compulsory attendance at schools. The study sample included 12- to 18-year-olds with JIA who wore an accelerometer (ActiGraph wGT3X-BT) on an elastic waist band during the waking hours of a 7-day period for measuring PA in categories, including sedentary behavior, as well as light, moderate and vigorous PA. Absolute and percentage intensity distributions were evaluated daily. Clinical parameters of participating patients were used from the National Paediatric Rheumatologic Database (NPRD). Results: Data of 54 patients (mean age 14.9 ± 1.8 years, female 71%, patients’ disease duration 7.9 ± 4.6 years, polyarthritis 41%, cJADAS-10 2.3 ± 2.4) were available for evaluation. Almost 70% of participating adolescents met the WHO-recommended minimum level of MVPA, while the average daily step count achieved was 7107. The average wear time was 857 min daily from Monday–Thursday with significant deviations from the mean on Friday (+46 min), Saturday (–40 min), and Sunday (–117 min). While the number of steps was slightly higher during the week than at the weekend, absolute MVPA times were lower. However, the percentage distribution remained constant over all days both for girls and boys. The average daily time spent sedentary was 10.8 ± 1.9 hours (no gender differences), with no relevant differences between weekdays and weekend. Conclusion: While a large proportion of patients achieve the WHO recommended minimum level of PA, they exhibit very pronounced sedentary behavior. The percentage distributions of the different physical behavior intensity categories are similar over all weekdays and weekend days. Interventions should generally try to shift activity away from sedentary behavior towards a more active lifestyle. Further results are expected with ongoing recruitment. References: [1] Gualano B:368-379. Acknowledgement: ActiMON is funded by the Federal Ministry of Education and Research (01EC1902F) Trial registration identifying number: German Clinical Trials Register DRKS00022258 Patient Consent: Yes, I received consent Disclosure of Interest : None declared
K. Mördrup 1 , J. G. Jungner 2 , E. Broström 3 , C. Bartholdson 3 1 Women’s and Children’s Health, Karolinska University Hospital, 2 Women’s and Children’s Health, Karolinska Institutet, 3 Women’s and Children’s health, Karolinska University Hospital, Karolinska Institutet, Stockholm, Sweden Correspondence: K. Mördrup Introduction: Juvenile Idiopathic Arthritis (JIA) is one of the most common acquired diseases during childhood in Sweden with approximately 200 children diagnosed every year 1 . Being diagnosed with JIA can be shocking and overwhelming for the child and the parents 2 , in addition all information given directly after the diagnose can be difficult to assimilate 3,4 . They therefore need supportive care by repeated information and communication between the scheduled visits 5 . A one-year support program, including scheduled visits and the possibility of direct contact with a nurse was therefore developed and offered to all newly diagnosed children and their parents to improve quality of care. Objectives: To explore parents experiences of participating in the support-program. Methods: When the child was diagnosed with JIA, the child and the parents were offered to participate in the support program from the time of diagnose to one year ahead. The program included seven structured person-centered visits. The nurse was coordinating and participating in all the visits (including visits with physicians) from the start. After completing the program, parents were invited to participate in semi-structured interviews. Fieldnotes were taken by the researcher who conducted the interviews and qualitative analysis was performed. Results: The interviewed parents were ten mothers and six fathers, and their children were between 1 and 16 years old when they were diagnosed with JIA. Data from the field notes show that the families greatly appreciated the possibility of direct contact by phone with the coordinating nurse i.e. good accessibility. They also found continuity as important at the unit and that they expressed great trust for the healthcare professionals. The parents described a professional and holistic care where their child have been listened to and treated from a child’s perspective. The family felt safe about the disease and medication. The coordination of the visits were described as good and the advantage to have a patient responsible nurse was mentioned. Some parents experienced that they felt they were included in a new familly. Conclusion: The 16 Swedish parents who had participated in the one-year support program after their child was diagnosed with JIA were very satisfied with the care they had received. We thus conclude that the new one-year program is a suitable way to improve quality of pediatric rheumatology care and supports the families during their first yeaNaddei 1 , F. Bovis 2 , F. Ridella 2 , C. Trincianti 2 , S. Pastore 3 , K. Minden 4 , M. Ekelund 5 , P. Barone 6 , S. Scala 1 , E. Patrone 1 , N. Ruperto 1 , A. Ravelli 1 , A. Consolaro 1 on behalf of The Paediatric Rheumatology International Trials Organisation (PRINTO) 1 Istituto Giannina Gaslini, 2 Università degli Studi di Genova, Genoa, 3 IRCCS Burlo Garofolo, Trieste, Italy, 4 German Rheumatism Research Centre, Berlin, Germany, 5 Ryhov County Hospital, Jonkoping, Sweden, 6 Catania University Hospital, Catania, Italy Correspondence: R. Naddei Introduction: Assessment of disease activity is a crucial component of the clinical management of children with juvenile idiopathic arthritis (JIA). According to the most recent requirements, both parent’s and children’s perception should be considered when evaluating the disease course and assessing effectiveness of therapy. Therefore, a new disease activity evaluation tool, based on parent/patient-centered outcome measures, is under development and named parent/child Juvenile Arthritis Disease Activity Score (par/childJADAS). Objectives: Aim of this study was to evaluate responsiveness to change of the parJADAS and to conduct an explanatory factor analysis (EFA) on the scores’ items. Methods: The parJADAS and childJADAS include 4 measures: 1) parent/child assessment of disease activity, rated on a 0-10 visual analogue scale (VAS); 2) assessment of pain intensity, rated on a 0-10 VAS; 3) self/proxy count of any swollen or painful joint up to a maximum of 10 joints; 4) assessment of morning stiffness (MS) on a Likert scale, ranging from no MS (0 points) to > 2 hours of MS (10 points). Responsiveness to change was assessed by computing the standardized response mean (SRM) in a subgroup of JIA patients enrolled in the PharmaChild registry, including subjects having a study visit at the time of biologic treatment initiation and a subsequent study visit no more than 6 months later, with a subjective rating of improvement by the attending physician. An EFA on the 4 items of the par/childJADAS was performed on a multinational dataset of JIA children enrolled in the study of Epidemiology, treatment and Outcome of Childhood Arthritis (EPOCA). The factors were extracted according to the principal factors method and the optimal number of factor extraction was based upon eigenvalues ≥ 1. The factors were rotated by the varimax method. Results: Since the amount of children’ observations in the corresponding dataset was not sufficient, responsiveness to change was assessed only for the parJADAS. Sixty patients met the requirements for SRM analysis. Second visit was at a median of 37 days after biologic treatment initiation. The SRM value obtained was 0.71. The EFA was conducted on data from 8,431 parents and 5,873 children of EPOCA dataset, who had the 4 items of the scores available. The Kaiser–Meyer–Olkin measure was 0.79 and 0.78 for the parents’ and the children’s samples, respectively, indicating that both samples were adequate, and the Bartlett’s test of sphericity resulted significant (p<0.0001) for both scores, indicating that a EFA may be useful. The EFA showed that one factor explained 59.0% of the variance in the parent sample and 61.0% in the child one. The factor loadings were high for both the samples, ranging from 0.60 (joint count and MS) to 0.90 (parent/patient assessment of disease activity, pain intensity level). Conclusion: The parJADAS SRM was in moderate range in patients judged as improved by the caring physician after starting a biological medication, suggesting that the responsiveness to change of the score is good. The EFA showed that the 4 items of both parJADAS and childJADAS work well together, indicating a good internal consistency. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
K. Nguyen 1 , J. Barsalou 2 , S. Benseler 3 , R. Berard 4 , G. Chédeville 5 , P. Dancey 6 , E. Dermikaya 4 , C. Duffy 7 , K. Houghton 8 , A. Huber 9 , N. Johnson 3 , D. Levy 10 , L. Lim 11 , K. Oen 12 , J.-P. Proulx-Gauthier 13 , A. Rosenberg 14 , D. Rumsey 11 , H. Schmeling 3 , L. Tucker 8 , R. Yeung 10 , J. Guzman 8 1 University of British Columbia, Vancouver, 2 Pediatrics, University of Montreal, Montreal, 3 Pediatrics, University of Calgary, Calgary, 4 Pediatrics, Western University, London, 5 Pediatrics, McGill University, Montreal, 6 Pediatrics, Memorial University, St. John’s, 7 Pediatrics, U of Ottawa, Ottawa, 8 Pediatrics, University of British Columbia, Vancouver, 9 Pediatrics, Dalhousie University, Halifax, 10 Pediatrics, University of Toronto, Toronto, 11 Pediatrics, University of Alberta, Edmonton, 12 Pediatrics, U of Manitoba, Winnipeg, 13 Pediatrics, Laval University, Quebec City, 14 Pediatrics, U of Saskatchewan, Saskatoon, Canada Correspondence: K. Nguyen Introduction: Treatments for juvenile idiopathic arthritis (JIA) have evolved at an accelerated pace over the last decade. It is important to document these changes and whether there has been concurrent improvements in patient outcomes. Objectives: To compare JIA treatments and outcomes in the first year after diagnosis from two cohorts in Canada, the 2005-10 Research in Arthritis in Canadian Children emphasizing Outcomes (ReACCh-Out) cohort and the 2017-21 Canadian Association of Pediatric Rheumatology Investigators (CAPRI) Registry cohort. Methods: To enhance the validity of the comparison, we included only patients recruited within three months of JIA diagnosis, used the same outcome criteria in all subjects, and compared Kaplan-Meier estimates at 70 weeks in both cohorts (1 year plus window for data capture). Inactive disease was defined as per Wallace criteria and inactive and minimally active disease were defined according to clinical Juvenile Arthritis Disease Activity Score 10 (cJADAS10) using the recently revised cut-offs [1]. Results: The 2005-10 cohort included 1128 subjects. The 2017-21 cohort 721. The cohorts were comparable in proportion of JIA categories and median cJADAS10 at subject enrollment, 8.2 (IQR 4.4, 14.6) and 8.0 (4.5, 13), respectively. Median age at disease onset was 8.5 years (3.2-12.2) and 8 years (4.1-13.2). The use of disease-modifying antirheumatic drugs (DMARDs) and biologics increased substantially (Table 1). By 70 weeks after diagnosis, 6% of subjects had started a biologic in the 2005-10 cohort compared to 26% in the 2017-21 cohort. The proportion of patients attaining inactive disease increased from 63% to 84% by Wallace Criteria. Conclusion: The main changes observed in JIA treatments in Canada between 2005-2010 and 2017-2021 were increased use of DMARDs and biologics. There was a concomitant increase in attainment of inactive and minimally active disease within 70 weeks of diagnosis, suggesting more aggressive treatment was associated with improved outcomes. [1] Trincianti C, et al. Arthritis Rheumatol 2021; 73: 1966-75. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
I. Nikishina 1 , S. Arsenyeva 1 , V. Matkava 1 , M. Kaleda 1 , S. Salugina 1 , E. Fedorov 1 , A. Shapovalenko 1 , T. Pachkoria 1 1 Paediatric, V.A. Nasonova ScientificTofacitinib (TOFA) is new therapy approach in pediatric rheumatology for management children with different rheumatic diseases (RD). In Russia TOFA was approved from September 2021 for the treatment of polyarticular juvenile idiopathic arthritis (JIA). The unique mechanism of action of Janus-kinases suggests that TOFA can be a useful option in the treatment of various RD in children. Objectives: To evaluate efficacy and safety of TOFA in children with different kind of RD. Methods: We analyzed data base of 52 patients (pts) who were treated by TOFA since 2018 to 2022. The data includes JIA, juvenile ankylosing spondylitis (JAS), chronic recurrent multifocal osteomyelitis (CRMO), juvenile dermatomyositis (JDM), and rare genetic syndromes such as fibrodi, camptodactyly-arthropathy-coxa vara-pericarditis syndrome (CACP)52 pts (18/35% boys; 34/65% girls). The largest number of pts had JIA: 26/63% RF-negative polyarticular subtype of JIA (7 was combined with psoriasis, 3-with uveitis, 2-with Blau syndrome, 1 – with Chron’s disease, 1 – with congenital insensitive to pain and CRMO, 1 – with Down’s syndrome, 1 – with CACP syndrome), RF-positive – 2/3%. 4/8% were diagnosed as systemic JIA (2 pts were in clinical trials, 2 – without current systemic features). JAS was presented in 3 pts, 1 of them was associated with CRMO. 1 pt has JDM with severe course of the disease. All other pts (16/31%) had rare genetic disorders: 2 pts with SAVI syndrome and 1 pt with CANDLE were treated by TOFA as first line with excellent effects (relief of fever, reduction of skin manifestations and pulmonary lesions); 13 pts with extremely rare genetic disease FOP (TOFA was administered due to strong uncontrolled progression of heterotopic ossification). Presence of synovitis, sacroiliitis and axial damage confirmed by X-ray/MRI/CT allowed us to establish alternative diagnosis of JAS in 6 pts and JIA in 7 cases for legal prescription8 months (from 1.5 to 43,9). The majority of TOFA administration was as a first-line treatment (27/52%), in 2-nd line – 12/23%, 3-d line – 4/7%, 4-th line – 3/6%, 5-th line – 5/10%, 6-th line – 1/2In most FOP pts new nodes formation immediately stopped and improvement of motions was registered. Also, we noticed regression of sacroiliitis and coxitis activity by MRI under TOFA therapy Drug tolerance was good in 51. 3 more withdrawals were due to primary inefficacy (2 pts with sJIA from clinical trial, 1 received TOFA as 5-th line therapy). Conclusion: We found that TOFA is highly efficient, well-tolerated medicine which may be a promising option for the difficult to treat variants of JIA (including psoriasis and CRMO- associated) and rare autoinflammatory diseases, such as FOP, Blau syndrome, CANDLE, SAVI, CACP. The anti-inflammatory action of TOFA seems to be preferable to the direct anti-cytokine effect of most biologic: Yes, I received consent Disclosure of Interest : None declared
, V. Matkava, M. Kaleda, A. ShapovalenkoS. Arsenyeva, I. Nikishina Introduction: Among the wide spectrum of Biologics used in pediatric rheumatology, abatacept (ABA) has a special place, which differs from other Biologics with direct anti-cytokine action. In addition to therapy with ABA in patients (pts) with juvenile idiopathic arthritis (JIA) in real clinical practice there is experience of its use “off-label” in such systemic rheumatic diseases (RD) as juvenile systemic scleroderma (jSS), juvenile dermatomyositis (JDM), systemic lupus erythematosus with juvenile onset (jSLE) and its overlaps. Objectives: To evaluate the drug survival of ABA therapy in children with different RD with focus on adverse events (AE). Methods: The study based on cohort of pts who were treated by ABA from 2010 to 2021 in our clinic. These pts are fulfilled the criteria for JIA and the criteria of above-mentioned RD. Data of the disease course were used to estimate drug survival with Kaplan-Meier and calculate AE rates. Results: The retrospective analysis includes data of all pts, who received ABA in our center, 205 pts in total. The largest number of patients were presented by RF-negative polyarticular subtype of JIA - 131/64%, RF-positive – 40/20%, persistent oligoarthritis – 8/4% (6 of them with uveitis). 15/7% were diagnosed as systemic JIA without current systemic features. 36/18% pts suffered from active uveitis. All other pts (11/5%) had systemic RD (jSS – 5, JDM – 3, jSLE – 3). In group of JIA pts we identified overlap-syndrome with JDM, jSLE, jSS features in 31/15% cases. 28/14% had Sjogren’s syndrome. The average age of disease onset was 5.9 years (from 1.25 to 17.2). The average age of ABA initiation (first-line) was 10,7 years (from 2 to 17.8 years). The mean duration of disease was 4.8 years. The mean duration of ABA treatment was 4,4 years. The majority of pts received ABA as first-line therapy (164), 2-nd line – 29, 3-rd line – 8, 4-th line – 4. There were 71 cases of ABA withdrawals. The most often reasons for drug discontinuation were inefficacy – 48/ 68%, secondary mostly (in 1 st line -30 from 164 (18%), 2 nd line- 10/29 (34%), 3 rd line- 4/8 (50%), 4 th line-4/4 (100%). Non-medical «organizational» problems led to the cancellation of therapy in 12/17%. Just in 1 pt ABA was stopped because of remission. AE were as reason ABA withdrawn in 10/15% (3 pts due to post-infusion reactions, 2 pts (the both with early oligoarticular onset) developed uveitis de-novo, one had verruca vulgaris, 2 - psoriasis de novo). In 1 girl we observed the development of cryptogenic epilepsy after 3-year ABA treatment, but therapy was continued. In 2 cases ABA was cancelled due to multiple sclerosis development. The first pt had 70-90% ACR response, however in 2 years of ABA-treatment the psoriasis was developed, ABA was continued. After 4 years of the therapy (at the age of 13) neurological symptoms appeared (headache, loss of sensitivity, ataxia, visual field defect) and multiple sclerosis was diagnosed. In 2-nd pt CNS demyelination was verified after 2 years of ABA therapy with appearance of dizziness, unilateral numbness. MRI-evidence was observed in both cases. ABA treatment survival at 1 year for the 1 st line was 88% and 2-4 th lines 84%, at 5 years 60% and 45% respectively. There were not significant differences in drug survival according to kind of RD. Conclusion: Our data showed great experience of ABA use in wide spectrum of pediatric RD. ABA often is the front-line therapy for certain subtype of JIA, especially in overlap-syndrome. It may be good option for the some pts with jSS, JDM, jSLE despite of indication «off label». Drug survival of ABA was higher in biologics-naïve patients. ABA had satisfactory safety profile in the long-term period, but rare adverse events, such as CNS demyelination and uveitis or psoriasis de novo were found. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
C. Niyasom 1 , C. Lertudomphonwanit 1 , S. Getsuwan 1 , S. Vilaiyuk 1 , A. Sobhonslidsuk 2 , P. Kaewduang 2 , S. Soponkanaporn 1 1 Pediatric, 2 Medicine, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand Correspondence: C. Niyasom Introduction: Monitoring serum transaminases is unreliable to detect liver stiffness and steatosis in patients with juvenile idiopathic arthritis (JIA) who have been treated with methotrexate. Transient elastography (TE) with controlled attenuation parameter (CAP) (FibroScan®) has emerged as a non-invasive tool to assess liver stiffness and steatosis. Objectives: To evaluate the prevalence and predictors of liver stiffness and steatosis in JIA patients treated with MTX by using TE with CAP. Methods: A cross-sectional study was conducted at Ramathibodi Hospital. Included subjects were JIA patients aged 1-18 years who received methotrexate (MTX) for at least 1 year, while patients with known chronic liver disease were excluded. Clinical features, medication information, and laboratory data were collected. TE measured liver stiffness and reported as liver stiffness measurement (LSM) with a cut-off >7 kilopascal (kPa) indicating significant liver stiffness. Steatosis was diagnosed if CAP >225 decibels/meter (dB/m). Results: Sixty JIA patients were enrolled with a median age (IQR) of 12.8 (10.6–15.0) years and a median disease duration (IQR) of 4.2 (2.1–7.8) years. The most common JIA subtype was systemic JIA (28.3%), followed by rheumatoid factor-positive polyarthritis (25%), and enthesitis-related arthritis (18.3%). A cumulative median (IQR) dose of MTX and steroids was 3,768 (1,806–6,466) mg, and 1,563 (0–7,694) mg, respectively. A median (IQR) duration of MTX and steroid usage was 45 (22–85) months, and 5.3 (0–29) months, respectively. None of them had significant liver stiffness with the median LSM (IQR) was 4.10 (3.43–4.58) kPa. Thirteen patients (21.7%) were diagnosed with steatosis. There was a significant higher median body mass index (BMI) Z-score in patients with steatosis (0.86, IQR 0.23–1.44) when compared to non-steatosis (-0.32, IQR -0.95–0.38) (p=0.007). Median cumulative dose of MTX in steatosis patients was 5,405 mg (IQR 2,760–9,761) which were significantly higher than in non-steatosis patients (3,370 mg, IQR 1,438–5,614) (p=0.017). Similarly, there were significantly higher median cumulative dose of steroids in steatosis patients 1,732 mg (IQR 0–26,657) VS 1,410 mg (IQR 0–6,545) than in non-steatosis patients (p=0.069). From multivariable logistic regression analysis, the only predictor of steatosis was BMI Z-score (OR 2.78 [95%CI 1.23– 6.13], p=0.013). Conclusion: Long-term low-dose MTX usage with median duration of 45 months does not increase the risk of liver stiffness in JIA patients. Since levels of liver enzymes can be normal in patients with steatosis, caution and monitoring for steatosis using TE in JIA patients with high BMI Z-score might be useful during MTX treatment. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
F. Ortiz-Márquez 1 , R. Redondo-Rodríguez 1 , L. Martín-Pedraz 2 , C. Padilla-Leiva 3 , G. Díaz-Cordovés 1 , R. Galindo-Zavala 2 , E. Núñez-Cuadros 2 1 Instituto de Investigación Biomédica de Málaga (IBIMA). UCG de Reumatología , Hospital Regional Universitario de Málaga, 2 UCG Pediatría, Hospital Materno-Infantil de Málaga, 3 Universidad de Málaga, Málaga, Spain Correspondence: F. Ortiz-Márquez 1 Introduction: Cognitive impairment is a comorbidity that affects rheumatological patients. Despite this, it hasn’t been studied in Juvenile Idiopathic Arthritis patients yet. Objectives: Prospectively evaluate changes in the cognitive function of patients with juvenile idiopathic arthritis (JIA) and associated factors. Methods: Design and protocol: We performed a prospective cohort study with JIA patients that participated in a previous cross-sectional study (2019) to evaluate cognitive function. After 24 months, the patients were administered the same test battery previously used through an established protocol, and data was collected from their clinical histories. The neuropsychological tests were corrected by a neurologist and neuropsychologist. Study population: Inclusion criteria: Patients aged ≥16 years with JIA classified according to the criteria of ILAR 2001. Patients with inflammatory or rheumatic diseases other than JIA, previous neurological disease not associated with the course of JIA, and patients with scores lower than the normal in the manual skill test were excluded. Outcomes: The main variable was cognitive impairment, defined as worsening of ≥2 scaled points after 24 months (V24) in any of the subtests used to evaluate each cognitive area in the Wechsler Adult Intelligence Scale (WAIS). The evaluated cognitive domains and their respective subtests were: Attention/concentration (Digit Span); verbal function (Vocabulary); visuospatial organization (Block Design); working memory (Letter-Number Sequencing); problem-solving (Similarities). Depression was evaluated by The Beck Depression Inventory-II (BDI-II): minimal (0-13), mild (14-19), moderate (20-28), and severe (29-63). Other variables: Clinical-epidemiological characteristics; treatments; and inflammatory activity evaluated as the C-reactive protein average (CRP) and JADAS-27 along the 2 years of follow-up. Statistical analysis: Descriptive analysis, followed by χ2 and paired T-test. Multivariate analysis to identify independent variables associated with impairment of cognitive function in JIA. Results: Fifty two patients with JIA were included. The clinical characteristics are shown in Table 1. Fifteen patients (28.8%) showed impairment in one or more cognitive functions. The most frequent impaired cognitive functions were working memory (17.3%) and attention/concentration (9.6%); followed by verbal function (7.7%), visuospatial organization (7.7%) and problem solving (3.8%). The variables independently associated with cognitive impairment were the mean CRP along the follow-up (OR [IC 95%], 1.291 [1.002-1.663]; p=0.047), depression (OR [IC 95%], 1.178 [1.001-386]; p=0.049) and biological treatment (OR [IC 95%], 0.196 [0.039-0.978]; p=0.049). This model would explain the 45% of the cognitive impairment in JIA (R2=0.45). Conclusion: Thirty percent of the patients with JIA showed cognitive impairment after 24 months of follow-up. Cognitive impairment was associated with higher inflammatory activity and depression. Biological therapy improves cognitive funcOzdemir Cicek 1 , N. Şahin 2 , A. Paç Kısaarslan 3 , M. H. Poyrazoğlu 3 1 Pediatric Rheumatology, University of Health Sciences, Kayseri City Research and Training Hospital, Kayseri/Melikgazi, 2 Pediatric Rheumatology, Kocaeli Derince Training and Research Hospital, Kocaeli, 3 Pediatric Rheumatology, Erciyes University, Kayseri/Melikgazi, Turkey Correspondence: S. Ozdemir Cicek Introduction: In recent years, attempts have been made to classify JIA into more homogeneous clinical groups. Objectives: We planned to classify the patients with oligoarticular, RF- polyarticular and undifferentiated groups according to ILAR criteria, according to their clinical and laboratory findings. Methods: Two hundred three patients with oligoarticular, RF- polyarticular JIA and undifferentiated arthritis included to the study. RF+ polyarticular JIA, enthesitis-related arthritis, psoriatic arthritis and systemic JIA patients were not included. Eighteen clinical and laboratory variables evaluated with TwoStep Cluster analysis. Categorical variables were sex, affected joints(hip, knee, ankle, shoulder, elbow, wrist, small joints of the hand and foot, cervical vertebra and temporomandibular joint), laboratory findings which determines the JIA subtype(ANA, RF and HLA B27 positivity), continuous variables were the age of disease onset, the number of affected joints, ESR and CRP values at disease onset. Clinical and laboratory features of the resulted clusters were then compared with each other. Results: Two hundred three JIA patients included to the study. The median age of patients was 13 (2.5-21.07) years, the age of diagnosis was 7 (1-16) years and 133 (65.5%) of the patients were female. Two clusters were generated as the result of cluster analysis. Cluster 1 had 94 (46.3%) and cluster 2 had 109 (53.7%) patients. The most important indicators in differentiating of the two clusters were small joint involvement, the number of involved joints, wrist, knee and elbow arthritis. In the first cluster, small joint involvement was observed, the number of affected joints was higher, TMJ, shoulder, wrist, elbow and ankle arthritis were higher, and initial acute phase reactants were found to be higher than the second cluster. Corticosteroids, DMARDs and biological treatments were at higher rates in the first cluster, and the remission rates at twelfth months and last visit were lower according to the second cluster. All of the patients in the second cluster had knee involvement and IAS was used more frequently compared to first cluster. Conclusion: Our results classified current oligoarticular, RF negative polyarticular and undifferentiated arthritis subgroups patients into two clusters. Small joint, wrist, elbow involvement and the number of involved arthritis were the most important factors for differentiating two groups. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
N. Palmou-Fontana 1 , A. Garcia-Rogero 2 , C. Redondo-Figuero 1 , A. Lopez-Sundh 1 , P. Mesa-del-Castillo 3 , G. Diaz-Cordoves 4 , B. Magallares-Lopez 5 , M. J. Cabero-Perez 6 , M. A. Gonzalez-Gay 1 , P. Collado-Ramos 7 on behalf of Sociedad Española de Reumatología Pediátrica (SERPE) 1 Servicio de Reumatologia, Hospital Universitario Marques de Valdecilla, Santander, 2 Gerencia de Atencion Primaria, Servicio Cantabro de Salud, Castro Urdiales, 3 Servicio de Reumatologia, Hospital Universitario Virgen de la Arrixaca, Murcia, 4 Servicio de Reumatologia, Hospital Regional Universitario, Malaga, 5 Servicio de Reumatologia, Hospital de la Santa Creu i Sant Pau, Barcelona, 6 Servicio de Pediatría, Hospital Universitario Marques de Valdecilla, Santander, 7 Servicio de Reumatologia, Hospital Universitario Severo Ochoa, Madrid, Spain Correspondence: N. Palmou-Fontana Introduction: Juvenile idiopathic arthritis (JIA) constitutes a heterogeneous group of inflammatory joint diseases. The International League of Associations for Rheumatology (ILAR) classifies and defines seven subtypes of JIA by specific and non-specific clinical features with exclusions. Among these diseases is Juvenile Psoriatic Arthritis (JPA). There are two pediatric diagnostic classifications based on clinical criteria: ILAR classification criteria and Vancouver classification criteria. Classification of JIA has been a great debate since this entity disappeared. (1) The CASPAR criteria are standard in adults. These criteria have a specificity of 91.4% and a sensitivity of 98.7%, making diagnostic classification easier. However, the peculiarities of childhood cause some differences to consider. Objectives: To compare the performance of the three different PsJIA classification criteria (ILAR, Vancouver and CASPAR criteria) in children identified as such in the routine rheumatology Clinic. Methods: Multicenter prospective observational study started in 2020. Patients seen in the pediatric rheumatology outpatient clinic with suspected PsJIA diagnosis were consecutively included. Sociodemographic, clinical characteristics and family history of Psoriasis were collected to assess compliance with diagnostic classification criteria (ILAR, Vancouver, and CASPAR). Results: Thirty-two children were included, predominantly female (24 girls). The median age at diagnosis was 13.5 (IQR: 4.5). Clinical onset was predominantly oligoarticular (19, 59.5%), followed by polyarticular (n:5, 18.6%), monoarticular (n:5, 18.6%), and onychopathy (n:2, 6.2%). Cutaneous Psoriasis was present in 20 children (62.5%). Family history of Psoriasis was recorded in 24 patients, 1st degree in 19 patients (54%), and a second degree in 5 (15.6%). Among the extra-articular manifestations, dactylitis stood out (n:14, 3.8%). Twelve patients (37,5%) had nail involvement. Rheumatoid factor (RF) was negative in 100% of the children, ANAS were positive in 11 children (34.4%), whereas HLAB27 was positive in 3 children (9.4%). Twenty-seven children met the VANCOUVER criteria: twenty children, defined dco (62.5%) and 7 (21.9%) probable diagnosis. Twenty children (62.5%) met the ILAR criteria. Twenty-seven (84.4%) children were diagnosed according to the CASPAR criteria. The diagnostic agreement between the ILAR and CASPAR criteria on the one hand, and between the ILAR and Vancouver criteria on the other hand, was weak (K=0.17 and K= 0.41). In contrast, the agreement was total (K=100) between the CASPAR and Vancouver criteria. Conclusion: Despite minimal changes between the Vancouver and ILAR criteria, the ILAR exclusion criteria limit the diagnosis of PsA in childhood. Given that CASPAR and Vancouver criteria were able to detect a more significant number of patients with PsA in our series (predominantly adolescents), the application of the CASPAR Criteria in the subgroup of children with late onset could be considered. (1) (1) Martini A, Ravelli A, Avcin T, et al, for the Pediatric Rheumatology International Trials Organization (PRINTO). Towards New Classification Patient Consent: Yes, I received consent Disclosure of Interest : N. Palmou-Fontana Consultant with: ABBVIE, AMGEN, PFIZER, Speaker Bureau with: ABBVIE, AMGEN, PFIZER, A. Garcia-Rogero Speaker Bureau with: GSK, C. Redondo-Figuero: None declared, A. Lopez-Sundh: None declared, P. Mesa-del-Castillo: None declared, G. Diaz-Cordoves: None declared, B. Magallares-Lopez: None declared, M. J. Cabero-Perez: None declared, M. A. Gonzalez-Gay: None declared, P. Collado-Ramos: None declared
E. Pardo Campo 1 , S. Burguer 1 , M. Pino Martinez 1 , I. Braña Abascal 1 , S. Murias Loza 2 , J. Rodriguez 2 , S. Alonso Castro 1 , S. Fernandez Aguado 1 , M. Alperi Lopez 1 , R. Queiro Silva 1 1 Rheumatology, 2 Pediatrics, Hospital Univ Central De Asturias, Oviedo, Spain Correspondence: E. Pardo Campo Introduction: Cervical spine involvement is often unrecognized in the early stages of juvenile idiopathic arthritis (JIA), as it is a rare manifestation. We know that JIA is the most common chronic rheumatic disease in childhood. However, cervical involvement is a rare manifestation in the early stages of the disease, and is extremely infrequent as the only manifestation. It is for this reason that the exact frequency, clinical features and evolution of these patients are not yet known. It has been described as a late complication and is considered to have a poor prognosis. Objectives: Therefore, our aim is to describe the clinical characteristics of patients with JIA who have experienced cervical involvement as a manifestation at the onset of the disease. Methods: An observational study was performed on patients with JIA who presented cervical involvement as the form of onset of the disease. Data was obtained from a total of 75 patients with JIA being followed by the Pediatric Rheumatology Unit of the Hospital Central de Asturias. Hospital Central de Asturias. We have reviewed the clinical histories and selected those with cervical involvement as a form of onset. Results: Three cases of JIA were detected in which cervical involvement was the form of onset of the disease in three different types of JIA. (Table 1) Conclusion: JIA in its different forms should be considered as a differential diagnosis in children presenting with cervical pain, cervical limitation or stiffness. This manifestation can go unnoticed in our patients and delay diagnosis. We should consider its occurrence in different categories within JIA. MRI can be of great help in the diagnosis of these patients. Early targeted therapy with anti-TNF inhibitors can help in the remission of cervical symptomatology. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
S. Pastore 1 , F. Corona 2 , A. Taddio 3 , A. Tommasini 3 1 Institute of Maternal and Child Health IRCCS “Burlo Garofolo, 2 Department of Medicine, Surgery, and Health Sciences, 3 Institute of Maternal and Child Health IRCCS “Burlo Garofolo”, University of Trieste, Trieste, Italy Correspondence: S. Pastore Introduction: Juvenile psoriatic arthritis (JPsA) has been a recognized entity since the 1970s. In 1989 the Vancouver criteria were proposed to identify cases of probable or definite diagnosis. Subsequently, the Vancouver criteria were replaced with ILAR criteria, revised for the last time in 2004 and still used in clinical practice. With these criteria JpsA is permitted also in the absence of frank psoriasis in children with arthritis accompanied by two of the following: dactylitis; nails pitting or oncycholysis, or psoriasis in a first-degree relative. Recently, the Pediatric Rheumatology International Trials Organization suggested new criteria for the diagnosis of Juvenile Idiopathic Arthritis (JIA), but psoriatic arthritis subgroup is missing. Objectives: To describe clinical and lab features of patients with JPsA, and therapeutic strategies. Methods: We retrospectively enrolled all patients cared at the Rheumatology Service of the Institute for Maternal and Child Health IRCCS “Burlo Garofolo” (Trieste) with JPsA diagnosis according 2001 ILAR criteria. For each patient we collected: age, sex, age at onset, familiar history, number of involved joints, inflammatory indexes at onset disease, anti-nuclear antibodies (ANA), human leukocyte antigen (HLA) B27 expression, presence of enthesitis, tenosynovitis and uveitis, concomitant autoimmune diseases. We collected also treatments and treatment response assessed by Wallace remission criteria. Results: 21 patients (16 F, 5 M) with juvenile psoriatic arthritis are enrolled. Seven out of 21 had a oligoarticular pattern, 14/21 had a polyarticular pattern (2/14 oligoextended). Out of 21, 3 patients had onset disease before age six. Thirteen out of 21 (62%) had high inflammatory indexes at onset disease: 7/13 had only high Erythrocyte Sedimentation Rate (ESR > 25 mm/h), 6/13 also had elevated C-Reactive Protein (CRP > 1 mg/L). Out of 21, 10 had ANA positivity and 2 had HLAB27 positivity. Seventeen out of 21 (81%) had fingers or toes dactylitis or both. Ten out of 21 patients (48%) had psoriasis, which in 9/10 appeared before arthritis. Six out of 21 (28%) had tenosynovitis of ankle. Two patients developed anterior uveitis. One of these is a girl with disease onset before the age of 6, positive ANA and HLAB27, with recurrent and symptomatic iridocyclitis unresponsive to first and second line therapies. Five patients had a concomitant autoimmune disease (3 celiac disease and 2 hypothyroidism). Seven out of 21 (33%) achieved disease remission with a modifying antirheumatic drug (DMARD): 6 with methotrexate and one with sulfazalazine. Nine out of 21 patients (43%) achieved disease remission with adalimumab; then one of these had loss of response. Three out of 21 (14%) achieved disease remission with etanercept. Two patients had a severe disease non responsive to different drugs, non-biologic and biologic DMARDs. Conclusion: In our patients with psoriatic arthritis tenosynovitis is the predominant clinical feature; most patients have dactylitis. More than half of the patients have high ESR at onset disease. In most patients disease remission was achieved with biologic therapy. Disclosure of Interest : None declared
M. Pavlenko 1 , D. Pavlenko 2 1 Universum Clinic, 2 Department of Ophthalmology, Bogomolets National Medical University, Kyiv, Ukraine Correspondence: M. Pavlenko Introduction: Juvenile idiopathic arthritis is the most common chronic rheumatic disease in children. Objectives: To describe the current state of research articles on juvenile idiopathic arthritis using bibliometric analysis. Methods: An advanced search was performed in May 2022 within all titles in the Core Collection of Clarivate Web of Science to identify all articles on juvenile idiopathic/rheumatoid arthritis until 2021. The predominant language, h-index of the research topic, and the number of citations were identified according to the built-in Analyze Result and Citation Report. Biblioshiny application of Bibliometrix (Aria, M. & Cuccurullo, C., 2017) R-package (RStudio, PBC, Boston, MA, USA) was used for scientific production, author, authors collaboration, and citation analyses. Results: Only articles, which comprised 46.7% (n=4,271) of all 9,144 publications between 1970 and 2021, were analysed. They were written by 13,018 authors, and published in 810 sources. Almost all articles were written in the English language (n=4,049; 94.8%). Singled-authored documents were rare (n=194; 4.5%) and were written by 1.1% (n=146) of all authors. The average years from publication was 14.8. The collaboration index was 3.2. There were 0.3 documents per author and 3.1 authors per document and 6.8 co-authors per document. The annual growth rate was 2.1%. The most productive year was 2021 (n=259; 6.1%). Source clustering through Bradford’s law revealed that Journal of Rheumatology (n=454; 10.6%; h-index=58), Arthritis and Rheumatism (n=253; 5.9%; h-index=83), Pediatric Rheumatology (n=239; 5.6%; h-index=26), Rheumatology (n=157; 3.7%; h-index=43), Clinical and Experimental Rheumatology (n=149; 3.5%; h-index=29), Annals of the Rheumatic Diseases (n=145; 3.4%; h-index=49), and Clinical Rheumatology (n=114; 2.7%; h-index=21) formed the core sources. Martini A (n=177; 4.1% ; h-index=57), Ravelli A (n=128; 3.0% ; h-index=49), and Ruperto N (n=112; 2.6%; h-index=48) contributed most and had the highest local impact. The most relevant affiliations included University of Toronto (n=244), University Genoa (n=199), and University Cincinnati (n=170). The h-index of the research field was 123. Totally, there were 110,668 citations with average citations per document of 25.9 and average citations per year per document of 2.0. Each publication had an average of 12.8 references. Top-5 cited papers are depicted in Table 1. The corresponding authors were most likely from the United States (n=871; 22.5%), Italy (n=328; 8.5%), and Germany (n=285; 7.4%). The United States (n=3426; 20.6%), Canada (n=1515; 9.1%), and Italy (n=1397; 8.4%) were the most productive countries. The United States, Italy, and the United Kingdom were cited mostly (30,558, 12,883, and 8,274 citations, respectively). When average article citations were considered, Italy, Canada, and Switzerland were in the top (39.28, 38.34, and 35.51 citations/article, respectively). Conclusion: In this study, the current state of research related to juvenile idiopathic arthritis was quantitatively characterized. The United States was the most prolific country and its research had the highest number of total citations. University of Toronto was the most common affiliation. Italy had the highest average article citation, E. Kaltsonoudis, P. Karagianni, P. V. Voulgari, A. A. Drosos University of Ioannina, Ioannina, Greece Correspondence: E. Pelechas Introduction: Polyarticular JIA (rheumatoid factor positive and rheumatoid factor negative), years after the first diagnosis, can affect significantly the patients’ quality of life (1). In the pre-biological era, conventional synthetic disease-modifying anti-rheumatic drugs (csDMARDs) and glucocorticoids (GCs) were the mainstay treatments affecting patients with the development of new comorbidities and complications at a very sensitive age (2). Objectives: Evaluation of the comorbidities and complications from csDMARDs and GCs in 23 patients with transition to adulthood in a tertiary university hospital in Greece. Methods: 23 patients (16 women) with a mean age 32 years and a mean follow-up of 5±2 years were evaluated. Average disease duration 12±3 years. Nineteen received biologic DMARDs (17 anti-TNFα, 1 Tocilizumab and 1 abatacept), and 4 csDMARDs with or without low dose GCs. Nine of the patients due to disease duration (pre-biological) received large doses of GCs for long periods of time (>10mg for >3 years) and the rest received short-term GCs. Twelve, had uncontrolled disease (DAS28 >3,2) for at least 3 years. Due to the nature of the data the JADAS score could not be used and the DAS28 has been used instead. In addition, the SF-36 (Short Form 36 Health Survey) has been used in order to evaluate health status of the patients. Results: osteoporosis resulted in 15 patients (9 due to high and prolonged GC intake and significant disease activity, and 6 only due to prolonged GC intake). Underdevelopment in 8 patients (height <1,55m), and 8 with emotional instability (all had received large doses of GCs and had significant disease activity). Two patients required total knee arthroplasty due to severe disease and one of them had to operate both knees. One patient had total hip arthroplasty due to osteonecrosis of the right hip. The SF-36 score was lower than 50% in most patients, and more specifically the components of the test with the worst scores were a) role limitations due to emotional problems (approx. 10%), b) general health (approx. 25%), c) energy/fatigue (approx. 30%). Nevertheless, the transition to the adulthood was without significant problems and the therapeutic interventions required were minimal. Conclusion: the advent of biological agents has significantly improved the quality of life and comorbidities of patients with polyarticular JIA. The increasing number of biological treatment options in recent years is expected to improve further the final outcomes of thRabhi 1 , K. Maatallah 2 , H. Ferjani 2 , W. Triki 2 , D. Ben Nessib 2 , D. Kaffel 2 , W. Hamdi 2 1 Rheumatology, 2 Kassab orthopedics institute, Ksar Said, Tunisia Correspondence: E. Rabhi. It’s an inflammatory condition that can lead to bone metabolism disturbance and osteoporosis (OP). We have increasing data on the negative effect of disease activity on bone mineral density (BMD) [1]. It is therefore essential to ensure optimal management of inflammation and a balanced diet to avoid adverse consequences on the bone. Objectives: Our study aimed to assess bone mineral density (BMD) and calcium intake in children with JIA. Methods: We conducted a retrospective study including children fulfilling the international league against rheumatism (ILAR) 2010 criteria. We collected epidemiological, clinical, juvenile arthritis disease activity score (JADAS), daily calcium intake, and therapeutic data. Results: Among 43 patients, thirteen underwent a bone mineral density measurement and were thus enrolled. There were 9 males and 4 females. The mean age was 13.1 ± 2.8 [10-20] years. The mean duration of the disease was 4.3 ± 3.2 years [1-11]. The mean body mass index (BMI) was 19.9 ± 5.9 [14-35.25]. The mean daily calcium intake was 574 ± 5.97 mg [252-725]. The mean JADAS was 2.7 ± 2.48 [0-6]. The main bone density was 0.975 ± 0.124 [0.73 -1,14] g/cm2 and the Z score ranged from -2.5 to 1.5 standard deviations (SD). Seven children had a low bone density (Z score<-2SD). Four patients had a history of bone fractures. Calcium intake was significantly correlated with BMD (p<0.05). However, there was no association between BMD and BMI (p=0.23) or JADAS (p=0.4).Conclusion: Our study showed that more than half of the patients had a low bone density and all of them had, according to the recommendation of the international osteoporosis foundation, a low calcium intake. These results emphasize the need to screen for calcium deficiency intake in order to avoid the aggravation of bone fragility.References 1. John Burnham, Justine Shults, Sarah E Dubner, Harjeet Sembhi, Babette S Zemel, Mary B Leonardi. Bone density, structure, and strength in juvenile idiopathic arthritis: Importance of disease severity and muscle deficits. 2008 Aug;58(8):2518-27 Disclosure of Interest : None declared
lph 1,2,3 , Y. Sanchez-Corrales 3,4 , T. Xenakis 3,4 , V. Alexiou 1,2,3 , C. Bolton 1,2,3 , S. Castellano 3,4 , L. Wedderburn 1,2,3 1 Infection, Immunity and Inflammation Department, UCL Great Ormond Street Institute of Child Health, University College London, 23 NIHR Great Ormond Street Hospital Biomedical Research Centre, 4 Genetics and Genomic Medicine Department, UCL Great Ormond Street Institute of Child Health, University College London, London, United Kingdom Correspondence: E. Ralph Introduction: CD161 is a C-type lectin receptor encoded by the gene KLRB1 and is expressed on a variety of immune cells. It has been shown that CD161+ cells are highly enriched in the synovial fluid of Juvenile Idiopathic Arthritis (JIA) patients, with CD161 expressed on several cell populations including Th17, Th17/1, and some Th1 cells (‘ex-Th17’ cells) 1 , regulatory CD4+ T cells 2 , some CD8 cells (e.g. MAIT cells), NK cells and innate lymphoid cells (ILC) 3 . Previous studies have generally relied on pre-selected populations and/or bulk studies, often using flow cytometry. Thus, there has not been a comprehensive survey of KLRB1 /CD161 expression in the different cell types present in synovial fluid with single-cell resolution. Objectives: To map the diversity of synovial inflammatory cells expressing CD161 using single-cell RNA sequencing to define expression of CD161 in immune populations in the joints of patients with JIA6, downstream analysis and average gene expression was performed using Seurat v4. Cell type annotation was performed using a single-cell multimodal PBMC reference 4 . Normalised data counts were used to calculate average gene expression per cell type. Units are transcripts per million (TPM) defined as TPM = (number of counts mapped to a gene x 10^6) / Total number of counts per cell). Results: KLRB1 expression was detected in 32% of the total SFMCs and was enriched in 7 of 30 clusters. Expression of KLRB1 was highest in MAIT cells, with 98.9% of these cells expressing KLRB1 , with an average expression level of 1903 TPM. The cluster identified as cytotoxic CD4+ T cells also expressed high levels of KLRB1, (89.8%, 1099 TPM), as did ILCs (80.3%, 1138 TPM), gamma delta T cells (70.4%, 911 TPM), natural killer (NK) cells (76.9%, 833 TPM), NK CD56 bright cells (70.1%, 697 TPM), and proliferating NK cells (58.8%, 314 TPM ). Central memory, effector memory and proliferating CD4+ T cells, as well as effector memory, naïve and proliferating CD8+ T cells, and regulatory T cells also expressed KLRB1 , but at lower levels. Conclusion: We have generated a single cell map of CD161-expressing populations in JIA synovial fluid cells. We have shown that KLRB1 is expressed in a variety of immune cells and quantify cell expression heterogeneity that has not been previously described. Future work will investigate the expression of CD161 on CD4+ T cell subsets, its correlation with inflammatory markers and clinical outcomes. REFERENCES 1. Nistala K.. 2010 Aug 17;107(33):14751-6. 2. Pesenacker A.M. et al. , CD161 defines the subset of FoxP3+ T cells capable of producing proinflammatory cytokines. Blood. 2013 Apr 4;121(14):2647-58. 3. Del Castillo Velasco-Herrera, M. et al. A novel innate lymphoid cell delineates childhood autoimmune arthritis. bioRxiv 416784. 4. Hao Y. et al. , Integrated analysis of multimodal single-cell data. Cell. 2021 Jun 24;184(13):3573-3587.e29. Disclosure of Interest : E. Ralph Grant / Research Support with: ER holds a personal Fellowship from NIHR Great Ormond Street Hospital Biomedical Research Centre, Y. Sanchez-Corrales: None declared, T. Xenakis: None declared, V. Alexiou: None declared, C. Bolton: None declared, S. Castellano: None declared, L. Wedderburn Grant / Research Support with: LW is a PI for the CLUSTER Consortium.LW declares in-, unrelated to this work, with Lilly and Novartis.
A. Kozhevnikov, V. Kenis, E. Melchenko, A. Ramazanova ational Medical Children’s Orthopedics and Trauma Surgery Research Center Named after H. Turner, Saint Petersburg, Pushkin, Russian Federation orrespondence: A. Ramazanova Introduction: Chronic undifferentiated progressive arthropathy (CUPA) of children is a large group of musculoskeletal diseases. A form of juvenile arthritis (JIA), the clinical finding of which includes not only chronic synovitis, but also progressive contractures of several joints, requires differential diagnosis with the genetic disorders e.g., skeletal dysplasia. Frequent misinterpretations of the results of examinations of children with recessive multiple epiphyseal dysplasia (rMED/MED4, gene SLC26A2 Mut, OMIM 229600) in favor of rheumatic pathology demonstrate the relevance of this problem. Objectives: The aim of the study is to determine the diagnostic criteria of skeletal dysplasia for differential diagnosis with JIA. Methods: We retrospectively analyzed clinical unit, laboratory findings, ultrasound, x-ray, MRI in our series of patients with CUPA (low laboratory activity, enthesopathy or synovitis with progressive idiopathic contracture). The study included 74 children who were treated at the National Medical children’s orthopedics and trauma surgery Research Center, Saint-Petersburg between 2005 and 2020. Results: We divided children into two groups: patients with rMED (52) and patients with JIA (22). Children with other monogenic mutations similar to arthritis and other forms of dysplasia were excluded from the study. Distinctive features in the clinical picture of the rMED were disproportionate constitution, absence of morning stiffness, symmetrical contracture formation, axial deformities of the lower extremities, and absence of physiological hypermobility at an early age with impaired motor activity. Laboratory data in children with rMED were normal or indicated moderate inflammatory response without any specific changes. Radiographic data demonstrated bilateral and symmetric flattening of epiphyse with epimetaphyseal widenings, double-layered patella, dystrophic spondylolisthesis at the L5-S1 vertebrae to 10-12 years of life. X-ray of the hip joints in young children with rMED showed symmetrical delay in ossification of the femoral heads, and some patients developed bilateral extrusion subluxation of the epiphyses and avascular necrosis. In JIA, we could see a staging of radiological phases: accelerated ossification of cartilaginous epiphyses, erosive-dystrophic changes, arthrosis-arthritis. Ultrasound and MRI are not helpful in making the diagnosis because signs of synovitis exist in both groups. Sensitivity to anti-inflammatory therapy was also not a pathognomonic criterion due to its effect on synovitis. Conclusion: Early diagnosis and non-surgical treatment rMED are important for recovery. Because in the opposite case, progressive arthritis leads to significant disability. A comprehensive approach in the evaluation of medical data and their dynamics, which takes into account the anatomical and physiological features of children, allows us to identify the true cause of arthropathP. Ramos, H. F. Menchaca-Aguayo, E. R. Mercedes-Pérez, N. De la Rosa-Encarnación, M. I. De la Cera-Rodríguez, A. Guzmán-Revilla, A. Barba-Aguilar, K. Primero-Nieto, H. Bermudez-Canales, S. Rodríguez-Aguayo, E. Faugier-Fuentes Reumatología Pediátrica., Hospital Infantil De México Federico Gomez, Mexico, Mexico Correspondence: P. P. Ramos Introduction: Juvenile idiopathic arthritis (JIA) unifies all forms of chronic arthritis, affecting not only the joints but also other systems. Psoriatic JIA (JPsA) is an uncommon category in childhood and there are few reports describing the characteristics and long-term outcome of patients with psoriatic JIA than other subtypes of JIA. Objectives: To describe the case and evolution in a patient with a diagnosis of JPsA. Methods: Description of a clinical case and review of the literature. Results: A 12-year-old female with 2 months of evolution with constitutional syndrome and arthralgias in the right hip and proximal interphalangeal joints associated with morning stiffness. Polyarticular JIA was diagnosed FR negative and methotrexate was started, and sulfasalazine was added due to persistent activity. He lost follow-up for four years. Subsequently, she presented with disease activity and clinical signs of sacroiliitis. MRI of the sacroiliacs was normal. She persists with arthritis of more than 10 joints, the change from methotrexate DMARD to leflunomide was performed, and the dose of sulfasalazine was increased. Approach for biologic therapy was performed. She lost follow-up for two years. She came again with disease activity, with arthritis and joint limitation, dermatosis in elbows and nail pitting; new laboratory studies, imaging and skin biopsy were performed. Due to the clinical features, a diagnosis of psoriasis was integrated and management with calcineurin inhibitor was started. Six years after the onset of symptoms, bilateral sacroiliitis was documented clinically and in sacroiliac MRI. Skin biopsy: psoriasis. Management with infliximab and double DMARDs was started. She does not present uveitis. Current treatment: methotrexate, sulfasalazine, infliximab, and topical tacrolimus. Conclusion: There are few reports describing the evolution of AIJps and it can be confusing when classifying it. In the case of the patient, regardless of the irregularity of adherence to treatment, the clinical expression presented different phenotypes: polyarticular JIA, spondyloarthropathy and psoriatic JIA; being a complex diagnosis at the time of classificaN. Ruperto 1 , E. Chertok 2 , J. Dehoorne 3 , G. Horneff 4 , T. Kallinich 5 , I. Louw 6 , M. Alessio 7 , S. Compeyrot-Lacassagne 8 , B. Lauwerys 9 , N. Martin 10 , K. Marzan 11 , W. Knibbe 12 , R. Martin 13 , X. Zhu 14 , S. Whelan 15 , L. Pricop 14 , D. J. Lovell 16 , A. Martini 17 , H. I. Brunner 16 on behalf of PRINTO/PRCSG 1 IRCCS Istituto Giannina Gaslini, Genova, Italy, 2 Voronezh State Medical University, Voronezh, Russian Federation, 3 University Hospital Gent, Gent, Belgium, 4 Asklepios Klinik Sankt Augustin GmbH, Sankt Augustin, 5 Charité-Universitätsmedizin Berlin, Berlin, Germany, 6 Panaroma Medical Centre, Cape Town, South Africa, 7 Università degli Studi della Campania Luigi Vanvitelli , Napoli, Italy, 8 Great Ormond Street Hospital for Children, London, United Kingdom, 9 Cliniques Universitaires Saint-Luc, Bruxelles, Belgium, 10 Yorkhill Hospital, Glasgow, United Kingdom, 11 Children’s Hospital Los Angeles, Los Angeles, 12 St Lukes Intermountain Research Center , Boise, 13 Novartis Pharmaceutical Corporation, 14 Novartis Pharmaceuticals Corporation, East Hanover, United States, 15 Novartis Ireland Ltd., Dublin, Ireland, 16 University of Cincinnati, Cincinnati, United States, 17 Università di Genova, Genova, Italy Correspondence: N. Ruperto Introduction: Juvenile idiopathic arthritis (JIA) categories of enthesitis-related arthritis (ERA) and juvenile psoriatic arthritis (JPsA) represent paediatric counterparts of adult non-radiographic axial spondyloarthritis and psoriatic arthritis, respectively. 1,2 JUNIPERA, a 2-year, randomised, double-blind, placebo (PBO)-controlled trial demonstrated significantly longer time to flare with secukinumab (SEC) vs PBO with sustained improvements up to Week (Wk) 104. 3 Objectives: To evaluate the effect of SEC on axial and peripheral manifestations in active ERA and JPsA patients (pts). Methods: Pts (2–<18 years age) with active disease (both ≥3 active joints and ≥1 active enthesitis site) were treated with open-label (OL) s.c. SEC (75/150 mg in pts <50/≥50 kg) in treatment period (TP)1. SEC was administered at baseline (BL) and Wks 1–4, 8 and 12. Responders at Wk 12 (JIA-ACR30 response) were randomised into the double-blind, withdrawal TP2 to continue SEC or PBO every 4 wks until a disease flare or up to Wk 100. The time to flare in ERA and JPsA pts in TP2 is reported here. JIA-ACR responses, resolution of enthesitis and dactylitis, JADAS-27, and axial, peripheral and skin manifestations were also assessed. Results: A total of 52/86 (60.5%) pts with ERA (mean age, 13.7 years) and 34/86 (39.5%) pts with JPsA (mean age, 12.2 years) were enrolled in the OL TP1. Flare risk reduction in TP2 was 55% (HR 0.45, 95% CI: 0.16–1.28, P =0.075) in ERA pts and 85% (HR 0.15, 95% CI: 0.04–0.57, P <0.001) in JPsA pts. Improvements in JIA-ACR responses, inactive disease and JADAS-27 were observed with SEC treatment at Wk 12 and at the end of TP2 (Table). Axial symptoms in ERA with SEC and PBO at the end of TP2 were: modified Schober’s test, 100% and 100%; inflammatory back pain, 100% and 50%; Flexion, ABduction and External Rotation (FABER) test, 100% and 83.3%; and clinical sacroiliitis, 100% and 50%. Pts with CHAQ score of 0 at the end of TP2 with SEC and PBO treatment were 9 each in ERA and 8 each in JPsA. Sustained improvements were also observed in enthesitis, dactylitis and other psoriatic skin manifestations (data not shown). Conclusion: In pts with active ERA and JPsA, SEC demonstrated a longer time to disease flare with flare risk reduction compared to PBO up to Wk 104 and exhibited rapid and sustained improvement of axial, peripheral and skin manifestations. References 1. Pagnini I, et al. Front Med 2021;8:6673052 2. Zisman D, et al. J Rheumatol 2018;94:11–16 3. Brunner H, et al. Arthritis Rheumatol 2021;73(10) Trial registration identifying number: NCT03031782 Patient Consent: Not applicable (there are no patient data) Disclosure of Interest : N. Ruperto Grant / Research Support with: Bristol Myers and Squibb, Eli Lilly, F Hoffmann-La Roche, Novartis, Pfizer and SOBI, Consultant with: Ablynx, Amgen, Astrazeneca-Medimmune, Aurinia, Bayer, Bristol Myers and Squibb, Cambridge Healthcare Research (CHR), Celegene, Domain therapeutic, Eli Lilly, EMD Serono, GlaxoSmith and Kline, Idorsia, Janssen, Novartis, Pfizer, SOBI and UCB, Paid Instructor with: Eli Lilly, GlaxoSmith and Kline, Pfizer, SOBI and UCB, Speaker Bureau with: Eli Lilly, GlaxoSmith and Kline, Pfizer, SOBI and UCB, E. Chertok: None declared, J. Dehoorne Grant / Research Support with: Abbvie, Roche, Consultant with: Abbvie, Roche, Pfizer, Speaker Bureau with: Abbvie, Roche, G. Horneff Grant / Research Support with: Pfizer, Novartis, Roche, MSD, Speaker Bureau with: Novartis, Pfizer, Janssen, T. Kallinich Speaker Bureau with: Roche, I. Louw Consultant with: Pfizer, Abbvie, Janssen, Amgen, Cipla, Speaker Bureau with: Pfizer, Abbvie, BMS, M. Alessio: None declared, S. Compeyrot-Lacassagne: None declared, B. Lauwerys Employee with: UCB Pharma, N. Martin: None declared, K. Marzan Grant / Research Support with: Novartis, Sanofi, W. Knibbe Speaker Bureau with: Novartis, Amgen, UCB, Abbvie, R. Martin Shareholder with: Novartis, Employee with: Novartis, X. Zhu Shareholder with: Novartis, Employee with: Novartis, S. Whelan Shareholder with: Novartis, Employee with: Novartis, L. Pricop Shareholder with: Novartis, Employee with: Novartis, D. J. Lovell Grant / Research Support with: Astra Zeneca, Boehringer Ingelheim, GSK, Hoffman LaRoche, Novartis, UBC, Consultant with: Astra Zeneca, Boehringer Ingelheim, GSK, Hoffman LaRoche, Novartis, UBC, A. Martini Consultant with: Eli-Lilly, EMD Serono, Janssen, Novartis, Pfizer, AbbVie, Idorsia, H. I. Brunner Grant / Research Support with: Novartis, Consultant with: Novartis
S. Arve-Butler 1,2,3 , E. Rydén 1,2 , A. Mossberg 1,2 , R. Kahn 1,2 1 Wallenberg Center for Molecular Medicine, Lund University, 2 Department of Pediatrics, 3 Department of Rheumatology, Clinical Sciences, Lund University, Lund, Sweden Correspondence: E. Rydén Introduction: Specific autoantibodies in Juvenile idiopathic arthritis (JIA) remain mostly unidentified, although they may serve as potential biomarkers for disease outcome and uveitis. Objectives: To investigate different strategies for autoantibody discovery of JIA-specific autoantigens. Methods: Serum (n =57) and plasma (n=6) were collected from JIA patients with oligoarticular or seronegative polyarticular JIA at the Department of Pediatrics at Lund University Hospital with informed consent and assent. Anonymized serum samples from pediatric controls (n=22) were available. Ethical permission was granted by the Regional Ethical Review board for southern Sweden (LU 2016/128). Plasma from six patients with ANA positive persistent oligoarticular JIA positive were included in two exploratory analyses to identify novel autoantigens. Two pools with three patients in each (with or without uveitis) was used. First, an assay covering 42 100 unique recombinant peptides of 50-150 amino acid each, from 18 000 different proteins was used. This explored approximately 94% of the human proteome by printing the peptides on glass slides which then were incubated with the two plasma pools. Second, immunoprecipitation (IP) by plasma incubated with mixed with cell lysates from the human epithelial cell line followed by liquid chromatography mass spectrometry at BioMS (Lund University) of captured proteins was used. Peptides or proteins identified in either of the two assays in combination with autoantigens found in literature made up a selection of peptides being further investigated in a targeted array (SciLifeLab, Stockholm). In this array, patient serum (n=57) was tested individually, as well as serum from controls (n=22). The array used color coded magnetic beads, individually coated with one of the peptides and was analyzed by flow cytometry (Luminex). The data was given as raw MFI and autoantigens were defined as reactive in <10% of controls and >10% of the patients after adjustment to background beads. Results: In the autoimmune profiling planar array, reactivity was detected to 332 peptides in at least one of the two plasma pools. Thirty-four peptides showed reactivity in both pools, 75 in the non-uveitis pool and 223 in the uveitis pool. Immunprecipitation followed by mass spectrometry showed reactivity for 131 full length proteins. Ninety-five of the precipitated proteins were overlapping in the two patient pools, 14 showed reactivity only in the non-uveitis pool and 22 in the uveitis pool. Unexpectedly, only two proteins showed reactivity in both the planar array and the IP. For targeted bead array, we selected 335 peptides for validation with an additional method and patient cohort. 174 from the planar array, 97 from IP and 102 from the literature. There was an overlap between peptides found in literature and in the IP or planar array. Of the 335 peptides, 4 were excluded due to weak coupling to the beads and 73 peptides due to high reactivity to control serum. Twenty antigens showed high reactivity in JIA patients. Fifteen of these were chosen from the planar array, three found in the literature as well as by IP and one each found exclusively in IP or in literature. Conclusion: Autoantibody discovery is highly dependent on the choice of method, as there was almost no overlap of autoantigens found by planar array and immunoprecipitation followed by mass spectrometry. The planar array identified 15 novel potential autoantigens and the IP identified one that was not previously mentioned in literature. Our findings show that established methods can be used to discover novel autoantigens in JIA. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
Sag 1,2 , Z. Balik 3 , S. Sener 3 , U. Akca Kaya 3 , S. Demir 3 , M. Kasap Cuceoglu 3 , E. Atalay 3 , S. Bocutcu 3 , T. Vural 1 , Y. Bilginer 3 , B. Deleuran 4 , S. Ozen 1,3 1 Pediatric Rheumatology Unit, Translational Medicine Laboratories, Hacettepe University, 2 Pediatric Rheumatology Clinic, Ankara Training and Research Hospital, 3 Pediatric Rheumatology, Hacettepe University, Ankara, Turkey, 4 Institution of Biomedicine, Aarhus University, Aarhus, Denmark Correspondence: E. Sag Introduction: Juvenile idiopathic arthritis (JIA) is the most common inflammatory joint disease in children, driven by continuous T-cell activation. T cell activation is counter-balanced by signals generated by co-inhibitory receptors (co-IRs) such CTLA-4, PD-1, LAG-3, and TIM-3. Objectives: We aimed to identify the role of co-IRs in the pathogenesis of different subtypes of JIA. Methods: In total, we included patients with oligoarticular JIA (n=67), polyarticular JIA (n=12), enthesitis related arthritis (n=17), systemic JIA (n=11) and healthy controls (HC, n=10). We collected plasma samples from the patients during the active phase of their disease. We measured the soluble plasma levels of co-IRs by commercial pre-defined cytometric bead array kits and their cellular expression by flow cytometry in blood mononuclear cells. We compared the plasma levels and cellular expressions of different coIRs within different JIA subgroups. Results: IL-2 levels were lower than HC in all JIA subgroups. The polyarticular JIA group distinguished from the four different JIA subgroups, by having different co-IR pattern. In this specific subgroup, CTLA4, PD-1 and 4-1BB levels were higher than other groups. Polyarticular JIA is the more chronic and severe form of JIA, especially when compared to oligoarticular JIA. We investigated the correlations between disease activity markers and plasma co-IRs. Plasma TIM3 levels correlated with erythrocyte sedimentation rate, C-reactive proteins and JADAS in the polyarticular JIA group. In oligoarticular JIA group, JADASs correlated with plasma PD-1 levels, C-reactive protein with PD-L1 plasma levels. Erythrocyte sedimentation rates correlated with IL-2, CD86, PD-L1 and PD-1 plasma levels. There was no correlation between disease activity markers and co-IRs levels in the systemic JIA group and enthesitis related arthritis group. Finally, we analysed the cellular surface expression of different co-IRs on the PBMCs of different JIA subtypes. Similar to plasma levels, both the percentage and the MFI (mean fluorescence intensity) of CTLA4 expression was higher in polyJIA subgroup. Conclusion: This is the first report studying the effects of different co-IRs in in different subtypes of JIA. Polyarticular JIA patients had a different co-IR profile, having more CTLA-4, PD-1 and 4-1BB in their plasma than the other subtypes of JIA, which may due to both increased degree of cellular activation and exhaustion of cells in this more resistent form of JIA. Acknowledgement This work was supported by a research grant from FOREUM Foundation for Research in Rheumatologyahin, S. Asadova 2 , S. N. Taşkın 3 , S. Doğantan 3 , S. Özdemir Çiçek 4 , E. Esen 3 , A. Paç Kısaarslan 3 , M. H. Poyrazoğlu 3 1 Pediatric Rheumatology, Derince Training and Research Hospital, Kocaeli, 2 Pediatrics, 3 Pediatric Rheumatology, Erciyes University, Faculty of Medicine, 4 Pediatric Rheumatology, Kayseri City Hospital, Kayseri, Turkey Correspondence: N. Sahin Introduction: Juvenile idiopathic arthritis (JIA) is a common chronic rheumatologic disease in children. In chronic diseases, comorbidities can adversely affect the course of the disease. Objectives: In this study, we aimed that investigated comorbidities in patients with juvenile idiopathic arthritis and their effects on the disease course. Methods: We retrospectively analyzed 204 patients under 18 years and with juvenile idiopathic arthritis. Patients were stratified into two groups with comorbidities and without comorbidities. Clinical findings, JIA subtypes, JADAS27, JSPADAS, JADI-A and E, and CHAQ scores were investigated in two groups. Results: The number of patients with comorbidity was 99 (48.5%). The most common comorbidity was familial Mediterranean fever (FMF) in 31 (31.3%) and uveitis in 23 (23.2%). The clinical and laboratory characteristics of the patients are summarized, and the comorbidities in table 1. There was no difference between patients with and without comorbidity in terms of age, age at diagnosis, duration of JIA, and sex. The most common JIA subtype in both groups was oligoarticular JIA, followed by enthesitis-related arthritis and polyarticular JIA. There was no significant difference in the number of comorbidities by JIA subtype (p=0.79). In addition, no correlation was found between the duration of JIA disease and the number of comorbidities (Rho=-0.69, p=0.49). According to the presence of comorbidity, there was no difference in the scores of JADI-A, JADI-E, CHAQ disability, CHAQ discomfort, and CHAQ pain (respectively, p= 0.52, p=0.33, p=0.77, p=0.44, p= 0.19). There was no significant difference in JSPADAS or JADAS 27 scores between the two groups (p=0.63, p=0.55, respectively). Conclusion: There was comorbidity in approximately half of the patients with juvenile idiopathic arthritis. Although the frequency was low, comorbidities involving all systems were observed. However, the presence of comorbidity did not affect the course of JIA. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
A. Boltjes 1 , A. Samat 1 , M. Plantinga 1 , M. Mokry 1 , B. Castelijns 2 , J. F. Swart 3 , B. J. Vastert 1,3 , M. Creyghton 2,4 , S. Nierkens 1,5 , J. V. Loosdregt 1,3 , F. V. Wijk 1,3 1 Center of Translational Immunology, University Medical Center Utrecht, 2 Hubrecht Institute, 3 Pediatric Rheumatology & Immunology, Wilhelmina Children’s Hospital, Utrecht, 4 Erasmus University Medical Center, Rotterdam, 5 Princess Maxima Center for Paediatric Oncology, Utrecht , Netherlands Correspondence: A. Samat Introduction: Dendritic cells (DC) are crucial for initiating and shaping immune responses. So far, little is known about the functional specialization of human DC subsets in (local) inflammatory conditions. Objectives: We profiled conventional (c)DC1, cDC2 and monocytes based on phenotype, transcriptome and function from the synovial fluid (SF) from Juvenile Idiopathic Arthritis patients (JIA). Methods: Paired peripheral blood (PB) and synovial fluid (SF) samples from 32 JIA and 4 RA patients were collected for mononuclear cell isolation. Flow cytometry was done for definition of antigen presenting cell (APC) subsets. Cell sorting was done on the FACSAria II or III. RNA sequencing was done on SF APC subsets. Proliferation assays were done on co-cultures after CD3 magnetic activated cell sorting (MACS). APC Toll-like receptor (TLR) stimulation was done using Pam3CSK4, Polyinosinic:polycytidylic acid (Poly(I:C)), Lipopolysaccharide (LPS), CpG-A and R848. Cytokine production was measured by Luminex. Results: cDC1, a relatively small DC subset in blood, were found to be strongly enriched in SF, and showed a quiescent immune signature without a clear inflammatory profile, low expression of pattern recognition receptors (PRR), chemokine and cytokine receptors, and poor induction of T cell proliferation and cytokine production. In stark contrast, cDC2 and monocytes from the same environment, showed a pro-inflammatory transcriptional profile, high levels of (spontaneous) pro-inflammatory cytokine production, and strong induction of T cell proliferation and cytokine production, including interleukin-17 (IL-17). Conclusion: At the site of inflammation, there is specific functional programming of human DCs, especially cDC2. Monocytes in particular seem to be the most pro-inflammatory, producing high levels of IL-6 and tumor necrosis factor alpha (TNF-a) and cDC2 also show a strong pro-inflammatory profile with high T cell activation capacity. In contrast, the enriched cDC1 remain relatively quiescent and seemingly unchanged under inflammatory conditions, pointing to a potentially more regulatory role. Patient Consent: Yes, I received consent Disclosure of Interest : A. Boltjes: None declared, A. Samat: None declared, M. Plantinga: None declared, M. Mokry: None declared, B. Castelijns: None declared, J. Swart Consultant with: Personal fees from Amgen outside of the relevant work, B. Vastert Consultant with: Personal fees from SOBI & Novartis outside of the relevant work , M. Creyghton: None declared, S. Nierkens: None declared, J. Loosdregt: None declared, F. Wijk Grant / Research Support with: ReumaFonds Nederland; grants from Sanofi, Regeneron and Leo Pharma outside of the relevant work, Consultant with: Johnson&Johnson and Takeda outside of relevant work
S. Samsonenko, T. Borysova Pediatric, “Dnipro State Medical University”, Dnipro, Ukraine Correspondence: S. Samsonenko Introduction: Juvenile idiopathic arthritis (JIA) is a chronic disease requiring years of therapy with non-steroidal anti-inflammatory drugs (NSAIDs), immunosuppressant’s, cytostatics, immunobiological agents. The aforementioned drugs, namely NSAIDs and cytostatics are potentially nephrotoxic. The above drugs, namely NSAIDs and cytostatics, are potentially nephrotoxic. About 8% of children with JIA have kidney damage, which develops on average 5 years after the onset of the disease. It has been established that the main risk factor for the development of kidney damage is the long-term exposure to NSAIDs and methotrexate in children with active forms of JIA. Early diagnosis of kidney damage will allow timely correction in the dosage of drugs and avoid their nephrotoxic effects. Objectives: To determine the effect of drug therapy in children with JIA on eGFR by using the Cystatin C-based equation and the Hoek formula based on the serum cystatin C study. Methods: 80 children with JIA participated in the study. The age of subjects was 10.4±4.41 (10.6-15.0) years. All children received methotrexate as a base drug. At the moment of examination 22 children received NSAIDs, 25 children received immunobiological preparations. Serum cystatin C content was determined by enzyme immunoassay. The Cystatin C-based equation 2012 and Hoek formulas were used to set the GFR by serum cystatin C levels. Results: Non-steroidal anti-inflammatory drugs led to a decrease in GFR as found by both the Cystatin C-based equation 2012 and the Hoek formula. The incidence of GFR reduction in patients treated with NSAIDs using the Cystatin C-based equation 2012 was 100%, and using the Hoek formula was 81.8%.The use of NSAIDs in children with JIA is a risk factor for the development of reduced GFR calculated by the Hoek formula. The incidence of reduced GFR in children with NSAID use was 54.5%, 6.7 times greater than in those without NSAIDs (OR = 12.9; CI: 3.76-44.25; p<0.001). There was a low chance of a Hoek formula decrease in GFR in children with JIA who received immunobiological therapy 9.1% vs 46.8% (OR = 0.11; CI: 0.03-0.42; p<0.001). Conclusion: Use of NSAIDs in children with JIA was more often associated with a reduction in GFR: by Cystatin C - based equation 2012 in 100% of cases p<0.01, by Hoek in 81.8%, p<0.001. The average of GFR was significantly lower in children treated with NSAIDs than in children without NSAIDs. Immunobiological therapy had a positive effect on the GFR value. The frequency of a decrease in GFR was significantly lower in the children treated with immunobiological therapy compared with those without immunobiological therapy 9.1% vs 46.8% (OR = 0.11; CI: 0.03-0.42; p<0.001)T. Schmidt 1,2 , E. Rydén 1,2 , A. Dahlberg 1,2 , S. Arve-Butler 1,2,3T. Schmidt Introduction: Synovial monocytes in oligoarticular juvenile idiopathic arthritis (oJIA) are polarized, displaying markers of both pro- and anti-inflammation. Objectives: To study the function of synovial monocytes, and to unravel how they obtain their phenotype. Methods: Synovial fluid (SF) and blood (which served as control) were collected from untreated oJIA patients upon therapeutic joint aspiration (total n=26). Surface markers (CD16, MerTK, HLA and CD86), cytokine production (IL-1ß, IL-6, IL-8 and TNF) and STAT phosphorylation (STAT1, STAT3 and STAT6) were analyzed in synovial- and circulating monocytes using flow cytometry. Isolated monocytes were used for efferocytosis assays using apoptotic neutrophils and co-stimulation of CD3 activated T cells from healthy donors. The influence of SF on healthy monocytes was analyzed by liquid-chromatography mass spectrometry and broad-spectrum phosphorylation assays. The mechanisms of how synovial monocytes obtain their functional phenotype was studied in vitro through stimulation of healthy monocytes using SF, migration using a transwell system or co-culture with fibroblast-like synoviocytes. Results: Monocytes from the joint of oJIA patients display functional alterations with pro- and anti-inflammatory features. Synovial monocytes, as compared to circulating monocytes, express markers of antigen presentation (HLA, CD86), induce proliferation and activation markers (CD25, HLA and CTLA-4) in healthy T cells and are primed for STAT1 phosphorylation. In contrast, synovial monocytes also express markers of clearance (MerTK, CD16), display less production of the pro-inflammatory cytokines (IL-1ß, IL-6, IL-8 and TNF) upon activation, and have increased efferocytosis. In healthy monocytes, SF from oJIA patients, as compared to serum, induces upregulation of biological processes involved in immune responses and regulation of lymphocyte proliferation, cell-cell adhesion, and endocytosis. At the phosphorylation level, synovial fluid induces mainly STAT3, which correlates with IL-6 levels in the synovial fluid (r=0.78, p<0.0011) and is fully blocked by pre-incubation with the anti-IL-6R antibody tocilizumab (p<0.0001) or the JAK inhibitor tofacitinib (p<0.0001). At the functional level, synovial fluid, mainly through an IL-6/STAT mechanism, induces the anti-inflammatory aspects observed in the patients’ synovial monocytes, such as surface markers (CD16, MerTK (p<0.0001), increased efferocytosis (p<0.0001) and resistance to production of IL-1ß, IL-6, IL-8 and TNF (p=0.0002) upon activation. The pro-inflammatory aspects are driven through cell-cell interactions in vitro , either through migration or co-culture, which result in increased expression of CD86, HLA and T cell activation (p<0.0001). Conclusion: Synovial monocytes from patients with oJIA display both pro- and anti-inflammatory functional alterations. This phenotype can be replicated in vitro , where SF induces the anti-inflammatory aspects mainly through IL-6/STAT signaling, whilst the pro-inflammatory aspects is replicated by cell-cell interactions through migration or co-culture with fibroblast-like synoviocytes. These data support a role of monocytes in the pathogenesis of oJIA and highlight potential impact of current and future drugs for the treatment of oJIA. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
V. Selmanovic 1 , A. Cengic 2 , A. Mustajbegovic 3 , D. Pokrajac 4 , T. Avcin 5 , M. Debeljak 6 , I. Sefic-Pasic 7 , M. Bukvic 7 , A. Dzananovic 7 1 Department for Allergology, Rheumatology and Clinical Immunology, Children’s Hospital University Clinical Center Sarajevo, 23 Department for Nephrology, 4 Department fo Nephrology, , Children’s Hospital University Clinical Center Sarajevo, Sarajevo, Bosnia and Herzegovina, 5 Departmentfor Allergology, rhjeumatology and Clinical Immunology, Childen’s Hospital University Clinical Center Ljubljana, 6 Department for Allergology, rheumatology and Clinical Immunology, Children’s Hospital University Clinical Center Ljubljana, Ljubljana, Slovenia, 7 Institut for Radiology, institut for Radiology, Sarajevo, Bosnia and Herzegovina Correspondence: V. Selmanovic Introduction: Renal disease is rare in children with juvenile idiopathic arthritis. The majority of children with nephrotic syndrome have minimal change disease, generally responsive to steroids. Only 10-20% are steroid-resistant. Objectives: to describe a case of rare association of early-onset polyarthritis, steroid-resistant nephrotic syndrom and MEFBgene mutation Methods: case report Results: boy,4,5y, first child of healthy non-consanguineous Caucasian parents, unremarkable family history. Disease onset: age 18mo with nephrotic sy. Treated accordingly with steroids; experienced 3 relapses by age 3y. At age 2y2mo, while od steroids, developed symmetrical arthritis of hips, knees, ankles. ANA, RF, HLAB27 were negative. There were positive Scl-70 antibody, repeatedly negative. Treatment was quickly escalated and cyclosporin A was succesfully added. Kidney biopsy showed minimal change disease, without signs of amiloidosis. No signs of other organ involvement. On follow up, at age 4,5y, child has preserved renal function, normotensive, mildly active left knee arthritis. Bilateral pes equinovarus was surgically corrected in infancy. Gene panels for nephrotic syndrome and arthritis revealed MAFBgene mutation NM_005461.5:c[125C>A];[=] in patient and mother, associated with hereditary osteolysis of carpal bones with and without nephropathy. Both are not fullfilling diagnostic criteria, so there is highly unlikely that the variant is disease causing. Conclusion: Pediatric rheumatologist treat early onset polyarthritis on everyday basis as well as nephrologist children with nephrotic syndrom of which 10-20% are steroid-resistant. Concomitant association of these two entities is rarely described, raising question whether are they part od the same still undefined disease and question of disease evolution. In this case, genetics were not diagnostic. Disclosure of Interest : None declared
vostyanov 1 , P. Lototskaya 2 , N. Babich 1 , D. Rassoha 1 , E. Zholobova 3 1 Department of Propaedeutics of Children’s Diseases, 2 Department of Rheumatology No. 1 of the University Children’s Clinical Hospital, 3 Department of pediatrics, I.M. Sechenov First Moscow State Medical University Ministry of Health of Russia (Sechenov University), Moscow, Russian Federation Correspondence: V. Sevostyanov Introduction: At present, the issue of studying effectiveness and safety of various genetically engineered biological drugs is of great interest. A clear analysis of the reasons for drug withdrawal and an algorithm for biology switching will allow a more balanced attitude towards the initiation of this type of therapy and development of individual plans for monitoring, including management of patients receiving genetically engineered therapy. Objectives: To analyze. Methods: An observational analytical cross-sectional study was conducted and included patients with juvenile idiopathic arthritis aged 0 to 17 years old, living in Moscow, who had history of switching and/or discontinuation of a biology in their anamnesis. This study assessed the following indicators: the period of biology therapy in months; the reason for changing or canceling therapy; the drug to which the switch was made. Results: The switching frequency was 9.9%. The main reasons for switching/cancellation were secondary failure - 57.8%, primary failure - 12.5%, therapy intolerance - 10.9%, and the development of “de novo” uveitis in 9.4% of cases. Cancellation of therapy due to the achievement of remission was noted in 6.2% of cases. There is a trend that more often patients were switched to adalimumab (29.3%); and when adalimumab was canceled, switching was carried out to tocilizumab and golimumab. When tocilizumab has been discontinued, the majority of patients were switched to canakinumab therapy. Conclusion: This analysis, being one of the first presenting the data from Russian Federation on the topic, shows that further accumulation and analysis of data, including cases of discontinuation of therapy, switching between drugs, as well as the duration of therapy before the event that caused the change or discontinuation of therapy are requiredS. J. W. Shoop-Worrall 1,2 , K. L. Hyrich 1,3 , L. R. Wedderburn 4,5,6 , N. Geifman 7 on behalf of CLUSTER, BSPAR-ETN Study, BCRD Study, CAPS, CHARMS 1 Centre for Epidemiology Versus Arthritis, 2 Centre for Health Informatics, The University of Manchester, 3 NIHR Manchester BRC, Manchester University NHS Foundation Trust, Manchester Academic Health Science Centre, Manchester, 4 Centre for Adolescent Rheumatology Versus Arthritis, GOS Institute of Child Health, University College London, 5 Paediatric Rheumatology, Great Ormond Street Hospital NHS Foundation Trust, 6 NIHR Great Ormond Street Hospital Biomedical Research Centre, NIHR Great Ormond Street Hospital Biomedical Research Centre, London, 7 School of Health Sciences, Faculty of Health and Medical Sciences, The University of Surrey, Guildford, United Kingdom Correspondence: S. J. W. Shoop-Worrall Introduction: Stratified medicine requires the identification of unique strata of a disease within which to base prognostic and treatment decisions. Juvenile idiopathic arthritis (JIA) offers a unique challenge in its inherent heterogeneity. The current ILAR classification, whilst useful for clinical categorisation, does not correlate with treatment outcomes. Therefore, further refinement, clustering and correlation of patient characteristics with treatment response are urgently required. Objectives: To identify novel, phenotypically consistent subgroups of children and young people (CYP) with JIA at the point of starting methotrexate, across 19 patient and disease characteristics. Methods: MTX-naïve CYP with JIA were selected if enrolled prior to April 2021 in one of four national JIA studies contributing to the UK CLUSTER consortium. Data from 19 harmonised study variables were extracted at point of starting MTX. Topological data analysis using a Gower similarity metric was used to identify clusters with distinct characteristics. Intervals and percent overlap between clusters were varied until an optimal model identified stable, potentially clinically plausible clusters. Significant differences in characteristics between identified clusters were tested using Kruskall-Wallis and Chi-Squared statistics. Results: Of 2915 CYP included, the majority were female (68%), of white ethnicity (90%); with the most common ILAR categories being oligoarthritis (35%) and RF-negative JIA (34%). The optimal TDA model identified six clusters which significantly differed across 16 of the 19 clinical variables at MTX initiation: Adolescents with low-moderate disease (Cluster 1, 41%), adolescents with predominantly sJIA and moderate-high disease (Cluster 2, 4%), children with predominantly sJIA and high disease (Cluster 3, <1%), children with oligo/RF-polyarthritis and low-moderate disease (Cluster 4, 43%) and two ANA-positive groups of largely females with moderate (Cluster 5, 11%) and high (Cluster 6, 1%) disease. Clustered groups also significantly differed in gender proportions (p<0.001), ethnicities (p<0.001), history of uveitis (p<0.001) and disease duration to both diagnosis (p<0.001) and MTX initiation (p<0.001), but did not differ in limited joint count (p=0.117), height (p=0.245) or BMI (p=0.394) z-scores. Conclusion: This study shows substantial heterogeneity in JIA at the point of MTX initiation, with six clusters identified across 19 demographic and clinical variables. ILAR categories across clusters were not always indicators of disease activity or symptom burden. Future analyses will correlate MTX treatment response within each cluster to understand what role these combined factors may have on initial treatment response. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
A. Smith 1,2 , C. Swindells-Macleod 1,2 , L. Kearsley-Fleet 1 , K. Hyrich 1,2 , S. Shoop-Worrall 1,3 1 Centre for Epidemiology Versus Arthritis, University of Manchester, 2 NIHR Manchester Biomedical Research Centre, Manchester University NHS Foundation Trust, 3 Centre for Health Informatics, University of Manchester, Manchester, United Kingdom Correspondence: L. Kearsley-Fleet Introduction: Oligoarthritis, a sub-type of JIA, can be categorised as persistent or extended. Children with persistent oligoarthritis, involving <5 joints, are often excluded from clinical trials as it is considered a milder form of JIA and managed less aggressively. Therefore, less is known about the longer term outcomes of those with persistent oligoarthritis. This knowledge could lead to better targeting of existing therapies for individuals and higher quality patient information. Objectives: To conduct a systematic literature review on the clinical and patient-reported outcomes of persistent oligoarthritis. This abstract presents the plans and current status of the review. Methods: Inclusion criteria for this systematic review are 1) inception cohorts with at least partial prospective data collection, 2) available in English language, 3) includes at least 50 patients with persistent oligoarthritis using ILAR criteria, 4) investigates clinical or patient-reported outcome. Exclusion criteria are 1) not specifically presenting or analysing outcomes in persistent oligoarthritis and 2) not reporting disease duration or follow-up time points from diagnosis at the point of outcome measurements. Search terms related to ‘oligoarthritis’, (variants of juvenile oligoarthritis plus outcomes of interest) were used in MEDLINE and Embase from 01/01/1997 to 30/05/2022 to cover the advent of current ILAR categories to present. These terms were also entered into PubMed from 30/05/2020 to 30/05/2022 to include publications published online ahead of print. Quality assessment, including common biases, of selected articles will be assessed using relevant questions from the Pasma et al. quality assessment (QA) tool. Primary outcomes to be extracted are the core JIA outcome variables (active joint counts, limited joint count, physician’s global assessment, parent global evaluation, erythrocyte sedimentation rate and additional functional status (CHAQ/HAQ). Additional outcomes are 1) remission status, 2) uveitis status, 3) pain, 4) Health-related Quality of Life and 5) treatment response. One reviewer will initially screen titles and abstracts. Two reviewers will then screen the full text of selected articles independently and agree the master list. The first reviewer will then extract data regarding study populations and outcome and another reviewer will check a selection of the extracted data. If there is disagreement or uncertainty between the two reviewers at any stage, a third reviewer will adjudicate. The review has been registered with Prospero (ID: CRD42022291943). Results: To date 1051 publications have been highlighted for initial review (title and abstract) by the first reviewer. The second review is now underway and preliminary results will be ready to present. Conclusion: There is an unmet need for improved understanding of outcome in persistent oligoarthritis. This systematic review will help inform treatment targets and future research in this common ILAR JIA categorSnipaitiene 1 , K. Snipaitiene 2 , I. Juskeviciute 2 , B. Skerbaite 2 , A. Baranauskaite 3 , S. Jarmalaite 2 1 Academy of Medicine, Pediatric Department, Lithuanian University of Health Scienses, Kaunas, 2 Human Genome Research Group, Life Sciences Center, Vilnius University, Vilnius, 3 Academy of Medicine, Rheumatology Department, Lithuanian University of Health Scienses, Kaunas, Lithuania Correspondence: A. Snipaitiene Introduction: New biomarkers for defining the disease activity in the juvenile idiopathic arthritis (JIA) patients are needed to predict disease course and tailor the individual treatment strategy. Several previous studies have shown that epigenetic regulation of inflammation through microRNAs (miRNAs) has significant impact on cytokine production and disease course in adult rheumatoid arthritis. Objectives: The aim of this study was to analyze if urine- and serum-derived inflammatory miR-16, -146a, and -155 could represent the disease activity in JIA patients. Methods: Twenty-three children diagnosed with JIA were included into the study (oligoarthritis and polyarthritis, excluding systemic JIA). For comparison, 5 healthy controls (HC) without any signs of inflammation were enrolled. Serum and urine samples were prospectively collected from Methotrexate- or anti-TNF-treated JIA patients and HC. JIA disease activity was evaluated using JADAS27, and JIA patients were divided into remission (R1, N=13) and active disease group (R0, N=10). The urinary and serum levels of selected miRNAs were evaluated by using quantitative reverse transcription PCR (RT-qPCR) method. Results: Eighty-seven % (20/23) of included JIA patients were female, and the mean age of JIA patients was 12.8 yrs (range 3-17 yrs). Significantly lower miR-16 serum levels were detected in JIA patients compared to HC (p=0.021). miR-16 levels were also significantly decreased in separate R0 and R1 groups vs. HC (p=0.023 and p=0.039, respectively). None of analyzed miRNAs were able to dichotomize JIA from HC in urine, however, significantly higher miR-16 levels were detected in R1 and R0 group patients vs. HC (p=0.013 and p=0.028, respectively). Furthermore, urinary and serum miR-16 levels were higher in those JIA patients who had the lowest patient global assessment (PGA) evaluation in compare to higher scores (PGA≤2 vs. ≥6, p=0.022 and vs. 3-5, p=0.041, respectively). Regarding the medications, serum miR-16 level was decreased in Methotrexate- vs. anti-TNF-treated group (mostly R0 vs. R1, p=0.067), resembling the lowest miR-16 abundancy in active disease. Moreover, male patients had lower urinary levels of miR-146a than females (p=0.011), and serum miR-155 was less abundant in younger patients (p>0.05). Bodily fluid comparison showed significantly higher levels of miR-146a in serum samples than in urine (p=0.005), whereas higher levels of miR-155 were detected in urine (p=0.013). Serum miR-16 demonstrated high diagnostic potential (AUC=0.85; p=0.017) with 85% sensitivity and 80% specificity. Conclusion: Findings of the study suggest that inflammatory miR-16 analysis in bodily fluids of JIA patients can be considered as potential diagnostic tool for disease activity and could be applied for non-invasive monitoring oftasiak 1 , A. Ryk 2 , J. Stańczyk 1 , E. Smolewska 1 1 Department of Pediatric Cardiology and Rheumatology, 2 Department of Biostatistics and Translational Medicine, Medical University of Lodz, Lodz, Poland Correspondence: A. Stasiakt is believed that children with JIA have lower cardiopulmonary capacity and worse exercise tolerance. The gold standard for assessing physical fitness is aerobic fitness, commonly referred to as the maximum or peak oxygen uptake volume (VO2 peak) measured during a maximum load exercise test. Reduced aerobic fitness may play a key role in predicting the health of JIA patients as it has been associated with cardiovascular diseases and increased adult mortality. The likely cause of this significant impairment is multifactorial and should be investigated to improve treatment strategy. Exercise programs should be applied and individualized or at least modified according to different types of the disease to improve aerobic fitness. Objectives: The aim of this study was to assess the oxygen capacity of adolescents with JIA along with echocardiographic and laboratory parameters in order to determine a group of patients with increased risk of developing cardiovascular diseases in comparison with healthy individuals. Methods: Determining cardiopulmonary parameters such as peak oxygen consumption, tidal volume, minute ventilation, exercise time, heart rate or oxygen pulse in combination with echocardiographic parameters and early markers of heart failure, such as NT-proBNP allowed to identify a group of patients with potential risk of developing cardiovascular disease, including heart failure. Patients were assessed based on parameters such as age, sex, BMI, type of JIA, disease activity, laboratory parameters, treatment. Results: Study consisted of 50 patients with JIA and 50 healthy patients who served as a control group. Patients with JIA had lower median values of peak VO2 (29.05 vs 38.02 ml/min/kg, p<0.001), O2 pulse (7.00 vs 11.40 ml/beat, p<0.001), minute ventilation (55.5 vs 84.5 l/min, p<0.001), oxygen uptake efficiency slope (1.62 vs 2.17, p<0.001), and cardiac output (8.25 vs 12.75 l/min, p<0.001) than in the control group. The ventilatory anaerobic threshold (VAT) was achieved earlier and at lower VO2 values in children with JIA (p=0.0001). Children with JIA also had lowered respiratory parameters such as maximum voluntary ventilation (p= 0.0031) and tidal volume (p=0.0002). Echocardiography revealed a significantly lower shortening fraction (p=0.0389); the ejection fraction was also lower in the JIA group, but it was not statistically significant. Fourteen patients (28%) had enlarged right ventricular dimensions in relation to BSA. Two patients had E/A on the mitral valve greater than 2, indicating a higher risk of left ventricular diastolic dysfunction . NT-proBNP levels of all but four patients were within norm range. Twenty-two patients attended any form of physical activity and they had significantly higher peak VO2 (p=0.0099) and ΔVO 2 /ΔWR relationship (p=0.0041) values than JIA patients who were not physically active. There were no statistically significant correlations between peak VO2 and disease duration and treatment. Conclusion: Children with JIA show moderate to severe physical impairment compared to healthy peers. Some of the patients had abnormalities in laboratory tests and echocardiographic examination, which may suggest an increased risk of cardiovascular complications. Patients who were physically active had significantly better aerobic capacity than those who were not physically active. Exercise programs tailored to the patient’s abilities should be implemented to reduce the risk of developing cardiovascular diseases. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
E. Tarakcı 1 , E. P. KısaIstanbul University Cerrahpasa, 2 Health Science Faculty, Department of Physiotheraphy and Rehabilitation, Biruni University, Istanbul, 3 Health Science Faculty, Department of Physiotheraphy and Rehabilitation, Medeniyet University, İstanbul, 4 Department of Pediatric Rheumatology, Cerrahpaşa Faculty of Medicine, Istanbul University Cerrahpasa, Istanbul, Turkey Correspondence: E. Tarakcı Introduction: Juvenile idiopathic arthritis (JIA) is a group of diseases that can affect many joints and systems with problems such as pain, joint stiffness, muscle atrophy and weakness. Pain is one of the most frequently reported conditions. The thought that pain may cause an exacerbation of the disease creates a fear and there may be significant barriers to increasing the level of activity. In the case of pain caused by the disease, kinesiophobia may develop in children. Objectives: The aim of the study is to evaluate the perception of kinesiophobia and proprioception due to pain status in children with knee involvement. Methods: Fifteen patients aged between 4 and 16 who were followed up with rheumatism diagnosis and treatment in Istanbul University Cerrahpaşa Health Sciences Physiotherapy and Rehabilitation Unit were included in the study. Sociodemographic information, involved joint location and disease duration of all cases who voluntarily accepted to participate in the study were questioned with a case report form. “Visual analog scale (VAS)” was used for pain, “Tampa Kinesiophobia scale (TKS)” for kinesiophobia, and “universal goniometer” for proprioception assessment. The target angle to be reached was determined as 60 degrees of knee flexion. The difference between the targeted angle and the achieved angle was recorded as the “achieved proprioception angle”The age of 15 children (4 boys, 11 girls) included in the study was calculated as 11.16±2.44, while the disease duration was calculated as 42.20±23.30 months. Pain status were determined as 4±2.39 (min:0- max:7). While there was a positional correlation between reaching the “achieved proprioception angle” by disease duration and TKS (p≤0.05), a positive correlation was found between reaching the proprioception angle determined by TKS and pain status (p≤001). No significant correlation was found between disease duration and pain status. Conclusion: It showed that the sensation of pain due to the disease in children with JIA may cause fear and avoidance behavior towards movement in children. Considering the duration of the disease, the importance of directing the children to movement with methods that will relieve pain in the early period and adding proprioception exercises to the treatment made us think. Although the small number of patients seems to be a limitation in the results of our study, it is not easy to use the TKS in children. The sample group of our study continues to be expanded. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
G. Tarantino 1 , D. Pires Marafon 1 , F. Comitini 2 , R. Simeoli 3 , A. Aquilani 1 , R. Nicolai 1 , E. Marasco 1 , F. De Benedetti 1 , S. Magni Manzoni 1 1 Rheumatology, Ospedale Pediatrico Bambino Gesù, Rome, 2 Paediatrics, Ospedale Di Cagliari, Cagliari, 3 Metabolic Disease, Ospedale Pediatrico Bambino Gesù, Rome, Italy Correspondence: G. Tarantinoof drug administration (1W vs 2Wto ADA6-month-period (Feb 2021-Jul 2021). Children with idiopathic uveitis were excluded. Samples furthest from last drug administration was defined as “trough level”. Data were analyzed via descriptive statistics (STATA 15.1). Results: Of 51 JIA patients treated with ADA (39% females), 28 had ANA positive oligoarthritis with almost one large joint involvement at disease onset (T0). None of them presented RF-positive polyarthritis, nor systemic JIA (s-JIA). The median age at T0 was about 3 years. Half of study cohort was b-DMARDS naive at ADA start (T1), while all children were on c-DMARDs. Chronic recurrent uveitis was the main reason for ADA starting, followed-by tenosynovitis and spine or hip active arthritis. At the first ADA/AAA sampling (T2), approximately 10% of patients had active disease, with elbows or wrists as the most frequently involved. Extreme variability was observed between AAA titers (median 8.4 AU/ml) and ADA levels (median 16.4 micrograms/ml), regardless from 1 or 2-weekly administration. ADA levels, compared to the days from last drug administration, reached plateau values corresponding to a pharmacokinetic steady state. Among both study groups, a possible inverse correlation between ADA and AAA was found. We identified 20 JIA patients with clinical (presence of arthritis or uveitis) or laboratory (ESR ≥15 mm/h; CRP> 0.5 mg/dL) disease activity. Among these in only 3 we found very high AAA titers (350.0 - 113.4 AU/mL). In the subgroup of 27 patients with “trough-level” sampling heterogeneous data about AAA titers and drug level distribution were observed. Conclusion: Our preliminary data showed a possible association between occurrence of AAA and lower ADA levels. However, a targeted risk analysis about high AAA titers and LOR incidence was not availablestudies JIA. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
, D. Pires Marafon, A. Aquilani, R. Nicolai, E. Marasco, F. De Benedetti, S. Magni Manzoni Italy Correspondence: G. Tarantino Introduction: IAGI allow precise release of steroids in inflamed sites, with proven efficacy in JIA. In younger children and in multiple injections procedures IAGI usually require general sedation, whereas local anesthesia in older patients and in single/few injection(s) procedures is well tolerated. It is not known whether COVID-19 pandemic conditioned the access of JIA children to IAGI and the procedure setting. Objectives: To observe the frequency and the features of IAGI procedures in JIA patients straddling COVID-19 pandemic. Methods: Records of JIA patients, who underwent IAGI from January 2019 to May 2021, with available information in the medical reports were retrospectively reviewed. Demographic and clinical features, including type, number and setting of intra/peri-articular injections, were registered. Data were analyzed through descriptive statistics. Results: Of a total of 297 Caucasian patients included in the study (78.8% females), 152 (51.2%) had persistent oligoarthritis, 46 (15.5%) extended oligoarthritis, 89 (30.0%) polyarthritis (only 4 RF-positive), 6 (2.0%) systemic arthritis, 2 (0.7%) psoriatic arthritis and 2 (0.7%) enthesitis-related arthritis (ERA). The median age at IAGI was of 9.4 years in patients undergone general sedation, and of 16.2 years in those in local anesthesia. At GCs injection, 167 patients (56.2%) were off therapy, 77 (25.9%) on treatment only with Methotrexate, 30 (10.1%) with TNF-inhibitors association. During the study period, 434 IAGI were practiced , without significant differences in frequency of procedures between the time interval before and after the COVID-19 pandemic outbreak (Nr.16/month and 14/month, respectively) and the setting (general sedation in 73% and 70%, respectively) . The median age of children at IAGI was of 9.4 yrs in those who underwent general sedation, whereas was of 16.2 yrs in those treated in local anesthesia. Most of patients (73%) underwent only one procedure, 19% two and 9% three or more. Of the 149 procedures with single injection, 69% were performed in local anesthesia. Of note, in 117 procedures with multiple injections (27%) 5 or more sites were injected. Of a total of 396 sites injected, 80% were large joints, 47% tendons/bursae, 40% small joints. As expected, the knee, the tibiotalar, the elbow and the wrist were the most frequently injected joints (55.3%, 36.9%, 16.4%, 14.8% respectively). Notably, all of hip injections (n=6) were performed after the COVID-19 outbreak. Conclusion: The frequency of IAGI procedures in JIA patients did not change with the COVID-19 outbreak at the study center. The knee, the tibiotalar, the elbow and the wrist were the most frequently injected joints. Hip injection were performed only during COVID-19 pandemia. Our results suggest that JIA patients and their caregivers were not limited by the pandemic in accessing the procedures and/or rely on the benefits of this treatment strategy despite potential logistic difficulties Patient Consent: No, I have not receive consent Disclosure of Interest : None declared
C. Tomé 1 , F. Oliveira-Ramos 1,2 , R. Campanilho-Marques 1,2 , A. F. Mourão 3,4 , S. Sousa 5 , A. T. Melo 1,6 , R. L. Teixeira 1,6 , A. P. Martins 7 , S. Moeda 8 , P. Costa-Reis 1,8 , R. P. Torres 3,4 , H. Fonseca 8 , M. Gonçalves 7 , M. J. Santos 1,5 , L. Graca 1,9 , J. E. Fonseca 1,6Lisbon Academic Medical Center, 2 Pediatric Rheumatology Unit, Centro Hospitalar Universitário Lisboa Norte, EPE, Hospital de Santa Maria, Lisbon Academic Medical Center, 3 Rheumatology Department, Centro Hospitalar Lisboa Ocidental, EPE, Hospital de São Francisco Xavier, 4 NOVA Medical School, Faculdade de Ciências Médicas, Universidade Nova de Lisboa, Lisbon, 5 Reumatology Department, Hospital Garcia de Orta, Almada, 67 Pediatric Surger8 PediatricLisbon, 9 Instituto Gulbenkian de Ciência, Oeiras, Portugal Correspondence: F. Oliveira-Ramos Introduction: Juvenile idiopathic arthritis (JIA) is the most common pediatric rheumatic disease and encompasses several different categories. Recently, our group has described that most patients diagnosed with the categories of extended oligoarticular JIA (eoJIA) and polyarticular JIA (pJIA) fulfil classification criteria for rheumatoid arthritis (RA) in adulthood. Moreover, we have also shown that B cell alterations and a B-cell related cytokine pattern are present since the first weeks of RA development. Therefore, we hypothesized that eoJIA and pJIA could also exhibit a cytokine profile in peripheral blood sustaining B cell activation. Objectives: The main goal of this study was to evaluate a panel of proinflammatory and anti-inflammatory cytokines and chemokines relevant for B cell triggering and function in serum samples of eoJIA and pJIA when compared to healthy controls and persistent oligoarticular JIA (poJIA). Methods: Blood samples were collected from eoJIA (n=15), pJIA (n=19) and poJIA (n=28) patients treated with disease modifying anti-rheumatic drugs. A group of age-matched healthy individuals (n=16) was used as control. Serum levels of APRIL, BAFF, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-17A, IL-21, IL-22, IFN-γ, PD-1, PD-L1, sCD40L, CXCL13 and TNF were measured by LEGENDplex TM multiplex bead-based immunoassay and/ or ELISA in all groups included. Results: Children with eoJIA and pJIA, but not poJIA, had significantly increased serum levels of APRIL and BAFF when compared to controls. Furthermore, APRIL and BAFF serum levels were significantly correlated in JIA patients. No significant differences were detected in serum levels of other cytokines between all groups analyzed. Conclusion: Patients diagnosed with eoJIA and pJIA, but not poJIA, have elevated serum levels of APRIL and BAFF in comparison to controls. These results indicate an involvement of these cytokines in the pathogenesis of these JIA categories, suggesting a B cell activation profile. Nevertheless, influence of treatment on the pattern of cytokine environment cannot be excluded. Further studies on B cell immune responses should be explored in JIA. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
C. Trincianti 1 , M. Backström 2 , M. Tarkiainen 3 , F. Bovis 1 , T. Qiu 4 , M. Esi 5 , D. J. Lovell 6 , N. Ruperto 7 , B. Gottlieb 8 , P. Vähäsalo 9,10,11 , A. Consolaro 1,7 1 University of Genova, Genova, Italy, 2 Department of Pediatrics, The Wellbeing Services County of Ostrobothnia, Vaasa, , 3 New Children’s Hospital, Helsinki University Central Hospital and University of Helsinki, Helsinki, Finland, 4 Department of Biostatistics and Epidemiology, Cincinnati Children’s Hospital Medical Center, Cincinnati, 5 University of Washington and Seattle Children’s Hospital, Seattle, Washington, 6 Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, United States, 7 Pediatric Rheumatology Unit, IRCCS Istituto G. Gaslini, Genova, Italy, 8 Cohen Children’s Medical Center, Lake Success, New York, United States, 9 Pedego Research Unit, University of Oulu, Oulu, 10 Department of Children and Adolescents, Oulu University Hospital, Oulu, 11 Medical Research Center Oulu, University of Oulu and Oulu University Hospital, Oulu, Finland Correspondence: C. Trincianti Introduction: The physician’s global assessment of disease activity (PhGA) is a key outcome measure of juvenile idiopathic arthritis (JIA). It consists of the rating of the overall level of child’s disease activity on a 100-mm or 21-numbered circle visual analogue scale (VAS), with anchors of ‘0 = no activity’ and ‘100 = maximum activity’. The PhGA is a complex construct that captures the examiner’s subjective appraisal of patient’s disease activity at the time of the visit. However, physicians do not always seem to use the VAS in a uniform way. Objectives: Aim of the study is to show the heterogeneity in PhGA scoring by comparing the assessment of a large international cohort of pediatric rheuamtologists evaluating multiple JIA patient’s case scenarios proposed through a web-based survey Methods: A questionnaire including 17 detailed JIA patient cases was sent electronically to all PRINTO and PR-COIN members. The cases were structured to represent a wide spectrum of clinical situations and the PhGA was assessed by the responders on a scale from 0 to 100. To demonstrate the consistency among the VAS scores provided by multiple physicians we assessed the inter-rater reliability using the intra-class correlation (ICC). ICC estimates and their 95% confidence intervals (CI) were calculated using the “psych” package available in R (version 3.5) based on a single-rating, 2-way random-effects model. ICC values less than 0.5 indicateMoreover, the variability of scoring in each case was compared by the interquartile range (IQR) of the PhGA scoring. Results: Out of 491 physicians who completed the survey, 418 (85.1%) completed the VAS scoring for all the 17 patients. The ICC of this group was 0.53 (95%CI: 0.38-0.72) indicating moderate to poor reliability. Considering the IQR of the single cases, those with the highest variability were: Case 1 (a polyarticular JIA patient with no joint involvement but with active uveitis, IQR= 37); Case 17 (an enthesitis related arthritis subject with 1 swollen joint, several restricted joints, complaining of important pain and morning stiffness, IQR= 30.5); Case 13: a RF positive polyarticular arthritis child with 10 active and 21 limited joints with 0.5 as pain VAS and no morning stiffness (IQR=28). Conclusion: The scoring of the PhGA seems to have a poor to moderate reliability throughout the world and, in some clinical pictures, the variability expressed as IQR of this measure result higher than the expected. Shared guidelines for scoring the PGA are therefore needed to obtain consistent patient assessment in clinical trials and routine practicTsitsami 1 , I. Sarrigeorgiou 2 , M. Tsinti 1 , P. Lymberi 2 1 First Department of Pediatrics, University of Athens Medical School, Children’s Hospital Aghia Sofia, 2 Laboratory of Immunology, Department of Immunology, Hellenic Pasteur Institute, Athens, Greece Correspondence: E. Tsitsami Introduction: Natural (auto)antibodies (NAAs) are primarily polyreactive antibodies encoded by germline V-gene segments. They detect autoantigens and new antigenic determinants with low affinity, and present prior to the body encountering cognate antigens. Therefore, NAAs provide a first line of defense allowing time for a specific antibody response to be elicited. Moreover, they play several roles in the immune system, including regulation of B-cell responses, control of B-cell development, selection of the B-cell repertoire, protecting thus the organism against autoimmune diseases. The IgM-NAAs are the most abundant in the body and the best studied while IgA-NAAs against lipopolysaccharides (LPS) have been considered as an endogenous homeostatic mechanism with innate specificity to microbiota. Objectives: Given the autoimmune nature of juvenile idiopathic oligoarthritis (JIO), this study was scheduled to examine the possible involvement of NAAs in its pathogenesis. Methods: Seventy JIO patients (aged 8,13±4,45 years, 14 males) were enrolled. The IgM antibody activity against the hapten trinitrophenyl (TNP) (proposed as a surrogate measure for polyreactivity because it is a synthetic molecule not present in the environment, to which individuals are not normally exposed), actin (a highly conserved cytoskeletal protein) and F(ab’) 2 IgG fragments (proposed as an index of immunoregulatory function) as well as concentrations of IgM ([IgM]) were measured by in-house ELISAs. Similarly, the IgA antibody activity against LPS and the concentration of IgA ([IgA]) were measured. Multivariate analysis was performed with ratios of specific IgM and IgA activities to the total IgM and IgA concentration as dependent variables and the clinical parameters of patients as independent variables. Results: Patients with active JIO presented with ratios of IgM activities against TNP, actin and F(ab’) 2 IgG fragment to the [IgM] as well as the ratio of IgA anti-LPS activity to [IgA] significantly lower than those with inactive disease. These findings were independent of patients’ gender and age, the age at disease onset, the duration of disease, the antinuclear antibodies (ANA) positivity and the presence of uveitis. Conclusion: Our results provide evidence supporting the autoimmune etiology of JIO. They also postulate a contribution of IgM-NAAs in the emergence of oligoarthritis flares. It is well known that the lower activity of IgM NAAs induces disturbances in tissue homeostasis, in the modulation of the immunological response and in the apoptotic clearance of cells that could drive disease flares. Finally, the observed correlation between IgA anti-LPS NAAs and disease activity might be related to the recently uncovered involvement of microbiota in the disease pathogenesi-K. Tuomi 1 , K. Rebane 2 , E. D. Arnstad 3,4 , L. Berntson 5 , A. Fasth 6 , M. Glerup 7 , T. Herlin 7 , H. Kautiainen 8,9 , E. Nordal 10 , S. Peltoniemi 11 , M. Rygg 12,13 , V. Rypdal 14 , M. Zak 15 , K. Aalto 2 1 Department of Pediatrics , New Children’s Hospital, Helsinki University Hospital, 2 Department of Pediatrics, New Children’s Hospital, Helsinki University Hospital, Helsinki, Finland, 3 Department of Pediatrics, Levanger Hospital, Nord-Trøndelag Hospital Trust, Levanger, 4 Department of Clinical and Molecular Medicine, NTNU, Trondheim, Norway, 5 Department of Women’s and Children’s Health, Uppsala University, Uppsala, 6 Department of Pediatrics, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden, 7 Department of Pediatrics, Aarhus University Hospital, Aarhus, Denmark, 8 Primary Health Care Unit Kuopio, Kuopio University Hospital, Kuopio, 9 Folkhälsan Research Center, Helsinki, Finland, 10 Department of Pediatrics, University Hospital of North Norway and UIT, Tromso, Norway, 11 Department of Pediatrics, Helsinki University Hospital, University of Helsinki, Helsinki, Finland, 12 Department of Clinical and Molecular Medicine, NTNU, Norway, 13 Department of Pediatrics, St. Olavs University Hospital , Trondheim, 14 Department of Pediatrics, University Hospital of North Norway and UiT, Tromso, Norway, 15 Department of Pediatrics, Rigshospitalet Copenhagen University Hospital, Copenhagen, Denmark Correspondence: A.-K. Tuomi Introduction: Obesity is a worldwide problem 1 but relatively little is known about its influence on the disease activity, functional ability, and quality of life (QoL) of patients with juvenile idiopathic arthritis (JIA). Objectives: To investigate the disease variables and QoL of patients with JIA divided into different body mass index (BMI) categories. Methods: This study is a part of the population-based Nordic JIA cohort study. All newly diagnosed JIA patients were recruited from defined regions of Finland, Sweden, Norway, and Denmark between 1997-2000 2 . Altogether 434 patients attended a follow-up visit (mean disease duration 17.5 ± 1.7 years) and 355 were included. Patients were categorized according to their (BMI): BMI<25.0, BMI 25.0-29.9, and BMI≥30.0. Disease characteristics were collected, and QoL (Fatigue Severity Scale FSS, Pittsburg Sleep Quality Index PSQI, SF36) was measured. Results: In total, 355 (81.7 %) patients were included (mean age 24,7 years, females 72 %). Fifty-five percent of patients in the group with BMI<25.0 had oligoarthritis, whereas in the group with BMI≥30.0 (obesity) 32 % belonged to either psoriatic or enthesitis-related JIA. BMI was also found to be related to higher disease activity score (DAS28). Higher BMI was related to lower functional ability, measured by Health Assessment Questionnaire (HAQ) (p<0.001). Patients with higher BMI reported significantly more pain (p=0.005) and had higher number of tender joints (p=0.006). Likewise, they had significantly higher CRP and ESR (p=0.002) and global VAS (p<0.001). They reported lower functional ability, experienced more fatigue, and their sleep quality was assessed to be inferior. Significant relationship was also found in the two domains of QoL: Bodily pain and general health. BMI was not found to be correlated with physical activity. Conclusion: BMI was related to disease activity, disability, and quality of life. The role of obesity in inflammatory conditions, like JIA, needs more attention in future research. References: 1) www.who.int/health-topics/obesity 2) Glerup M, Rypdal V, Arnstad ED, Ekelund M, Peltoniemi S, Aalto K,and Research. 2020;72(4) Patient Consent: Yes, I received consent Disclosure of Interest : None declared
J. W. van Straalen 1 , S. de Roock 1 , V. Stanevicha 2 , L. Lamot 3 , A. Estmann Christensen 4 , I. Rumba-Rozenfelde 5 , C. Lazar 6 , Y. Uziel 7 , T. Arkachaisri 8 , N. M. Wulffraat 1 , N. Ruperto 9 , J. F. Swart 1 on behalf of Paediatric Rheumatology INternational Trials Organisation (PRINTO) 1, 2 Riga Stradins University, Children University Hospital, Riga, Latvia, 3 University Hospital Center Zagreb, University of Zagreb School of Medicine, Zagreb, Croatia, 4 Department of Pediatrics, Odense University Hospital, Odense, Denmark, 5 Pediatric Rheumatology, University of Latvia, Riga, Latvia, 6 Spitalul Clinic de Urgenta pentru Copii, Cluj-Napoca, Romania, 7 Pediatric Rheumatology Unit, Department of Pediatrics, Meir Medical Centre, Kfar Saba, Israel, 8 Translational Immunology Institute, DukeNUS Medical School, SingHealth Academic Medical Center, Singapore, Singapore, 9 Clinica Pediatrica e Reumatologia, IRCCS Istituto Giannina Gaslini, Genoa, Italy Correspondence: J. W. van Straalen Introduction: Autoimmune diseases (ADs) tend to cluster within families. However, little is known about the disposition to AD among children diagnosed with juvenile idiopathic arthritis (JIA). Objectives: To determine the prevalence of and factors associated with ADs in parents of children with JIA. Methods: Data were collected from the international Pharmachild register. Characteristics were compared between patients with and without a family history of AD. Prevalence rates of familial ADs were calculated and compared with general population prevalence rates as reported in the literature. Results: The most common ADs in parents of the included JIA patients (n = 8,673) are listed in Table 1. For several ADs, prevalence was higher compared to reported numbers in the general population. Clinical Juvenile Arthritis Disease Activity Scores (cJADAS) at study entry and last follow-up were not significantly different between patients with (n = 1,231) and without a family history of AD (n = 7,442) ( P = 0.61 and P = 0.82, respectively). Factors associated with a family history of AD were older age at JIA onset ( P < 0.01), Scandinavian residence ( P < 0.01), enthesitis-related arthritis, psoriatic arthritis and undifferentiated arthritis ( P < 0.01), ANA positivity ( P = 0.03) and HLA-B27 positivity ( P < 0.01). Conclusion: Several ADs have an increased prevalence in parents of children with JIA. Familial AD therefore proves to be a risk factor for JIA development and certain diseases should not be overlooked during family health history at the diagnosis stage. A family history of AD is associated with the JIA subtype but does not influence the severity or disease course. References 1. Parisi R, Iskandar IYK, Kontopantelis E, Augustin M, Griffiths CEM, Ashcroft DM. National, regional, and worldwide epidemiology of psoriasis: systematic analysis and modelling study. BMJ . 2020;369. 2. Vanderpump MPJ. The epidemiology of thyroid disease. Br Med Bull . 2011;99(1):39-51. 3. Almutairi K, Nossent J, Preen D, Keen H, Inderjeeth C. The global prevalence of rheumatoid arthritis: a meta-analysis based on a systematic review. Rheumatol Int 2020 415 . 2020;41(5):863-877. 4. Stolwijk C, Onna M van, Boonen A, Tubergen A van. Global Prevalence of Spondyloarthritis: A Systematic Review and Meta-Regression Analysis. Arthritis Care Res (Hoboken) . 2016;68(9):1320-1331. 5. Ng SC, Shi HY, Hamidi N, et al. Worldwide incidence and prevalence of inflammatory bowel disease in the 21st century: a systematic review of population-based studies. Lancet . 2017;390(10114):2769-2778. 6. Thierry S, Fautrel B, Lemelle I, Guillemin F. Prevalence and incidence of juvenile idiopathic arthritis: A systematic review. Jt Bone Spine . 2014;81(2):112-117. doi:10.1016/j.jbspin.2013.09.00ermé, J. Granhagen Ljugner, C. Bartholdson Karolinska Institutet, Stockholm, Sweden Vermé Introduction: In Sweden, approximately 1500-2000 children suffers from Juvenile Idiopathic Arthritis (JIA). The transfer from pediatric rheumatology care to adult rheumatology care is often perceived as difficult and creates a great deal of concern for the adolescents and their parents. Releasing control and allowing the adolescents to take greater responsibility is, for example, one if the difficulties in the transition process. It´s important that parents feel confident and ready to handover the responsibility to facilitate and give support to the adolescent during the transition process. Objectives: The purpose of the study is to investigate parent’s perspective of how ready adolescents with Juvenile Idiopathic Arthritis (JIA) are to transfer to adult care and to take responsibility for their own health. Methods: Parents to members of The Swedish National Organization for Young Rheumatics and parents to patients at the pediatric rheumatology clinic at Astrid Lindgren´s Children´s hospital in Sweden, aged 14-18, received an invitation to participate in the study by responding to questions included in the Readiness for Transition questionnaire . Results: Key results: - Most of the parents felt that their adolescents took responsibility for talking to the healthcare professionals when they visit the clinic but few took talked with healthcare professionals via phone. - Only a few percent (3%) of the parents reported that the felt that their adolescents where fully ready to transfer to adult care. The data consist of 96 parents who answered the questionnaire (women n= 67, men n= 28). The results show that parents perceive that adolescent’s responsibility varies greatly depending on the area of responsibility. When it came to areas concerning their own care parents reported that the adolescents took greater responsibility than when it came to for example taking contact whit the clinic in different ways. Furthermore, the results show that a little more than a quarter of the parents felt that their adolescent didn’t take any responsibility for regularly blood test, but it was also little more than a quarter of the parents who felt that their adolescents almost always took responsibility for regularly blood test. The parents additionally reported that the adolescents often took responsibility for their medications, showing up at booked visits at the clinic and talking to the healthcare professionals. The areas that the parents reported that the adolescents took little responsibility for were booking visits to the clinic, talking on the phone with healthcare professionals and renewing prescriptions. When the parents were asked to tell how ready they felt their adolescent where to take full responsibility for their health only 4 % reported that the adolescent where fully ready and 43% reported that their adolescent was not ready at all. Similar results were reported when the parents where asked how ready the adolescents where to transfer to adult care. Only 3% reported that their adolescent was fully ready for transfer and 47% was not ready at all to transfer to adult care. Conclusion: The result demonstrates that parents to Swedish adolescents with juvenile arthritis didn´t perceive that their adolescents where ready to transfer to adult care. Parents need support to be able to find strategies to help the adolescents to increase their participation and knowledge in their own care. One possible way to meet those needs is to introduce a structed transition program in pediatric rheumatology care in Sweden. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
086. Aortic stiffness comparison between healthy and juvenile rheumatoid arthritis children P087. Polyserositis as the initial presentation of systemic JIA P088. Vaccination with SRP as a trigger of systemic juvenile idiopathic arthritis P089. Prolonged fever, rash, arthritis and hyperferritinemia in a child: a case of systemic idiopathic juvenile arthritis with suspicion of occult MAS P090. Comparative analysis of serum biomarkers and peripheral blood gene expression in systemic juvenile idiopathic arthritis and macrophage activation syndrome P091. Speaking the same language: international cross-validation of emerging biomarkers for juvenile idiopathic arthritis - the 2022 CARRA-PRES collaborative research award P092. Predictors of remission in children with systemic juvenile idiopathic arthritis receiving tocilizumab: a retrospective cohort study P093. SARS-COV-2 in systemic juvenile idiopathic arthritis: can ANTI-IL-1 drugs prevent MIS-C? A monocentric data collection P094. The emerging role of cardiovascular magnetic resonance in the assessment of cardiac involvement in systemic juvenile idiopathic arthritis: early treatment for an early resolution P095. Fever with rash - the path leads us here P096. Atypical skin manifestations at onset of systemic juvenile idiopathic arthritis are associated with failure of first-line corticosteroid treatment and difficult-to-treat disease P097. Clinical features of systemic juvenile idiopathic arthritis associated with interstitial lung disease: single center’s experience P098. Dynamics of vascular condition and endothely functions in patients with systemic form of juvenile idiopathic arthritis on the treatment P099. Fever of unknown origin: an unexpected outcome P100. Establishing the CARRA Registry research network for systemic juvenile idiopathic arthritis-associated lung disease (CARE-NETS) P101. Patterns of disease course in systemic juvenile idiopathic arthritis – preliminary data from the CAPS cohort P. Avramovski 1 , S. Stoilova 2 , M. Lazarevski 3 , S. Talev 4 , M. P. M. P. Avramovska 5 1 Internal medicine, Clinical hospital D-r T. Panovski - Bitola, 2 High medical scholl, St Klemet of Ohrid - Bitola, Bitola, 3 Internal medicine, GOB 8th september, Skopje, 4 Chirurgie, 5 Gynecology and obstetric, Clinical hospital D-r T. Panovski - Bitola, Bitola, North Macedonia Correspondence: P. Avramovski Introduction: Aortic pulse wave velocity (PWV) is indicator of arterial stiffness. It is advances with age and seems accelerated in certain disease conditions like rheumatoid arthritis (RA), diabetes and chronic kidney disease. Objectives: The aim of this study was to find the difference between PWVs between healthy and RA patients. Methods: arotid-femoral PWV was assessed by pulsed-Doppler ultrasound synchronized with electrocardiography (ECG) in 65 healthy and 87 juvenile rheumatoid arthritis children aged 10.3 ± 4.1 years. Demographic data: age, height, weight, heart rate (HR) and high-sensitive C-reactive protein (hs-CRP) were measured or taken from a health record. Results: The median PWV were: 4.52 (3.24 to 5.21) vs. 4.71 (3.47 to 5.56) in healthy and RA patients. PWV is significantly higher in RA patients (P = 0.0007). PWV significantly correlates with age (r = 0.245, P = 0.022), height (r = 0.312, P = 0.003), but not with HR (r = 0.176, P = 0.102) and weight (r = 0.187, P = 0.082) in RA patients group. PWV significantly correlated with CRP in juvenile RA patients (r = 0.241, P = 0.024), but not in healthy control (r = 0.179, P = 0.154). Conclusion: Arterial stiffness or precisely PWV was more pronounced in the child with juvenile RA than in the healthy control. Hs-CRP as a marker of acute and low-grade inflammation is correlated with artery elasticity in juvenile RA patients Patient Consent: No, I have not receive consent Disclosure of Interest : None declared
J. N. Bathia 1 , P. Pal 1 , I. R. Chaudhury 2 , P. P. Giri 3 , R. KapoorIntensive Care, Institute of Child Health, Kolkata, India Correspondence: J. N. Bathia Introduction: In systemic onset juvenile idiopathic arthtitis (sJIA), polyserositis may precede the development of arthritis but they constitute rare and incidental findings Objectives: to describe a three years old who presented with polyserositis to be later diagnosed as sJIA on development of arthritis. Methods: Following a trauma to the left ankle this 3 year old boy presented with high grade fever for 15 days. This was associated with shortness of breath for 5 days. there was history of a transient erythematous rash over the trunk extremities 2 days prior to admission. On examination, the child was febrile, there was pallor, air entry was decreased in the right chest, abdominal examination revealed hepatosplenomegaly, ascites, and there a tender swelling over the left groin. Initial investigations revealed: Hb-7.9 gm/dL, total leucocyte count was 43. 6 x 10 3 /μL neutrophils 79%, lymphocytes 18%, Platelet-120 x 10 3 /μL ,serum albumin 2.3 g/dL, serum globulin 5.8gm/dL, serum ferritin 534 ng/ml, CRP- 168 mg/L. Initial Chest Xray was apparently normal however a ultrasonography of the chest done a few days later revealed right sided pleural effusion. USG guided pleural tap was done which revealed fibrinopurulent collection. Ascitic tap was also done. Blood and pleural fluid culture revealed no growth Results: Considering sepsis the child was started on broad spectrum antibiotics. The fever was persistent despite adequate antibiotic coverage of 2 weeks. Bone marrow and echo-cardiogram were normal. Tuberculosis ruled out. However, the fever still persisted. With a suspicion of an autoinflammatory disease child given pulse methyl prednisolone followed by oral prednisolone following which the fever subsided and the child’s condition improved. The child was then discharged on oral oral steroids. On follow up, as the steroid were tapered the child developed arthritis of bilateral ankle joints. At this juncture the diagnosis of systemic onset juvenile idiopathic arthritis (sJIA) was made. Subcutaneous methotrexate was added and the child currently remains in remission Conclusion: Systemic onset juvenile idiopathic arthritis may present with persistent fever and polyserositis that may resemble a disseminated sepsis without any arthritis or specific sign to establish diagnosis. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
H. Bermúdez Canales, E. Faugier, S. Rodríguez Aguayo, K. Primero Nieto, A. Barba Aguilar, A. Guzmán Revilla, N. De la Rosa Encarnación, P. Ramos Tiñini Reumatología, Hospital Infantil de México Federico Gómez, Ciudad de México, Mexico Correspondence: H. Bermúdez Canales Introduction: Vaccines have been considered triggers of autoimmune diseases in genetically predisposed individuals through various mechanisms, such as molecular mimicry, the formation of immune complexes and the activation of polyclonal lymphocytes. However, for most patients, viral vaccines don’t carry a risk of systemic autoimmune disease and should be administered according to current recommendations because of their remarkable efficacy in reducing morbidity and mortality in all age groups. Objectives: Describe the case of a patient who triggered Systemic Juvenile Idiopathic Arthritis after vaccination with SRP. Methods: Caso clínico Results: One year old female. Her condition began 5 days after administration of the vaccine (SRP). Clinical manifestations: evening fever (39°C) associated with salmon-colored and evanescent macular exanthema. She was diagnosed with Kawasaki disease and was treated with intravenous immunoglobulin (2grkgdo), acetylsalicylic acid (50mgkgday) and methylprednisolone (2mgkgday). Referred for treatment due to persistent symptoms and arthritis of the knees, carpals and ankles of 4 months of evolution. At evaluation, febrile, arthritis in shoulders, elbows, carpus, knees and ankles, lymphadenopathies in right anterior cervical region of 2 x 2 cm and bilateral inguinal region of 1 x 1 cm, in addition to erythematous macules predominantly in trunk, upper extremities and pelvic limbs, 2 to 5 mm in size were evident. Laboratory: leukocytosis with neutrophilia; anemia and thrombocytosis. Elevation of acute phase reactants (ESR and CRP). Renal and hepatic function preserved. Chest X-ray and echocardiogram without alteration. Negative infectious approach (blood cultures, urine culture, procalcitonin and viral serology). Oncological approach, bone marrow aspirate negative for malignancy. Diagnosis of Systemic Juvenile Idiopathic Juvenile Arthritis was integrated and treatment with prednisone (2mgkgday) and Tocilizumab (162mg subcutaneous every 2 weeks) was started, achieving clinically inactive disease. Conclusion: There is limited data on the relationship of immunization and systemic autoimmunity. We report the exceptional case of Systemic Juvenile Idiopathic Juvenile Arthritis following vaccination with SRP. The comprehensive and thorough evaluation of the patient with systemic manifestations allows a timely diagnosis and initiation of appropriate treatment to control the disease without complications or sequelae. Disclosure of Interest : None declared
M. E. Caseiro Alves 1 , A. Sampaio Mesquita 2 , H. Sousa 2 , M. Paula Ramos 3 1 Departamento de Pediatria Médica, Hospital De Dona Estefania - Centro Hospitalar e Universitário de Lisboa Central, 2 Serviço de Pediatria, Hospital de Vila Franca de Xira, 3 Unidade de Reumatologia Pediátrica, Hospital De Dona Estefania - Centro Hospitalar Universitário de Lisboa Central, Lisboa, Portugal Correspondence: M. E. Caseiro Alves Introduction: Systemic Juvenile Idiopathic Arthritis (sJIA), mediated by autoinflammatory cytokines, is an unique form and the most severe one of Juvenile Idiopathic Arthritis. Its diagnosis is usually challenging and a high index of suspicion is needed. Macrophage activation syndrome (MAS) is a known potentially fatal complication of sJIA which needs prompt treatment. Objectives: To describe a case of severe SJIA complicated with MAS and to explore therapeutic options Methods: An 11 year-old african male child, brought to the emergency room of a second level portuguese hospital, for a 1 month daily fever, anorexia, fatigue and joint pain with 8-Kg weight loss. The patient also reported feeling illness, with intermittent low-grade fever and an evanescent erythematous exanthem in the previous six months, already evaluated in a medical appointment. Upon observation, the patient appeared fatigued, febrile with an evanescent exanthema (face and trunk), hepatomegaly and bilateral arthritis of small joints of the hands and feet, wrists and ankles. Blood tests revealed microcytic hypochromic anemia (Hb 9.4 g/dL) with leukocytosis, neutrophilia, thrombocytosis, hypoalbuminemia, with elevated C-Reactive protein (15 mg/dL), erythrocyte sedimentation rate (VS 113 mm/h), fibrinogen and ferritin (1703 ng/mL). Cardiac evaluation was normal. Infectious, neoplasic and others auto-immune diseases were excluded. Results: Ceftriaxone and ibuprofen were started. Progressive worsening in the next 24 hours with unremitting fever and prostration. Blood tests revealed a slight decrease of hemoglobin and increase in ferritin levels (15.637 ng/mL) and soluble CD 25. Concerning of installation of MAS, bolus of methylprednisolone was initiated. Treatment was maintained with prednisolone (PRD) and methotrexate (anti-IL1 was not authorized for the hospital administration) with improvement of systemic manifestations but refractory arthritis. Six months later, still in PRD, he was referred to a central hospital and interleukin-6 (IL-6) inhibitor was started with good disease control, allowing complete weaning of PRD 6 months later. Conclusion: Diagnosis of sJIA may be rather difficult with unspecific manifestations, as reported in this case with several months of disease evolution and a severe presentation on admission. Even though not fulfilling the 2016 criteria of MAS in sJIA, the clinical deterioration with extreme hyperferritinemia (>10,000ng/mL), strongly suggested rapidly evolving MAS. Prompt and life-saving treatment prevented the child of a high morbi-mortality risk. Nonethless, in sJIA the precocious treatment with anti-IL1/6 is usually related to better prognosis. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
C. Hinze 1 , M. Saers 1 , C. Kessel 1 , G. Prencipe 2 , F. de Benedetti 2 , D. Föll 1 , C. Bracaglia 2 1 Paediatric Rheumatology and Immunology, University Hospital Münster, Münster, Germany, 2 Paediatric Rheumatology, Ospedale Pediatrico Bambino Gesù, Rome, Italy Correspondence: C. Hinze Introduction: Systemic juvenile idiopathic arthritis (SJIA) is characterized by severe inflammation and may be complicated by life-threatening macrophage activation syndrome (MAS) which is driven by activation of the interleukin (IL)-18-interferon (IFN)γ-axis. How peripheral blood gene expression and respective serum biomarkers are related in SJIA and MAS is incompletely understood. Objectives: To evaluate the relationship of peripheral blood innate immunity-driven gene expression and related serum biomarkers in a cohort of patients with SJIA in different disease states. Methods: Seventy-five whole-blood-derived RNA and parallel serum samples from 45 patients (median age at sample 6.2 years) from 2 centres were analyzed using a 24 gene custom NanoString panel including IL-1/NFkB, type 1 interferon (IFN), and type 2 IFN-related genes, and a 23-plex Luminex panel. The panels included 9 related targets, i.e., both gene in the NanoString panel and related protein in the Luminex panel (CXCL9/CXCL9, CXCL10/CXCL10, IL1A/IL-1a, IL1B/IL-1b, IL1RN/IL-1RA, IL6/IL-6, IL18/IL-18, S100A9/S100A9, and S100A12/S100A12. Four different disease states were considered (18 clinically inactive disease [CID], 37 active disease [AD], 9 MAS [as defined by ACR/EULAR], and 11 pre-MAS, i.e., prior to the development of full-blown MAS). Normalized NanoString counts and composite IL-1/NFkB-related, type 1 interferon (IFN) and type 2 IFN scores, and Luminex MFI values were used for comparisons. Correlation analyses were performed. Groups were compared using non-parametric statistics. Results: There was mild to moderate correlation between the respective gene expression and serum protein levels as measured by Spearman rank correlation across all samples for the following genes/proteins: CXCL10 (r=0.40), IL6/IL-6 (r=0.40), IL1A/IL-1a (r=0.59), IL1B/IL-1b (r=0.45), IL18/IL-18 (r=0.40), S100A9/S100A9 (r=0.50) but not between CXCL9/CXCL9 and IL1RN/IL-1RA. When comparing the individual parameters across the different disease states, significant differences across disease states were seen for most of the serum proteins, and especially prominent for CXCL9, CXCL10, and IL-18. Strong correlations were seen, for example, between the following composite gene expression scores and serum proteins: IL-1/NFkB-related score with CXCL9 (r=0.60), IL-1a (r=0.64), IL-6 (r=0.72), S100A9 (r=0.55), type 1 IFN score with CXCL10 (r=0.58), and type 2 IFN score with FasL (r=0.85), CXCL10 (r=0.78), TNF (r=0.71) but not with CXCL9 (r=0.08). Conclusion: In patients with SJIA in different disease states, a marked dysregulation of inflammation-related serum proteins is observed, and especially IL-18 and CXCL9 in pts with MAS or pre-MAS. There was a strong correlation between IL-1/NFkB-related, type 1 IFN, and type 2 IFN gene expression signatures with several of the serum proteins assessed. IL-1RA serum levels were strongly confounded by anakinra (recombinant IL-1RA) therapy, explaining a lack of correlation between IL1RN expression and IL-1RA levels. In summary, both assessment of serum biomarkers and peripheral blood gene expression signatures may be instructive when assessing different disease states in SJIA. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
Kessel 1 , R. Marsh 2 , C. Wouters 3 , P. Proost 4 , P. Matthys 4 , D. Foell 1 , S. Canna 5 , G. Prencipe 6 , C. Bracaglia 6 , F. De Benedetti 6 , D. Dissanayake 7 , R. Laxer 7 , S. Vastert 8 , F. Minoia 9 , K. L. Brown 10 , D. A. Cabral 11 , G. Schulert 12 on behalf of PReS sJIA/MAS working party 1 Department of Pediatric Rheumatology & Immunology, University Children’s Hospital Muenster, Muenster, Germany, 2 Division of Bone Marrow Transplantation and Immune Deficiency, Cincinnati Children’s Hospital, Cincinnati, United States, 3 Department of Pediatrics, University Hospitals Leuven, 4 Department of Microbiology, Immunology and Transplantation, Rega Institute KU Leuven, Leuven, Belgium, 5 Children’s Hospital of Philadelphia and University of Pennsylvania Perelman School of Medicine, Philadelphia, United States, 6 Bambino Gesù Children’s Hospital, Rome, Italy, 7 Department of Pediatrics, Hospital for Sick Children, Toronto, Canada, 8 Department of Pediatric Rheumatology and Immunology, University Medical Center Utrecht, Utrecht, Netherlands, 9 Pediatric Rheumatology, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy, 10 Department of Pediatrics, British Columbia Children’s Hospital Research Institute, 11 BC Children’s Hospital University of British Columbia, Vancouver, Canada, 12 Cincinnati Children’s Hospital, Cincinnati, United States Correspondence: C. Kessel Introduction: : Over the past decade, several studies have validated the use of key biomarkers such as IL-18, CXCL9 and S100 proteins in diagnosis and follow-up of children with polyarticular course and systemic juvenile idiopathic arthritis (JIA). Despite the promise of these biomarkers, their utility is limited by their availability in only some specialized labs and medical centers. In addition, as opposed to CRP the appropriate reference ranges for these biomarkers is not standardized. Finally, even though good correlation of biomarker levels acquired in different centers using different assays may occur, absolute values can vary substantially. Collectively, this hampers a wider introduction of these markers into routine clinical care. Objectives: In this project we set out to cross-validate emerging JIA, systemic JIA and MAS biomarkers across different measurement platforms and different international centers involved in CARRA and PReS. Methods: We have assembled an international consortium of four CARRA Registry sites (Cincinnati, Philadelphia, Toronto, Vancouver) and four PReS (Leuven, Muenster, Rome, Utrecht) clinical research centers. In a first step we will distribute healthy donor serum samples spiked with defined concentrations of recombinant S100 proteins, CXCL9 and IL-18 to all participating centers. IL-18, S100 proteins, and CXCL9 will be determined using locally used platforms including specific ELISA, luminex, mesoscale and Ella assay. In a second step patients’ samples enrolled in the FROST study will be shipped to co-investigators labs for respective biomarker analyses. Following this all data will be analyzed for correlation coefficients and spike recovery to determine agreement across identical platforms in different labs. Linear fit models will be used to determine conversion of results across different platforms. Results: Data collection is still in progress and ongoing. Conclusion: The findings of this project will be highly significant because broad standardization of biomarker levels and interpretation across centers is essential for international collaborative research. Our efforts can strongly impact any future (clinical) research study in the field and beyond, that involves IL-18/CXCL9/S100 protein-driven differential diagnosis, disease activity assessment or guided treatment. T. Disclosure of Interest :Proost: None declared, P. Matthys: None declared, D. Foell Grant / Research Support with: Novartis, SOBI, Pfizer, Consultant with: Novartis, SOBI, Chugai-Roche, S. Canna Consultant with: Novartis, SOBI, AB2 Bio, Johnson & Johnson, Simcha Therapeutics, G. Prencipe: None declared, C. Bracaglia Consultant with: Novartis, SOBI, F. De Benedetti Consultant with: Hoffmann-La Roche, Novartis, AbbVie, Novimmune, and SOBI, D. Dissanayake Grant / Research Support with: Gilead Sciences, Consultant with: Novartis, R. Laxer Consultant with: SOBI, Novartis, Eli Lilly Canada, Sanofi, S. Vastert Grant / Research Support with: Novartis and SOBI, Consultant with: Novartis and SOBI, F. Minoia: None declared, K. Brown: None declared, D. Cabral: None declared, G. Schulert Consultant with: Novartis
E. Krekhova 1,2 , E. Alexeeva 1 , T. Dvoryakovskaya 1 , K. Isaeva 2 , R. Denisova 2 , A. Chomakhidze 2 , O. Lomakina 2 , A. Mamutova 2 , A. Fetisova 2 , M. Gautier 2 , C. Chibisova 2 , I. Kriulin 1 , I. Tsulukia 2 1 Pediatric, Sechenov First Moscow State Medical University, 2 Rheumatology, National Medical Research Center of Children’s Health, Moscow, Russian Federation Correspondence: E. Krekhova Introduction: Tocilizumab (TOC) is a highly effective biological drug for therapy of systemic juvenile idiopathic arthritis (sJIA). The identification of early prognostic factors of response to TOC is necessary to increase treatment efficacy and predict long-term outcomes in children with sJIA. Objectives: This study aimed to identify possible predictors of response to the TOC treatment in patients with sJIA. Methods: We analyzed retrospectively the 258 prescribing of TOC to the patients with sJIA treated at the National Medical Research Center of Children’s health, Moscow, Russia (initiation between July 2009 and February 2021). Predicted outcomes included inactive disease after 12 months of therapy with TOC (according to C. Wallace criteria and JADAS71). Probable prognostic factors (n=115) included baseline demographic, clinical, laboratory variables, previous or concomitant therapies and changes some of them on the 1 st and 3 rd month of the therapy. We determined predictors of response using multivariate logistic regression analysis. Results: After 12 months of TOC therapy, in 179 cases (76%) was achieved inactive disease according to C. Wallace criteria and in 170 cases (72%) was achieved JADAS remission. The predictors of achieving the stage of inactive disease/remission according to C. Wallace criteria by the 12 month of TOC therapy were: duration of morning stiffness at sJIA onset, its dynamics after 1 and 3 months of treatment, the physician global assessment of disease activity after 1 month of therapy, and the patient/parent global assessment of overall well-being after 3 months of therapy. The duration of morning stiffness at sJIA onset and upon treatment initiation, the physician global assessment of disease activity after 1 and 3 months, and number of painful joints after 3 months were associated with remission according to JADAS71 index by the 12 month of TOC therapy. Conclusion: Our results showed that the characteristics of joint syndrome before and 1–3 months following treatment initiation, dynamics of objective and subjective assessment of disease activity after 1-3 months of therapy, could predict the effectiveness of TOC by 1 year of therapy in sJIA patients. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
M. C. Maggio, G. CorselloThe worldwide pandemic of SARS-CoV-2 continues to have a serious impact on global health care across the world. The risk of severe COVID-19 is of concern for patients with rheumatic diseases and especially systemic Juvenile Idiopathic Arthritis (sJIA) using disease-modifying anti-rheumatic drugs (DMARDs) including biologics. Even if COVID-19 shows to be milder in children than in adults, multisystem inflammatory syndrome in children (MIS-C) is a fearful and a life-threatening complication. Objectives: The international literature showed that COVID-19 underwent without serious complications in sJIA patients controlled by biologics, but not by steroid therapy. Many studies sustain the hypothesis that uncontrolled sJIA and its underlying inflammatory state are a risk factor for severe COVID-19 in children, and that immunosuppression associated with prolonged corticosteroid treatment represents another risk factor for severe disease. Methods: We describe the case series of 5 patients affected by sJIA, acquiring SARS-CoV-2 infection. 4/5 was treated for sJIA with anti-IL-1 beta biologic drug, at the dosage of 4 mg/kg/4 weeks and showed a good control of the sJIA. One patient, with a concomitant mutation of NLRP-3, was treated with anakinra and prednisone for the difficult control of the disease. 60% of the patients previously received 2 doses of the SARS-CoV-2 vaccine. Results: All the patients showed persistent fever (100%), arthralgia and or arthritis (80%), respiratory symptoms (100%), with a good saturation in air, abdominal pain (40%). They were treated with ibuprofen, and they continued anti-IL-1 treatment. The acute phase resolved in a variable period of 5-10 days and the haematological parameters returned to the normal range. The patient with the worse control of the disease and the NLRP3 mutation, 4 weeks later, showed fever, rash, cheilitis, conjunctivitis, significant increase of AST, ALT, gamma-GT, pro-BNP, troponin. The diagnosis of MIS-C was defined, and the treatment choice was with IVIG (2 gr/kg), methylprednisolone (2 mg/kg/day i.v.) and the increase of anakinra dose to 4 mg/kg/day. The acute phase of the disease was controlled, with the complete recovery of the child. Conclusion: Our case series confirm a good outcome of the disease in children with SARS-CoV-2 infection, when the rheumatic disease is well controlled. The NLRP3 gene mutations increase IL-1 beta synthesis, with and underlying amplification of the risk of MIS-C. furthermore, the undergoing treatment with steroids in a patient with a bad control of sJIA is a further risk factor for a poor outcome. The vaccination with the SARS-CoV-2 vaccine can be a further protective factor for patients with sJIA. Disclosure of Interest : None declared
M. C. Maggio 1 , A. Alaimo 2 , F. Finazzo 3 , G. Corsello 4 1 University Department PROMISE “G. D’Alessandro”, University of Palermo, 2 U.O.C. of Paediatric Cardiologic Division, Children Hospital “G. Di Cristina”, 3 U.O.C. of Paediatric Radiology, Children Hospital “G. Di Cristina”, ARNAS, 4ystemic Juvenile Idiopathic Arthritis (sJIA) may seriously damage various structures of the cardiovascular system. However, cardiovascular disease (CVD) is understudied and, than, underestimated in patients with sJIA, but chronic systemic inflammation associated with cardiovascular risk factors are causes of CVD. The initial evaluation of sJIA patients is based on non-invasive modalities, however frequently they fail to detect subclinical forms of CVD. Objectives: Cardiovascular magnetic resonance (CMRI) can offer early and non-invasive information about CVD in sJIA, allowing timely starting of cardiologic and anti-rheumatic drugs. Methods: A 11-year-old girl presented with flu-like symptoms, systemic inflammation with myocarditis, and cardiomyopathy. She was treated with paracetamol and FANS, however, for the worsening of clinical conditions, she was transferred to our paediatric rheumatic unit. She showed fever, with 2-3 spikes over the day > 39°C, associated with evanishing rash, and with spontaneous defervescence during the day. She showed arthralgia at the legs, back pain, with functional limitation, with a polyarticular localization, without swelling. ECG and echocardiography were normal in the first days of the disease. During the first weeks of the follow-up, she presented infero-lateral negative T wave. She showed enhanced troponin levels, and for the suspicion of myocarditis, CMR was performed. The delayed myocardial enhancement exhibited focal myocardial fibrosis. Results: She was treated with steroids and after a gradual tapering, with canakinumab, a biological drug against IL-1 beta, at the dosage of 4 mg/Kg/every 4 weeks. Her condition improved dramatically, with a complete recovery of ventricular function. ECG documented the complete resolution of negative T wave and CMR showed the resolution of myocardial fibrosis. She is still followed in our unit. She reached complete remission, and canakinumab is now administered every 10 weeks. Conclusion: Myocarditis is a well-documented complication of sJIA. This unusual case emphasizes the important role of canakinumab in the treatment of sJIA and the efficacy of a early treatment with biological drugs in these cases. Furthermore, we can speculate that the early treatment with canakinumab contributed to the prompt remission of the patient, to the complete recovery of the myocarditis and to the sJIA remission of the disease. Disclosure of Interest : None declared
S. Maity P D Hinduja National Hospital and Medical Rsearch Centre, Mumbai, India Correspondence: S. Maity Introduction: 8 year old male child, 1 st by birth order , born out of non-consanguineous union presented with complaints of high grade fever upto 102-103 degree F , 2-3 spikes/day , since last 2.5 months with intact activity in inter-febrile period. History of rash over face , usually at the time of fever and subsiding with fever . Weight loss 3-4kgs. No history of joint pain/ swelling/ redness. Objectives: 1) To determine the etiology of the illness 2) To start necessary treatment /intervention at the earliest . Methods: On examination , the child had Left upper cervical and bilateral inguinal lymph nodes palpable. No systemic localizing sign ,No hepatosplenomegaly. Laboratory Evaluation : CBC-Hemoglobin 11.3 gm%, Total Leucocyte count 27900, N92L6, PLATELET 2.66 LAKHS, PCV 32, ESR 37. Given antibiotics- azithromycin and amoxiclav-clavulinic acid , without resolution. Blood and urine cultures were negative ,sputum for Tuberculosis work-up was negative. CSF- Protein-112, sugar- 64, WBC- 10/hpf, CSF for TB work-up- Negative. Shifted to tertiary care center .CT chest done, no significant abnormality found. Broncho alveolar lavage fluid sent for evaluation , negative for TB workup and malignant cells . 2D Echo showed no vegetations. ANA, ANCA, C3 -normal. Ferritin was high- 1354 (<140). Bone marrow study -normal. MRI brain with spine s/o no brain demyelination or meningeal enhancement , bilateral Atlantoaxial joint effusion was noted. Results: In view of Quotidian fever for more than 6 weeks associated with evanescent rash lymphadenopathy and Radiological evidence of joint involvement , Diagnosis of SYSTEMIC JUVENILE IDIOPATHIC ARTHRITIS (SJIA) was considered. Started on steroid and weekly methotrexate, responded well to the treatment. Conclusion: High grade fever with evanescent rash for prolonged duration in an otherwise well child should raise the suspicion of SJIA and the child should be worked-up accordingly. The take home message is , subtle radiological evidences of joint involvement can help in the diagnosis of the same. Statement : I tried to include the image in this submission but unable to do so, kindly note that i can communicate the necessary image via amail or any appropriate portalL.-A. Eveillard 1 , P. Quartier 2 , N. Ouldali 3 , B. Badder-Meunier 2 , F. Aeschlimann 2 , C. Abasq-Thomas 4 , C. Ballot 5 , P. Bouric 6 , A. Desdoits 7 , C. Dumaine 3 , C. Galeotti 4 , V. Hentgen 8 , A. Lefèvre-Utile 9 , A. Chausset 10 , T. Hubiche 11 , I. Kupfer-Bessaguet 12 , S. Leclerq-Mercier 13 , S. Mallet 14 , I. Melki 3 , E. Merlin 15 , J. Miquel 16 , M. Piram 17 , D. Talmud 18 , N. Garcelon 19 , C. Vinit 3 , A. Welfringer 20 , E. Bourrat 21 , U. Meinzer 3 1 Department of Infectious Disease and Internal Medicine, Reference centre for Rheumatic, AutoImmune and Systemic diseases in children (RAISE), Robert Debré University Hospital, Assistance Publique Hôpitaux de Paris, 2 Pediatric Immunology-Hematology and Rheumatology Unit, Rare Disease Reference Centre (RAISE), IMAGINE Institute, Necker-Enfants Malades Hospital, Assistance Publique Hôpitaux de Paris, Université de Paris, 3 General Paediatrics,Université de Paris, Paris, 4 Department of Pediatric Rheumatology Reference Centre for Autoinflammatory diseases and amyloidosis (CEREMAIA), Hôpital Bicêtre, Assistance Publique Hôpitaux de Paris, Le Kremlin-Bicêtre, 5 Paediatric haematology, Jean-Minjoz Hospital, Besançon, 6 Department of Biochemistry and Molecular Biology, CNRS, Recombinaisons genetiques, Rennes, Rennes, 7 Department of pediatric surgery, AutoImmune and Systemic diseases in children (RAISE), Reference Centre for Autoinflammatory Disorders (CEREMAI), CHU de Caen, Caen, 8 Reference Centre for Autoinflammatory Disorders (CEREMAI), Centre Hospitalier de Versailles, Le Chesnay Cedex, 9 General Pediatrics and Pediatric Emergency Department, Jean Verdier Hospital, Université de Paris, INSERM, U976 HIPI Unit, Institut de Recherche Saint-Louis, F-75006,Assistance Puplique-Hôpitaux de Paris (APHP), Paris, 10 Inserm CIC 1405, service de pédiatrie, CHU Estaing, Clermont-Ferrand, 11 Department of Dermatology, Université Côte d’Azur, Nice, 12 Department of Dermatology, Centre Hospitalier de Quimper, Quimper, 13 Pathology Department, Necker-Enfants Malades Hospital, Assistance Publique-Hôpitaux de Paris, Paris, 14 Department of Dermatology, Timone Hospital, Marseille, 15 Department of Pediatrics, Clermont-Ferrand University Hospital, Clermont-Ferrand, France, 16 Department of Pediatrics, CHU Reunion Saint Pierre, Saint-Pierre, Réunion, 17 Pediatric dermatology unit, Department of Pediatrics, CHU Sainte Justine, CHU Sainte Justine Research Centre, University of Montreal, Montréal, Canada, 18 Pediatric Department, American Memorial Hospital, CHU de Reims, Reims, 19 Institut Imagine, Centre de Recherche des Cordeliers, UMR 1138, INSERM, 20 Department of Dermatology, Reference Centre for Genodermatoses and Rare Skin Disease (MAGEC), Necker-Enfants Malades hospital, Assistance Publique Hôpitaux de Paris, Université de Paris, 21 Department of DermatologyFrance Correspondence: L.-A. Eveillard Introduction: The rash in systemic juvenile idiopathic arthritis (SJIA) is usually transient, but cases of atypical skin lesions because of their fixed nature have been reported. Objectives: To describe the spectrum of typical and atypical skin lesions present at onset of SJIA in children and examine whether their presence may be an early clinical marker associated with difficult-to-treat patients. Methods: A retrospective French multicenter study from 2002 to 2020, including pediatric patients with SJIA meeting the PRINTO classification criteria with documented skin lesion. Results: 80/112 (71%) patients presented typical and 32/112 (29%) atypical skin lesions. Patients with atypical skin lesions had a higher CRP level (p=0.002) and higher ferritin (p=0.0015). The presence of atypical skin disease was associated with a risk of first-line treatment failure at 1 year (OR 10.6 CI[3.4; 46.8]), failure of glucocorticoids as first-line treatment at 1 year (OR 5.6 CI[2.2; 16.4]), need for biotherapy treatment at one year (OR 5.5 CI[1.9; 19.7]) and at two years (OR 8.2 CI[2.2; 53.3]). Conclusion: Initial atypical skin lesions are associated with a poorer disease evolution, and may require the use of first-line biotherapies. Initial diagnostic evaluations for SJIA should include an examination by a dermatologist. Trial registration identifying number: CNIL 1980120 Patient Consent: Yes, I received consent Disclosure of Interest : None declared
M. Kaleda, I. Nikishina, E. Nikolaeva, S. Salugina, E. FedorovM. Kaleda, I. Nikishina Introduction: It’s now well known, that systemic juvenile idiopathic arthritis (sJIA) may be associated with lung disease (LD), which albeit insufficiently defined and characterized. Objectives: to characterize the clinical features all cases of LD associated with sJIA (sJIA-LD) based on long-term experience of the single center. Methods: Retrospective study of all consequently pts with sJIA-LD in pediatric department of single center for the last 10 years. Results: sJIA-LD is rare pulmonary complications which found in 5.0% of our cases of sJIA - 6 pts in total (4 were girls). The median (Me) age of sJIA at onset was 2.7 y.o. [IQR 1.8; 5.9]. The Me of the number of systemic manifestations at onset was 4.5 [3;6], 83.3% of pts had typical rash. The Me of the number of active joints at the time of sJIA verification was 8.0 [6; 18]. The Me of CRP level at onset was 72.5 mg/l [IQR 45.2; 112.4]), the Me of ferritin level - 892.5 ng/ml [IQR 349.0; 7256.0]). 5 pts had the history of macrophage activation syndrome (MAS) – 13.5% from all pts with MAS in our cohort of sJIA. All of them had MAS at onset, 4 – recurring MAS. There was a known family history of lung abnormalities in 1 patient – 2 of her older sisters died in the neonatal period from unidentified pulmonary pathology. 1 patient had the history of repeated episodes of eosinophilia (max 16%) and atypical urticarial rash that occur before LD detection. The therapy in all pts included steroids (100%), DMARDs (methotrexate (MTX) alone – 2 pts, MTX and cyclosporine consecutive – 4 pts), biologics (B) – all pts. The Me disease duration prior to start of treatment with B was 11.0 months [IQR 3.0; 18.0]. All pts were treated by tocilizumab (TCZ) as the 1 st line of B. 1 patient received TCZ only. 5 pts had anaphylactic reactions on TCZ so they have got from 2 to 5 B consecutively (in 3 pts TCZ switched to canakinumab (CAN), in 1 – to rituximab-anakinra-CAN, in 1 – to adalimumab-abatacept-CAN-sarilumab). The reasons for substitution therapy for the other B were loss of efficacy. The median of disease duration at the time of sJIA-LD verification was 4.0 years [IQR 2.8; 6.25]. At LD diagnosis, respiratory signs and symptoms were subtle in 4 pts, absent in 2 pts, although hypoxia was in 2 pts. The clinical signs of pulmonary hypertension were absent. All pts had bulbous deformity with erythematous clubbing of fingers. All pts had pulmonary involvement on chest CT scans (4 - septal thickening involving the periphery of multiple lobes, most marked in the lower lung zones, with ground-glass opacities, 1 – predominance of peribronchovascular consolidation, 1 - crazy-paving). 2 pts died, the duration of the disease at the time of death was 4.0 and 4.5 years. They had progressive LD with multiple organ failure, but without new episodes of MAS, 1 patient received TCZ, 1 - CAN. Other pts continued to receive B in stable mode with steroids in a low dose without evidence of LD progression and without of recurrent MAS during the last year of follow-up. Conclusion: According to our data, patients with sJIA-LD mostly had an early onset of sJIA, the history of MAS, and an anaphylactic reactions on TCZ. The vast majority of pts had difficulties with the choice of therapy of sJIA. Usually lung involvement had hidden course for the years just with small subclinical signs. The establishment of predictors for early recognition of sJIA-LD, prevention strategies and special approaches to the choice of therapy are very important to ensure a better prognosiT. Marushko 1 , O. Onufreiv 1 , Y. Marushko 2 1 Pediatrics-2, Shupyk National Healthcare University of Ukraine, 2 Pediatrics of postgraduate education, Bogomolets National Medical University, Kyiv, Ukraine Correspondence: O. Onufreiv Introduction: The course of systemic juvenile idiopathic arthritis (JIA) is characterized by damage to small vessels. Diagnosis of vascular changes in people with risk factors for their early development, including the systemic form of JIA, in childhood in the subclinical stage is important for the prevention of cardiovascular disease in these patients in adulthood. Objectives: To analyze the condition of blood vessels and endothelial function in patients with the systemic form of JIA on the background of basic therapy and treatment with immunobiological drugs. Methods: We observed 24 patients with the systemic form of JIA, 15 received methotrexate, 9 patients were prescribed tocilizumab due to insufficient effectiveness of basic therapy. The mean patient age was 9.2±0.5 years. The average duration of the disease was 2.8±0.9 years. At the time of tocilizumab administration systemic glucocorticoid therapy (GC) (methylprednisolone) was administered for 15 (62.5%) over an average of 2.3 years, with a mean dose of 0.2 mg/kg for prednisolone. To assess the effect of immunobiological drugs on the condition of blood vessels, patients underwent duplex sonography to determine the thickness of the intima-media complex of the common carotid arteries (CCA), the IMT of the abdominal aortic, the stiffness index of CCA, differences in diastolic velocities and diameters of the brachial artery using the test for endothelium-dependent dilatation of the vessel and the index of left ventricular myocardial mass (LVM). The difference between the values of these indicators before investigation and after 6 months of therapy was also calculated. Results: The inclusion of tocilizumab in the complex therapy of patients with systemic JIA, who had insufficient effect from treatment with methotrexate and GC, led to the elimination of systemic manifestations of the disease within six months, reducing the severity of joint syndrome, average number of joints with synovitis per patient and normalization of laboratory parameters. In patients with JIA who received tocilizumab in combination therapy, the magnitude of the changere significantly smaller than in children receiving methotrexate. In the group of children receiving methotrexate, in all patients the value of these indicators increased over 6 months of follow-up, which indicated the progression of subclinical vascular damage despite a decrease in JIA activity. The mean differences in the difference in diastolic velocities and brachial artery diameter in children receiving tocilizumab before to immunobiological administration were significantly lower than those in the methotrexate group. After six months of therapy with immunobiological drug, the value of these indicators increased significantly compared to their initial values. These data indicate that before starting treatment with immunobiological drugs in patients with JIA there was endothelial dysfunction, which after 6 months of therapy was not detected again. Conclusion: In children with JIA on immunobiological therapy, the rate of progression of subclinical vascular wall lesions and increase in LVM mass was significantly lower than in patients receiving basic treatment. In patients with systemic JIA on complex therapy with tocilizumab for six months the rate of increase in the IMT of CCA, the IMT of the abdominal aortic, the stiffness index of CCA and the index of LVM mass was significantly lower than in patients with JIA who received standard base therapy. There was observed normalization of endothelial function during 6 months of using tocilizumab. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
M. Rodriguez Ortiz 1 , S. Murias Loza 1 , L. Calle Miguel 1 , M. A. Alonso Álvarez 1 , J. Rodríguez Suárez 1 , E. Pardo Campo 2 , J. Zanabili Al-Sibai 3 , S. Rodríguez Ovalle 1 1 Pediatrics Department, 2 Rheumatology Department, 3 Hematology Department, Hospital Universitario Central de Asturias, Oviedo, Spain Correspondence: M. Rodriguez Ortiz Introduction: Most of fever of unknown origin (FUO) cases are caused by infectious diseases, but autoinflammatory entities, such as Systemic onset Juvenile Idiopathic Arthritis (SoJIA) must be included in the differential diagnosis of FUO, since early therapeutic approach is key to improve prognosis. Objectives: We herein present a 15-month-old male patient admitted for FUO. We describe the initial stages of the disease, and the unexpected outcome after diagnosis. Methods: Medical chart was reviewed. Results: A 15-month-old male patient presented with fever >39,5°C for 11 days; evanescent maculopapular erythematous rash which intensifies with fever; generalized adenopathies; carpus and right knee arthritis; increased acute phase reactants (APR), and anemia. Infectious and neoplastic aetiology were ruled out by complementary examinations (including PET-CT and bone marrow aspirate). SoJIA was then diagnosed, and corticosteroids were started on 19th day of fever with rapidly good response, which allowed hospital discharge with oral prednisolone (2 mg/kg/day dosage with subsequent tapering) and prophylactic trimethoprim-sulfamethoxazole. After 12 days, while receiving prednisolone 0.6 mg/kg/day, inflammatory symptoms reappeared. Given SoJIA relapse he was hospitalized and anti-IL1 biological agent (Anakinra) was started. However, no response was observed and the child showed clear clinical worsening which led to Anakinra withdrawal after 3 days. Analytical criteria for Macrophage Activation Syndrome (MAS) secondary to SoJIA were then met. Megaboluses of methylprednisolone and cyclosporine were started, followed by frank progressive clinical and analytical improvement. Unexpectedly, he developed severe neutropenia (up to 10 neutrophils/μL), being the only altered analytical parameter. Filgrastim was thus started but no increase of neutrophils count was reached after 9 days. Review of all medications was then performed, leading to trimethoprim-sulfamethoxazole discontinuation due to its known myelotoxicity. Three days later, laboratory examinations showed recovering of neutrophils count up to 13,000/μL, with good parallel outcome of MAS and SoJIA. Conclusion: When suspecting SoJIA, early treatment must be started, and close clinical and analytical monitoring should be carried out to assess response and identify the possibility of MAS as a potentially serious complication. In the case of neutropenia with no clear origin after conventional studies, it is key to collect information on treatments, suspending those that are potentially myelotoxic. Trimethoprim-sulfamethoxazole is a widely used antibiotic as prophylactic agent, thus its potential adverse events should be always taken into account. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
G. Schulert 1,2 , E. Verweyen 1 , S. Thornton 1,2 , C. S. Lages 1 , E. Eloseily 1 , M.-L. Chang 3 , M. E. Riordan 4 , A. Russell 5 , M. Natter 3 , Y. Kimura 4 on behalf of CARRA Registry SJIA-LD Cohort Investigators 1 Cincinnati Children’s Hospital, 2 University of Cincinnati College of Medicine, Cincinnati, 3 Boston Children’s Hospital, Boston, 4 Hackensack Meridian Health, Hackensack, 5 Duke Clinical Research Institute, Durham, United States Correspondence: G. Schulert, and currently affects as many as 1 in 20 SJIA patients. Despite recent advances, there remain key unanswered questions regarding disease prevalence, influence of biologic treatments, pathogenesis, and outcomes. Objectives: The central objective of this project is to establish a cohort of patients with SJIA-LD within the CARRA Registry to accelerate clinical and translational research to understanding SJIA-LD. Methods: Existing or newly enrolled CARRA Registry patients with SJIA and suspected, probable, or definite SJIA-LD were enrolled in the cohort. In addition to standard registry data, lung disease specific clinical data was obtained at baseline and at 6 month follow-up. In addition, CARRA standard biosamples were collected at baseline and follow-up. Multiparameter flow cytometry panel was developed and validated using peripheral blood mononuclear cells (PBMC) previously collected locally using CARRA standard biosamples kits and standard operative procedures (SOP). Flow cytometry data was collected using the Aurora spectral cytometer (Cytek) and analyzed using FlowJo (v10.7.1). Results: As of May 1, 2022, 24 patients were enrolled in the SJIA-LD cohort from 12 CARRA Registry sites. Patients were 58% female and median age at enrollment was 4.5 (IQR 6.5). 55% of patients had definite (biopsy-proven) SJIA-LD, 36% probable SJIA-LD, and 9% suspected SJIA-LD. Median SJIA duration at time of lung disease diagnosis was 1.8 years. Most common clinical features were clubbing (60%), tachypnea (60%) and cough (40%). Most common radiographic findings were bronchial wall or peribronchovascular thickening (63%), ground-glass opacities (60%), and septal thickening 56%). Patients had a median Physician Global Assessment of Lung Disease (PGALD) of 3.5 (range 0-8/10) at time of cohort enrollment. Baseline biosamples have been obtained from 19 patients collected at 10 sites. To allow for immunophenotyping of PBMC from patients with SJIA-LD, we have developed a 27-color flow cytometry panel including lineage markers, surface activation markers, and intracellular cytokines. Validation experiments showed this panel could reliably identify monocyte (classical, non-classical, intermediate), dendritic cells, T cell (CD4 vs CD8, Th1/2/17, resting, memory, effector), B cells (naïve, regulatory, plasma), and NK cell subsets, as well as differences in expression of activation markers and cytokines in cellular subpopulations. Conclusion: The CARRA SJIA-LD cohort is actively enrolling patients and collecting biosamples across the network. We have developed a multiparameter flow cytometry panel suitable for analysis of longitudinal biosamples. Ongoing goals include characterizing clinical features of the SJIA-LD cohort, disease progression over time, and defining cellular populations associated with disease trajectory. The SJIA-LD cohort will serve as an ongoing prospective cohort study for future clinical and translational research in this emerging clinical entity. Patient Consent: Yes, I received consent Disclosure of Interest : G. Schulert Consultant with: Novartis, E. Verweyen: None declared, S. Thornton: None declared, C. Lages: None declared, E. EloseilyY. Kimura: None declared
P. Sinnappurajar 1 , S. Shoop-Worrall 2 , A. Ramanan 1 , K. Hyrich 2 , G. Cleary 3 , C. Ciurtin 4 , F. McErlane 2 , L. R. Wedderburn 4 , A. Chieng 5 1 Paediatric Rheumatology, Bristol Royal Hospital for Children, Bristol, 2 Arthritis Research UK Centre for Epidemiology, University of Manchester, Manchester, 3 Paediatric Rheumatology, Alder Hey Children’s Hospital, Liverpool, 4 Centre for Adolescent Rheumatology Versus Arthritis, UCL UCLH GOSH, London, 5 Paediatric Rheumatology, Royal Manchester Children’s Hospital, Manchester, United Kingdom Correspondence: P. Sinnappurajar Introduction: Systemic juvenile idiopathic arthritis (sJIA) is thought to follow a monocyclic, polycyclic or persistent course. The reported proportions of children following each course varies in the literature, with the percentages ranging from 15% to 83% for a persistent course in different cohorts. This study examined the clinical course of children with sJIA in the Childhood Arthritis Prospective Study (CAPS), a UK multi-centre cohort. Objectives: To determine the patterns of disease course in sJIA patients in the CAPS cohort. Methods: Data from children with sJIA were extracted from the CAPS database, which captures clinical outcome data at presentation (baseline), 6 months, 1 year then annually to 5 years. Cases were included from the cohort inception in 2001 until 2016, ensuring all cases had opportunity for 5 years of follow up. Clinically inactive disease (CID) was defined at each time point using the 2004 Wallace criteria (no active joints, no fever/rash/serositis/splenomegaly/lymphadenopathy, no active uveitis, normal ESR/CRP, physician’s global assessment (PGA) indicating no disease activity), with modifications that reflected the core clinical outcomes seen in systemic JIA. Wallace criteria 1 excluded uveitis outcomes, whilst Wallace criteria 2 excluded both uveitis outcomes and PGA. CRP data was also excluded from both criteria as the results are not standardised between different laboratory assays. If CID status could not be generated due to missing data at individual time points, it was replaced using last-observation carried forward. Children were classed as having a persistent course if they never attained CID by the above definitions, a monocyclic course if they attained CID then maintained it throughout the 5-year follow up period, and a polycyclic course if they attained CID and subsequently flared during the follow up. Children who only first attained CID at the last follow up visit were excluded, due to the inability to further define their clinical course. Results: There were 96 cases of systemic JIA identified in the CAPS database between 2001 and 2016. 59% were female, and the mean age at onset was 7.5 years (SD 4.8). Based on Wallace criteria 1 (excluding uveitis and CRP), two cases that first reached CID on the last visit (5 th year), were excluded. Of the remaining 94 patients, 82% (77/94) were defined as having a persistent course (never achieved CID), 12% (11/94) had a monocyclic course (reached CID and subsequently maintained remission throughout the follow up period) and the remaining 6% (6/94) had a polycyclic course (achieved CID and subsequently flared). Using Wallace criteria 2 (excluding uveitis, CRP and PGA), three cases that attained CID on the last visit were excluded, leaving 93 patients for analysis. By this definition of CID, the proportions of children were similar across the 3 clinical patterns with 39% (36/93) having a persistent course, 34% (32/93) having a monocyclic course and 27% (25/93) exhibiting a polycyclic course. Conclusion: Following this preliminary analysis of the CAPS database, we found that similar percentages of children with sJIA will follow either monocyclic, polycyclic or persistent disease patterns over a 5-year time period, which is broadly in keeping with the literature. A higher percentage of children attain CID when PGA is excluded. This cohort will be further examined to identify whether the proportions following the three disease patterns have changed over time, and whether any variables at disease onset correlate to the disease patterns, which could ultimately help guide early treatment decisions. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
102. Childhood-onset sacroiliitis: rheumatic causes and the correlation between clinical findings and magnetic resonance imaging P103. Inflammatory marker profiles in pediatric familial Mediterranean fever and accompanying spondyloarthritis: siblings or distant cousins? P105. MRI evaluation of active sacroiliitis in juvenile spondyloarthritis treated with TNF inhibitors P. O. Avar-Aydin 1 , Z. B. Ozcakar 1 , S. Kaynak-Sahap 2 , F. Aydin 1 , C. Arslanoglu 1 , N. Cakar 1 , O. S. Fitoz 2 , F. Yalcinkaya 1 1 Department of Pediatric Rheumatology, 2 Department of Pediatric Radiology, Ankara University Faculty of Medicine, Ankara, Turkey Correspondence: P. O. Avar-Aydin Introduction: Childhood-onset sacroiliitis is a challenging condition that requires to consider several causes in the differential diagnosis including infections, malignancies, medications, and rheumatic diseases. Objectives: To evaluate the etiology and the features of MRI-proven sacroiliitis in pediatric patients with rheumatic diseases in a referral center from an Eastern Mediterranean country. Methods: Demographic and clinical data were extracted from the electronic medical records of the patients with sacroiliitis followed in the last 5 years. Active inflammatory changes of the sacroiliac joint (SIJ) were scored by the inflammation score according to the degree, intensity, and depth of the bone marrow edema (BME) and the presence of enthesitis, capsulitis, and structural damage lesions was evaluated by the modified SPARCC scoring system. Results: A total of 46 patients were found to have MRI-proven sacroiliitis with a mean follow-up of 3.13±2.15 years. There were three groups of patients diagnosed with sacroiliitis when classified according to etiology: JIA (n:17), FMF (n:14), and CNO (n:8). Seven patients, FMF and JIA (n:6) and FMF and CNO (n:1) had a co-diagnosis that might cause sacroiliitis and they were excluded from the group analyses. Thirty patients (65.2%) were male. The mean age at disease onset was 10.97±3.93 years and six patients diagnosed with FMF or CNO were younger than 6 years. The prevalence of sacroiliitis in the relevant cohorts was 11.9% for JIA, 3.8% for FMF, and 34.6% for CNO groups. On the MRI of the SIJ, capsulitis was present in 17 patients (37.0%) and enthesitis in 14 patients (30.4%) in addition to BME with a mean total inflammation score of 22.50±13.80. Besides, structural damage lesions were present in 73.9% of the patients at the onset of sacroiliitis. Nonsteroidal anti-inflammatory drugs were used in all patients as the first-line treatment. Disease-modifying anti-inflammatory drugs were given to 67.4% and TNFi to 54.3% of the patients. In the comparative analyses of three groups of patients, the weight and height percentiles were significantly lower in FMF group and HLA-B27 was more frequent in JIA group. Other findings of demographics, family history, differential blood counts, acute phase reactants, and the usage of TNFi were similar. BME intensity score on MRI and the frequency of capsulitis and enthesitis were significantly higher in CNO group. There was a significantly strong or moderate correlation between total inflammation score and inflammatory back pain, JSpADA score, ISSF score, clinical CNO score, and CRP; BME intensity score and inflammatory back pain, JSpADA score, and CRP; BME depth score and ISSF score, clinical CNO score, CRP, and ESR; and the presence of sclerosis/erosion and clinical CNO score. Conclusion: This is the first study to evaluate the etiology of sacroiliitis in children with various pediatric rheumatic diseases and the relationship between clinical and MRI findings of newly-diagnosed sacroiliitis. We demonstrated that JIA, FMF, and CNO were the causes of childhood-onset sacroiliitis in our region. Characteristic findings on MRI might help the differential diagnosis of patients with sacroiliitis. Moreover, inflammatory and structural damage lesions found on MRI correlated with several clinical and laboratory features. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
S. Bocutcu Cetin 1 , E. Sag 2 , K. Cankirili 3 , M. Kasap Cuceoglu 1 , E. D. Batu 1 , O. Basaran 1 , Y. Bilginer 1 , S. Ozen 1 1 Pediatric Rheumatology, Hacettepe University, 2 Pediatric Rheumatology, Ankara Training and Research Hospital , 3 Translational Medicine Laboratory, Hacettepe University, Ankara, Turkey Correspondence: S. Bocutcu Cetin Introduction: Familial Mediterranean fever (FMF) is a hereditary disease characterized by recurring, self-limited inflammatory episodes. The disease is caused by a gain-of-function mutation in MEFV (Mediterranean FeVer) gene, causing an excessive activation of pyrin inflammasome and thus overproduction of proinflammatory cytokines. 1 Spondyloarthropathies accompanying FMF include inflammatory arthritides, sacroiliitis, spondylitis, enthesitis, and extraskeletal manifestations, and in children, enthesitis-related arthritis (ERA). 2 Although the evidence supporting the role of innate immunity is overwhelming, the exact pathogenesis of the aforementioned diseases is yet to be determined. Objectives: In this study, chosen inflammatory marker levels are evaluated in order to gain further knowledge on the pathogenetic mechanisms of FMF and accompanying spondyloarthritis (SpA). Methods: Between December 2020 and June 2021, we collected four groups of patients with FMF-SpA with high disease activity, ERA with high disease activity, FMF during the attack period, and FMF remission period. Having collected 20 samples from 16 patients, the inflammatory marker levels of these groups were compared with each other and the healthy donors. Results: TNF-α levels are significantly increased in all the patient groups compared to the healthy controls. No statistically significant difference in IL-1β levels was found among the patient groups or the healthy controls. However, transcriptomic studies found a significant difference between the patient groups and the healthy controls. IL-6 levels showed a significant decrement in remission period in FMF patients compared to the attack period. IL-18 levels were significantly increased in the FMF-SpA group, suggesting the possibility of a potential inflammatory biomarker for ERA/spondyloarthritides in the FMF group. Conclusion: This study is the first to investigate the inflammatory pathogenesis of FMF and accompanying spondyloarthropathy. Corroboratory studies are needed to further evaluate the role of these cytokines and their incorporation into personalized medicine. References 1. Chae JJ, Cho Y-H, Lee G-S, Cheng J, Liu PP, Feigenbaum L, et al. Gain-of-function Pyrin mutations induce NLRP3 protein-independent interleukin-1β activation and severe autoinflammation in mice. Immunity. 2011;34(5):755-68. 2. Sönmez, H. E., Batu, E. D., Demir, S., Bilginer, Y., & Özen, S. (2017). Comparison of patients with familial Mediterranean fever accompanied with sacroiliitis and patients with juvenile spondyloarthropathy. Clinical and experimental rheumatology, 35(6), 124-127. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
K. Yamazaki 1 , A. Fujikawa 2 , T. Nozaki 3 , Y. Motonaga 4 , M. Mori 4 1 Division of Rheumatology and Allergology, Department of Internal Medicine, 2 Department of Radiology, St. Marianna University School of Medicine, Kawasaki, 3 Department of Radiology, St. Luke’s International Hospital, Tokyo, 4 St. Marianna University School of Medicine, Division of Rheumatology and Allergology, Kawasaki, Japan Correspondence: K. Yamazaki Introduction: Even though magnetic resonance imaging (MRI) assessment of the sacroiliac joints (SJ) is useful in the early diagnosis of spondyloarthritis (SpA), the evaluation of MRI features in juvenile SpA has not been well established. Objectives: We aimed to evaluate JSpA-specific MRI features of SJ for use in the early diagnosis of sacroiliitis. Methods: We collected seven cases clinically diagnosed with JSpA and successfully treated with TNF inhibitors. Two pediatric musculoskeletal radiologists retrospectively evaluated the MRI features of sacroiliac joints in the patients before and after the treatment. Results: Seven JSpA patients (5 males, 2 females; Median age 14 (12 to 20)) are included in this study. The median time from onset to diagnosis is three years. Before TNF-inhibitors treatment, inflammatory back pain, SJ tenderness, and peripheral enthesitis were present in all patients. One of the patients has HLA-B27. MRI features at diagnosis showed active inflammatory lesions of sacroiliitis such as bone mallow edema (3/7), capsulitis (0), enthesitis outside of sacroiliac joint (1/7), and structural lesions such as erosion (3/7), sclerosis (3/7), backfill (0), fat metaplasia (0) and ankylosis (0). The clinical severity of inflammatory back pain and peripheral enthesitis did not correlate with the MRI findings. MRI findings of bone marrow edema improved in all three patients 6-12 months after initiation of TNF inhibitors treatment. Conclusion: The assessment of SpA International Society (ASAS) definition for adult has previously been reported to be less sensitive in children. In the early stages of sacroiliitis, inflammatory changes in the SJ are so subtle that they may not be detected on MRI. Since the early diagnosis of active sacroiliitis is important in terms of treatment options, a practical definition of active sacroiliitis specific to children and adolescents is required for pediatric rheumatologists. Disclosure of Interest : None declared
106. First case with a possible diagnosis of haploinsufficiency of A20P107. Successful treatment with infliximab in a child with deficiency of interleukin-36 receptor antagonist (DITRA)P108. A two-year-old libyan boy with recurrent abdominal pain and fever: familial mediterranean fever P109. Plasma MIR-520B overexpression in patients with rheumatoid arthritis P110. DADA2 in Malaysian children - novel mutations and “heterozygous presentation” P111. Chronic non-bacterial osteomyelitis in children: a multi-center cohort from Saudi Arabia 112. Colchicine response in TRAPS – a molecular enigma P113. DADA2 with 2 different phenotypes - our first experience P114. Efficacy and safety of anti-interleukin-1 treatment in large colchicine resistant familial Mediterranean fever cohort: single center experience P115. A score for predicting colchicine resistance at the time of diagnosis in Familial Mediterranean Fever (FMF): data from the TURPAID registry P116. Menstruation-triggered attacks in adolescent girls with Familial Mediterranean Fever (FMF) P117. The performance of a gene panel analysis among patients with systemic autoinflammatory diseases P118. A novel missense mutation in NLRP3 causing inflammasome hyperactivation and subsequent sensorineural hearing loss as a part of atypical CAPS P119. A family with pulmonary deficiency, polyarthritis, glomerulonephritis and vasculopathy: diagnosis in 3rd generation P120. Experience with IL-1 inhibitors in patients with monogenic autoinflammatory diseases in a tertiary hospital’s pediatric rheumatology unit P121. Acute febrile neutrophilic dermatosis -sweet syndrome- case presentation P122. Effects of unopposed IL-18 in mixed inflammatory environments P123. DNA methylation and gene expression signatures in CD14+ monocytes separate cno patients from healthy controls P124. Evaluation of activity and performance using Canada occupational performance measure in children with Familial Mediterrenian Fever P125. Determination of participation in children with Familial Mediterrenian Fever P126. Damaging variants in P2X7R associate with chronic nonbacterial osteomyelitis (CNO) P127. Type 1 interferon pathway and Aicardi-Goutieres syndrome: a case report P128. Effect of the janus kinase inhibitor baricitinib in the treatment of COPA syndrome P129. Characterization of AR-CGD female patient with novel homozygous deletion in CYBC1 gene presenting with unusual clinical phenotype P130. The preferential use of anti-interleukin 1 therapies in various pediatric rheumatic disorders: outcomes of treat-to-target approach P131. Predict-CRFMF score: a novel model for predicting colchicine resistance in children with Familial Mediterranean Fever P132. Can the diagnosis of PFAPA syndrome be confirmed? data from the long-term follow-up of a single-centre cohort 33. Long-term efficacy and safety of canakinumab in patients with Familial Mediterranean Fever (FMF) - interim analysis of the reliance registry P134. Impact of coronavirus outbreak restrictions on behaviors of patients with Familial Mediterranean Fever P135. Blood levels of interleukin-18 (IL-18) in patients with systemic juvenile arthritis and monogenic autoinflammatory diseases (FMF, CAPS, TRAPS) 36. Renal involvement and amyloidosis in autoinflammatory diseases: data from the Eurofever registry P137. Bisphosphonates in chronic recurrent multifocal osteomyelitis: pamidronate vs zoledronate P138. Use of canakinumab in children with cryopyrin-associated periodic syndrome: a single center study P139. A20 haploinsufficiency identified in a girl initially diagnosed with systemic juvenile idiopathic arthritis P140. Evaluation of gastrointestinal system complaints in pediatric Familial Mediterranean Fever patients P141. Chronic nonbacterial osteomyelitis and immune checkpoint molecules P142. Familial case of PSTPIP1-associated myeloid-related proteinemia inflammatory (PAMI) syndrome P143. Withdrawn P144. Long-term safety and efficacy of canakinumab in cryopyrin-associated periodic syndromes (CAPS) – 36-month data from the reliance registry P145. Gblau syndrome from India P146. Multiple autoinflammatory and autoimmune features in families with haploinsufficiency of A20 P147. Do Familial Mediterranean Fever (FMF) patients treated with interleukin-1 inhibitors adhere to colchicine treatment? A population-based study P148. IL-1antagonist treatment in recurrent aseptic meningitis related to Familial Mediterranean Fever 49. Music for CAPS: the experience of the centre for paediatric rheumatic diseases of Palermo P150. FMF or SURF: a diagnostic dilemma in children with recurrent febrile episodes P151. Pediatric SAPHO syndrome: single entity or different diseases? 52. Validation of the pediatric Behçet disease criteria (PEDBD): a consensus-based approach 3. Pericarditis in children: systemic and isolated recurrent pericarditis P154. Idiopathic chronic pericardial effusion in pediatric age: the occasional finding in an asymptomatic patient P155. A case of recurrent pericarditis in a patient with Bardet-Biedl syndrome P156. Current practice and research priorities in chronic non-bacterial osteomyelitis – an international survey directed to patients, families, and clinicians P157. Sacroiliitis as a presenting symptoms in a young patient with FMF P158. A case of adenosine deaminase 2 deficiency (DADA2) with an uncommon complex phenotype: a diagnostic challenge P159. Treatment of hematologic manifestations in a group of patients with monogenic autoinflammatory diseases (AID): single center experience P160. Long-term efficacy and safety of canakinumab in patients with hids (Hyper-IGD Syndrome) - interim analysis of the reliance registry 61. The usefulness of the genetic study in the clinical practice of monogenic autoinflammatory diseases: from diagnosis to the application of precision medicine P162. Adenosine deaminase 2(DADA2) deficiency in five indian children : a single center experience P163. Chronic non-bacterial osteomyelitis (CNO): a study from a single center in India P164. Pyoderma gangrenosum- mirror of active autoimmune disease P165. Criteria of remission and risk factors for disease severity in long-term follow-up of children with chronic non-bacterial osteomyelitis P166. Alone we can diagnose so little; together we can diagnose so much! when multispecialty collaboration spotlights PFIT and autoinflammation P167. Human type I interferonopathy with mutations in CTNNA3 68. Chronic Recurrent Multifocal Osteomyelitis (CRMO): new insights into extra-osseous manifestations P170. The Russian cohort of patients with TRAPS according to the federal rheumatology center P171. Long-term efficacy and safety of canakinumab in patients with tumor necrosis factor receptor-associated periodic syndrome (TRAPS) - interim analysis of the reliance registry 72. Outcomes and post-acute sequelae of COVID-19 in patients with systemic autoinflammatory diseases P173. Genotypes and phenotypes patterns in patients with NLRP3 gene variants P174. Netosis dysregulation in adenosine deaminase 2 deficiency P175. Type I interferon signature: a standardized method for clinical application P176. Recurrent fever without molecular diagnosis: lessons from autoinflammatory disease registries P177. Real-life data on autoinflammatory diseases: what do we learn from international patient registries? 8. Flow cytometric analysis of ASC specks as a novel biomarker for monitoring autoinflammatory diseases P179. Significant reduction in time to diagnosis of chronic non-bacterial (CNO) and Chronic Recurrent Multifocal Osteomyelitis (CRMO) over the past 13 years in 45 children from one clinical center 80. Encephalitis due to Anti-N-Methyl-D-Aspartate receptor antibodies in a pediatric patient first case report andliterature? Review F. L. Zaldumbide-Alcocer, Y. R. Loyola on behalf of Hospital de especialidades del niño y la mujer, Querétaro, México Pediatría, Hospital de especialidades del niño y la mujer, Querétaro, Mexico Correspondence: F. L. Zaldumbide-Alcocer Introduction: Autoinmune encephalitis (AD) due to anti-NMDA receptor antibodies (anti-NMDAr) isa subacute encephalopathy that can progress to severe encephalopathy. In Mexico, the incidence is unknown, however the world literature? reports cases associated with paraneoplastic syndrome with a predominance in females. It is the most common AD in pediatric patients, although the paraneoplastic association is less frequent. Objectives: The objective of this research is to present the first case of autoinmune encephalitis due to anti NMDA antibodies in our Hospital, as well as to realize a review of the pathology. Methods: We present the case of a 10 year old male patient who began suffering from infection in the upper airways (sinusitis) characterized by fever spikes and general malaise, receiving antibiotic therapy with second-generation cephalosporins with remission of the condition. Ten days later, he begins with disorganized thoughts, bradilalia, bradypsychia, psychomotor agitation, delusional ideas, visual hallucinations, insomnia, anxiety, facial dyskinesias, behavioral and conductive alteration, inattention, rapid and incongruous speech, bilateral dysmetries. Physical examination Roomberg doubtful positive, stiff neck and hiperreflexia. Results: Lumbar? puncture was realized and showed 63% NM, 37% PMN, glycorrhachia 59, proteinorachia <2, DHL 23. Negative results for BAAR, Gram stain, india ink, cerebrospina fluid culture, respiratory viral panel and hepatitis. Positive anti -NMDA antibodies were demonstrated. Electroencephalogram with abnormal activity in the right fronto-temporal region frequently, sometimes bilateral, slowing of the base activity. Magnetic resonance T2 FLAIR cortical restriction in both temporo-occipital lobes, predominantly left, with inflammatory changes of edema. Encephalic and extrapyramidal syndrome was integrated. During hospitalization, the patient presented symtoms of neurological deterioration with status epilepticus requiring intensive care management and phase III ventilation. Extensive? studies were performed with the intentional search for associated tumors that weren´t demonstrated. Testicular ultrasound did not report seminoma. Phatology of endocrine origin was ruled out. The management with antipsychotics, benzodiazepines, anticonvulsants and steroids with pulses 30mgkgdose for 5 days associated with gammaglobulin 2grkgdo. Given with favorable response to first line management, he was discharged 31 days after the onset of symptoms with reduced steroid management, anticonvulsant, antipsychotics and maintenance inmunossupressants. Conclusion: In patients with encephalic syndromes, it is important to consider autoimmune encephalitis due to anti -NMDA receptor antibodies as a differential diagnosis, since this pathology may be under-diangosed in our enviroment, timely diagnosis and treatment can considerably reduce morbidity and mortality, Libya Correspondence: Y. Elfawires Introduction: Monogenic autoinflammatory disorders (AID) and primary immunodeficiencies can present early in life with features that may be mistaken for Behçet’s disease (BD). These disorders involve a growing multisystem inflammatory diseases associated with defects in the innate immune system. The symptoms appears to range through unprovoked inflammation (fever, rash, ulcers, and elevation of acute phase reactants ).We herein report a case under workup for Haploinsufficiency of A20 in a young Libyan female that is suffering from recurrent mouth and genital ulcers. Objectives: to highlight the importance of considering the monogenic AID in children presenting with signs and symptoms mimicking BD Methods: a case report Results: A 7 yrs old Libyan female born to consanguineous parents, presented to our clinic as a referral from the GI clinic,with the complaint of having recurrent mouth and perianal ulcerations. The problem started at age of five, six months before presentation. With the appearance of the ulcerations about three/month, lasting for about a week and resolved spontaneously with no recognized trigger, the ulcers then started to involve other areas including the genitalia, behind ears, around umbilicus with discharging fluid/pus. Systemic review showed that these ulcers are not associated with fever, good general wellbeing, good appetite, no significant wt loss ( although her wt fall below 3rd centhile for age and sex) , arthralgia ( both knees/ no arthritis PGALS was normal), skin rash follicular/postular involving pressure areas initially then progressed to involve the face, no nail nor hair changes, she had H/O recurrent URTI and skin infections that were managed with local Rx as they appear, also H/O constipation initially that then progressed to altered bowel habit. F/H her grandmother know case of IDDM & RA. The girl was approached with the DD of BD, IBD, SLE, FMF, hyper IgD and Haploinsufficiency of A20. Her blood tests showed: CBC WBC 10.2 ( 5.11 neutro, 4.39 lympho, 0.58 mono, EO 0.10), Hgb 11.9 g/dl, PLT 477+. ESR 107, 70, 78 ( always high). CRP never been +ve, LDH 369, Ferretine 16.37 ( low ), urine for Pr:Cr ratio N, ALT 10, AST 23, Bil 0.3, serology : viral screen negative, Immunoglobuline assay IgA, IgE, IgD, IgG, IgM all within normal levels, ENA screen 7.3 ( > 1.2 +ve), ANA, ds DNA, p-ANCA and c-ANCA all negative, EBV serology negative, TTG negative. Vitamin B12 normal level, Zinc serum level normal, S Amyloid normal. Molecular genetic analysis of the MEFV and MVK gene came with no significant pathologic variant detected, HLA B51 positive. She had an upper and lower GI endoscopy done for her with an ilial biopsy taken all came normal with no abnormality. U/S abdomen also normal. Ophthalmological examination Normal no uveitis. Furthermore complement level ( C1-C9 &C50 levels), T cell function assessment ( CD4,CD8 level & ratio, CD 18, CD 45 & CD56 ), immunoglobuline response for vaccination ( anti tetanus and diphtheria IgG levels) neutrophils function ( DHR flow cytometry ), genetic testing ( TNF AIP3 gene mutation +/- WES), biopsy from the ulcers for histopathology but unfortunately due to lack of resources and lack of financial support these test are not done yet.she is on colchicine tab 1mg once daily, local steroids for mouth and genital ulcers and systemic steroids has been used for short courses with no much improvement, Azathioprine has been added waiting for the response, she is also on oral hygiene care and septrine as prophylactic Abx. Conclusion: Due to the wide variety of disorders that present with symptoms that mimic BD, especially when presenting early in life, consideration of monogenic AID and immune deficiencies with early implementation of gene testing is crucial to shorten the duration of patients suffering and to come up with appropriate management planchlidren’s hospital , 2 Paediatric Rheumatology, Faculty of Medicine, Tripoli university, Tripoli Children’s hospital , Tripoli , Libya Correspondence: A. A. Abushhaiwia Introduction: General pustular psoriasis (GPP) is a rare form of psoriasis and is clinically characterized by widespread eruptions of sterile pustules and bright erythematous skin accompanied by periods of fever, chills, neutrophilia, and elevated serum C-reactive protein. In 2011, Marrakchi et al3 reported a subgroup of GPP patients with a specific genetic defect: a deficiency of interleukin-36 receptor antagonist (DITRA). We report for the first time DITRA in a Libyan child successfully treated with infliximab. Objectives: To report the efficacy of treatment with 5 mg/kg infliximab intravenous infusion at Weeks 0, 2, and 6, followed by a monthly regimen, was effective and in inducing complete or almost complete responses in a child with Methods: case report is described Results: A 4 year-old Arab Libyan boy, born from non-consanguineous parents came to our attention due to a suspicion of a systemic autoinflammatory condition. Of note, the index patient had two healthy brothers and an uncle diagnosed with psoriatic arthritis. At the age of 2 years recurrent and severe episodes of generalized pustular psoriases requiring many hospital admissions started. Initially, he was managed as of pustular psoriasis after a 6-month history of appearance. Diffuse pustules involving cheeks, perioral area associated with mouth ulcers, characterized skin lesions. A rapidly progression of his condition could be observed characterized by worsening of general condition and generalization of the rash over different body parts. Other clinical pictures frequently observed was fever; psoriatic nail changes (e.g. Pitting and onychomadesis) and ichthyosis altogether with joint involvement limiting his activity. Rash became severe with discharging pustules and failure to thrive could be observed. At his admission, widespread presence of scaly erythematous plaques with pinhead pustules involving the scalp, face, extremities, torso, and buttocks. The palms and soles were also affected. Nail pitting and onychomadesis. At his admission a complete blood count demonstrated anaemia HGB 8.1 g and leukocytosis. However, during febrile episodes the patient’s white blood cell count increased to 17,000/ll with neutrophilia of 92%, ESR 50mm, and elevated CRP 20mg level. No other condition, such as infection, could be observed over the time. A skin biopsy demonstrated parakeratosis, spongiosis, and psoriasiform acanthosis; we could also observe an elevated level of vitamin B12 757.90 pg (reference range). Due to a suspicion of a systemic autoinflammatory condition, a genetic sequencing was requested. A homozygous already described mutation in the IL36RN was found (c.80T>C;. pLeu27Pro), thus confirming the diagnosis of DITRA. Once neither anti-IL1 (anakinra) neither anti-Il12/23 (secukinumab) is available in Libya , high doses of systemic steroids was initiated and could achieve clinical control. In order to avoid prolonged use of steroids, the patient’s dose was gradually tapered. However, cutaneous exacerbation could be observed and a steroid spared agent, methotrexate, at a dose of 7.5 mg once per week was therefore added. Under this treatment no improvement was seen .Treating him by anti-TNF (inflixmab) 5mg IV, 2 weeks after starting infliximab IV reveals near complete resolution of psoriatic skin lesions. He is in total remission, “completely cleared skin”. Since October 2021 Conclusion: DITRA is a rare disease. We demonstrate in this a child with DITRA that TNF-alpha inhibition with infliximab dramatically improved the dermal changes and could normalize the skin within 2 weeks and his quality of life clearly improved after the use of infliximab treatmentElfawires 1 , A. Ateeq 2 , S. Elazragh 3 1 Paediatric Rheumatology , Faculty of Medicine,Tripoli university, Tripoli Children’s Hospital , 2 Paediatric Rheumatology , 3 General Paediatrics , Tripoli Children’s Hospital , Tripoli , Libya Correspondence: A. A. Abushhaiwia, characterized by recurrent self-limited attacks of fever accompanied with peritonitis, pleuritis, or arthritis. Inherited in an autosomal recessive fashion. Occurs as a result of pathogenic variants in Mediterranean fever (MEFV) gene. Rarity of these diseases exposure during medical practice and lack of genetic testing for confirmation makes diagnosis challenging in Libya and in other middle-income countries Objectives: To report for the first time the clinical and genetic findings of FMF in a two-year -old boy from Libya. Methods: is a target gene panel commercially available (ISU university Germany) was requested. The results: clinical presentation was and we found a heterozygous mutation on MEFV gene (c.2177T>C p.Val726Ala Results: A 2 year-old Libyan boy from the North Africa, born of non-consanguineous healthy parents, came to our attention because of recurrent episodes of fever since at age 4 months. That’s required him many hospitalizations and several and no specific diagnosis being made and could be achieved, recurrent attacks of fever that reached as high as 39c daily, lasting from 5-8 days, every 2-8 weeks sometimes accompanied by abdominal pain, vomiting, joint pain and diarrhea. The initial attacks were occurring as frequently as every 3 weeks. He had neither history of previous surgeries except he had undescended testes on May 2020, ultrasound scrotum&inguinal regions that showed retractile right testicle and left testicle seen within medial third of left inguinal canal and LH, testosterone both were within normal range, nor a family history of same illness or immunodeficiency. His physical examinations were unremarkable only he is under weight 10.5kg (failure to thrive). Laboratory tests during attacks revealed mild anaemia. (Haemoglobin as low as 9 -10.5g/l) during the attacks with platelets were within normal limits, white blood cells count were as high as 24,400.Remarkable leukocytosis (15,000 mm3-24, 000 mm3) mainly neutrophil 66.3%, blood film revealed mild microcytic hypochromic anemia, always with fluctuant levels of acute reactant C-reactive protein (values between 50 and 283mg/L) markers as erythrocyte sedimentation rate (ranged from 40 to 80 mm/h), all cultures included blood, urine and CSF were negative, vitamine B12 was elevated 1230pg ,immune profile tests all of them were within normal range included immunoglobulin assay was normal .Evaluation of lymphocyte immunophenotyping test ( quantitation of total B cell numbers (CD19) and total T cell numbers (CD3), and quantitation of T cell subsets (CD4, CD8) and natural killer (NK) cells (CD16), total CH50,C3,C4 all were within normal range, serum amyloid was normal <3mg , With normal laboratory work-up during wellbeing was a constant finding. Chest radiography, echocardiography and abdominal ultrasound were always normal. Based on his clinical background, physical examinations and his above some lab tests he is most likely the diagnosis of FMF or MVK. Molecular analysis for FMF (MEFV) revealed heterozygous c.2177T> p. V726A. Therefore the clinical and diagnosis of FMF was established for the child. The patient responded well to colchicine therapy started at a dose of 0.5 mg/day Conclusion: FMF is frequent in many Mediterranean countries, Libya is one of the Mediterranean countries but rarely reported in Libya or delay in diagnosis due to we don’t have those particular tests in public hospitals, it is one of the greatest challenges that these diseases require so many investigations that people can’t afford it. Furthermore; the prevalence of hereditary autoinflammatory diseases particular FMF remains unknown in Libya. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
Y. Achour, S. Chaabane, L. Keskes Laboratory of Human Molecular Genetics, Faculty of Medecine of Sfax, Sfax, Tunisia Correspondence: Y. Achour Introduction: Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disorder, affecting 0.5 to 1% of the population worldwide. It is characterized by unknown etiology in which genetics, environmental, and epigenetics aspects are considered as the main risk factors that contribute to the development of this chronic disease. MicroRNAs are endogenous small noncoding sequences of single-stranded RNA, which cause alteration of gene expression by post-transcriptional modifications, destabilize target mRNAs to inhibit protein synthesis and regulate crucial pathways and cellular processes, such as cell growth, differentiation, proliferation, and cell death. Some microRNAs have been associated with a higher risk and progressionto RA, as well as with clinical variables of RA (tender joint and disease activity score (DAS28) -erythrocyte sedimentation rate (ESR)). Moreover, miRs such miR-520b is recognized as a critical regulator by suppressing endothelial cell inflammation and might serve as a potential therapeutic target for atherosclerosis. Objectives: The current study aimed to investigate the expression levels of circulating plasma miR-520b in Tunisian RA patients and to determine possible correlations between expression levels and clinical and immunological features. Methods: Total RNA was isolated from plasma of 40 RA patients and 10 age-gender-matched healthy controls. Expression of miR-520b was measured using quantitative real-time PCR methods by TaqMan qRT-PCR. Correlation analyses was performed using Mann-Whitney U test and Spearman’s coefficient test. Results: The results shows that the expression level of miR-520b was considerably increased in RA patients compared to the control group (p-value = 0.0005). A positive correlation was observed with high activity disease (DAS28 > 5,1) (p-value = 0.008) and overall C-reactive Protein (p-value = 0.031). Moreover, a significant association of the expression of miR-520b with high CRP levels ( CRP > 10mg/L) was revealed. There was no significant correlation of miR-520b overexpression and seropositivity to anti-CCP and RF patients. Conclusion: To the best of our knowledge, this is the first report of miR-520b expression in RA. Our study demonstrates that plasma miR-520b is associated with the pathogenesis of RA and it may act as a novel diagnostic marker for disease. Disclosure of Interest : None declared
S. A. Affendi 1 , S. P. Tang 1 , S. C. Lim 2 , B. S. Koay 1 , S. K. Ng 1 , P. Nadarajah 1 , S. Y. Lee 1 , L. R. Sangaran 1 1 Paediatric Rheumatology, Selayang Hospital, 2 Paediatric Rheumatology, MARA University of Technology Malaysia (UITM), Selangor, Malaysia Correspondence: S. A. Affendi Introduction: Deficiency of adenosine deaminase 2 is a recently described monogenic autoinflammatory condition characterised by multi-system vasculopathy that often presents in early childhood. It has a wide heterogenous presentation which poses a diagnostic challenge to treating clinician. The aim of this case series is to highlight novel mutation associated with DADA2, the clinical spectrum and treatment outcome at our centre. Objectives: We describe here a case series of children with proven DADA2. Methods: Five children with DADA2 presenting with various forms of vasculopathy and immunodeficiency were identified in a single cetre tertiary hospital over 6 years follow-up duration. Their clinical data and treatment outcomes were analysed. Results: this case series we share our experience in managing children with DADA2 in the only paediatric rheumatology centre in Malaysia. 5 DADA2 patients were reported in 6 years follow-up duration period since 2016. Their age range from 2 months to 9 years old. There are 3 males and 2 females (with a pair of siblings) within this case series and all of them are of Malay ethnicity. 3 children were diagnosed within less than a year, whilst 2 other children took more than a year highlighting the diagnostic challenge posed by this condition. Neurological symptoms namely stroke was the dominant clinical picture (n=4) and 1 patient presented with right retinal artery occlusive vasculitis at 2 months of age. 2 children had skin manifestations (1 with livedo reticularis whilst 1 had morphea-like skin condition). Recurrent fever (n=3) and orchitis (n=1) with abdominal pain (n=1) are other clinical symptoms that were reported. 1 child went on to developed low IgM with recurrent CMV infection. Of note, haematological involvement were not observed in our case series The diagnosis of DADA2 were made via genetic panel for autoinflammatory (Invitae panel) . Via this genetic panel, the pair of siblings showed homozygous novel mutation in ADA2 gene leading to their clinical symptoms. The novel mutation c.369G>T (p.Trp123Cys) has not been reported in the literature, highlighting the potential genomic heterogeneity of DADA2 . All of the children in this case series has ADA2 plasma level activity of 0 mU/g protein and 1 child had level of 5.6 mU/g protein. All 4 children were treated with anti-TNF except for 1 child who is on MMF whilst awaiting for funding approval for anti-TNF. 3 of these children have shown good inflammatory control with anti-TNF recovery whilst 1 patient has non-sustainable respond to anti-TNF. 4 of these children has had significant morbidities due to uncontrolled inflammation, steroid toxicity and sequelae from the recurrent stroke. Conclusion: The spectrum of clinical presentation in DADA2 continues to expand in tandem with discoveries of novel mutation in ADA2 gene. More studies are needed in the future to highlight genotype-phenotype correlation and this will perhaps help to tailor a more target therapy for these children, High index of suspicion amongst clinician is vital in order to clinch the diagnosis and to prevent accrued organ damage. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
L. Alahmadi 1,2,3 , J. Almasoud 1,2,3 , W. Alsuwairi 1,2,3 , A. Alrasheed 1,2,3 , S. Al-Mayouf 4 , F. Alkhars 4 , A. Asiri 5 , D. Alwakeel 5 , A. Alenazi 6 , S. Alenazi 6 , R. Bakry 7 , K. Alsufyani 8 , O. Aldibasi 2 , J. Alqanatish 1,2,3 1 Pediatric Rheumatology, King Saud bin Abdulaziz University for Health Sciences, 2 King Abdullah International Medical Research Center (KAIMRC), 3 King Abdullah Specialist Children’s Hospital,National Guard Health Affairs, 4 Pediatric Rheumatology, King Faisal Specialist Hospital and Research Center, 5 Pediatric Rheumatology, Prince Sultan Military Medical City, 6 Pediatric Rheumatology, King Fahad Medical City, Riyadh, 7 Pediatric Rheumatology, East Jeddah Hospital, Jeddah, 8 Pediatric Rheumatology, Maternity and Children’s Hospital, Mecca, Saudi Arabia Correspondence: L. Alahmadi Introduction: Chronic non-bacterial osteomyelitis (CNO) is an auto-inflammatory disorder that mainly affects the bone and occurs in children and adolescents. It is a chronic condition with a spectrum of presentation ranging from a mild self-limiting mono-focal disease to a sever chronic recurrent multifocal or so called Chronic Recurrent Multifocal Osteomyelitis (CRMO). Objectives: The objective of the study was to evaluate demographic, clinical, laboratory, imaging,histopathology characteristics, and treatment responses of pediatric CNO patients seen in Saudi children. Methods: The clinical records of 58 patients diagnosed with CNO between 2015 and 2021 at 6 centers in 3 major cities in Saudi Arabia were reviewed. Results: We reviewed a total of 58 children (n=36, 62% females), with median age of 8 years at diagnosis of CNO. The most common clinical manifestation was bone pain (n=51, 88%) followed by bone swelling (n=32, 55%). Fever was the most frequent systemic manifestation (n=8, 13%), followed by skin rash (n=6, 10%) and GI symptoms (n=5, 9%). Eighty five percent (n=49) of patients had elevated ESR while CRP was positive in (n=24, 41%). X-ray was the most frequent imaging modality (n=49, 85%) followed by Regional-MRI (n=41, 71%), bone scan (n=33, 57%), CT-scan (n=24, 41%), whilst a whole body MRI was the least used modality. The most common sites of bony lesions were lower limb (n=37, 64%), and bone biopsy was done in 34/58 (59%) patients. The pelvis and clavicle were involved in 18/58 (31%) in our cohort. NSAIDs were the most used medications (n=51, 86%), followed by Bisphosphonate and Anti-TNF in 55% (n=32), and Methotrexate and systemic steroids in 36% (n=21) of patients. Clinical remission was achieved in 44/58 (83%) of CNO patients. Conclusion: This study describes the phenotype of a large-scale sample of CNO in our pediatric population and the practice of our pediatric rheumatologist in treating these patients. It is a step forward towards developing a local consensus in treating this rare entity in our SaudiBalan, S. Raj, M. CB Rheumatology & Clinical Immunology, Amrita Vishwavidyapeetham, Kochi, India Correspondence: S. Balan Introduction: TNF receptor associated Periodic Fever Syndrome (TRAPS) is an autosomal dominant, monogenic, autoinflammatory disease, characterized by mutations in TNFRSF1A gene coding for TNFR1 , which results in aberrant downstream signalling, resulting in widespread systemic inflammation and increased risk of reactive amyloidosis. Lack of response to colchicine is one of the defining features of TRAPS. Objectives: To present a child and a family with a TRAPS mutation who are responsive to Colchicine, an unusual observation in patients with this illness Methods: We present here a case of 9 year old female child, who presented with recurrent episodes of fever lasting for 7 to 12 days, associated with abdominal pain, maculo-papular rash, arthralgia, conjunctivitis every 3-4 months once since the age of 9 months, with strong family history. She was noted to have neutrophilic leucocytosis, grossly elevated inflammatory markers during febrile episodes. She was previously treated elsewhere with NSAIDs and glucocorticoids during flares with some response. As genetic testing was not a viable option when she first presented ,she was subsequently started on colchicine at a dose of 1mg/day with excellent clinical response. Genetic testing finally performed 4 years after the initial presentation ,revealed pathogenic TNFRSF1A variant. Her aunt ,also in the pediatric age group as well as her father, uncle and grandfather have the same mutation and all of them are currently responsive to Colchicine. She continues to be under our follow up since 3 years and is doing well on colchicine Results: Following commencement of Colchicine, she went down from 3-4 episodes/year to none and from 40-60 days of symptomatology a year to about 6-8 days of minor symptoms/year Inflammatory markers have stayed in the normal range. Her 17 year old aunt, father and great uncle with the same gene have shown similar clincial response with Colchicine The mutation this family carries-TNFRS1A Exon3 c.236C>T, Heterozygous and pathogenic . Conclusion: In resource limited settings, Colchicine can be considered as a therapeutic option in patients with TRAPS in those patients who show good clincial response Trial registration identifying number: none Patient Consent: Yes, I received consent Disclosure of Interest : None declared
B. Balažiová 1 , P. Čižnár 1 , D. Moravčíková 2 , I. Hulínková 1 , T. Freiberger 3 , E. Froňková 4 , T. Dallos 1 1 Department of Paediatrics, Comenius University Medical School in Bratislava, Bratislava, 2 2nd Department of Paediatrics, Slovak Medical University, Banská Bystrica, Slovakia, 3 Genetic laboratory, Centre for Cardiovascular and Transplant Surgery, Brno, 4 Department of Paediatric Haematology and Oncology, 2nd Faculty of Medicine, Charles University, Prague, Czech Republic Correspondence: B. Balažiová Introduction: Deficiency of ADA2 (DADA2) is a rare multisystem monogenic autoinflammatory disease with a constantly increasing spectrum of clinical phenotypes and disease-causing mutations in the ADA2 gene (formerly CECR1). Objectives: To analyse the phenotypes of our first two Slovak patients with DADA2. Methods: Demographics and clinical phenotype of 2 patients with DADA2 were analysed in retrospect by review of their medical records. Results: Patient 1 is a Caucasian boy of unrelated parents, who presented acutely with diplopia and ataxia due to a mesencephalic brain lesion at the age of 4.5 years. Soon after neurological restoration, he developed intermittent fever, livedo racemosa, bilateral hand thrombophlebitis after cannulation, splenomegaly with multiple focal lesions considered to be due to embolism and generalized lymphadenopathy. Persistently increased inflammatory markers (CRP 50-60 mg/l), B-lymphopenia, hypogammaglobulinemia with low post-vaccination antibodies were noted. Inflammation responded promptly to corticosteroids (CS); he was substituted with immunoglobulins regularly. Two heterozygous mutations in the ADA2 gene were confirmed at the age of 5.3 years (pathogenic missense mutation c.506G>A;p.Arg169Gln(1) and a missense variant of uncertain significance c.505C>T;p.Arg169Trp(2)). Biologic therapy with etanercept resulted in control of inflammation, the immunological disorder persists. Patient 2 is a Caucasian girl of unrelated parents, treated for autoimmune haemolytic anaemia (AIHA) at 8 months of age, who developed persistent firm maculopapular lesions after CS discontinuation (11 months). She developed arthralgias without apparent arthritis and intermittent fever (up to 38.5°C) at 21 and 23 months, respectively, splenomegaly and permanently increased inflammatory parameters (CRP 50-110 mg/l) were noted and the patient was treated for suspected systemic JIA with pulse CS and methotrexate and later cyclosporine A. With CS tapering intermittent fever and joint pain recurred, livedo racemosa was noted and a defect in B-cell differentiation without hypogammaglobulinaemia was identified (28 months). NGS analysis revealed two heterozygous mutations in the ADA2 gene (pathogenic missense mutation c.140G>C;p.Gly47Ala(3) and a new variant c.881+1G>C predicted to considerably reduce the efficacy of gene splicing), each inherited from one parent. Treatment with etanercept was recommended. Conclusion: Even in our limited experience, our first two Slovak patients with DADA2 presented with highly variable phenotypes and with two novel, likely pathogenic mutations identified in each patient. A high degree of suspicion is required to identify DADA2 among patients with persistent inflammation, immunological and/or haematological disorders, as well as neurologic manifestations. References: 1. National Center for Biotechnology Information. ClinVar; [VCV000120303.323/ 2. National Center for Biotechnology Information. ClinVar; [VCV000956376.3] [Internet]. [cited 2022 May 23]. Available from: https://www.ncbi.nlm.nih.gov/clinvar/variation/956376/ 3. National Center for Biotechnology Information. ClinVar; [VCV000120301.14] [Internet]. [cited 2022 May 24]. Available from: https://www.ncbi.nlm.nih.gov/clinvar/variation/120301/ Patient Consent: Yes, I received consent Disclosure of Interest : None declared
K. Barut, I. Ulkersoy, N. Gucuyener, F. Haslak, M. Yildiz, A. Gunalp, G. Yalcin, S. Sahin, A. Adrovic, O. KasapcopurK. Barut Introduction: Familial Mediterranean fever (FMF) is the most common hereditary autoinflammatory disease, characterized recurrent episodes of fever peritonitis, pleuritis, arthritis and rash, Colchicine is the mainstay of FMF treatment, although colchicine is the first line treatment in FMF, 5-10% of patients do not respond, another 2–5% do not tolerate the drug well. Interleukin (IL)-1 antagonists are the treatment of choice in resistant or intolerant cases. The FMF50 score is used in evaluation of disease activity, defined as 50% improvement in all of 6 criteria. Complete clinical and laboratory response is characterized with absence of clinical features and normal laboratory findings Objectives: We aimed to evaluate anti IL-1 effect (anakinra and canakinumab) on colchicine resistant FMF cases by comparing the FMF50 score and complete clinic and laboratory response. Methods: In this study all FMF patients followed up at tertiary pediatric rheumatology clinic assessed by examining patients’ files and network information, among these cases who met definition of resistant FMF were included in the study. in the first step colchicine dosage increased to maximum tolerated dose, after maximum dosage colchicine, if still had considered Cr FMF, then started other colchicine form (opacalcium colchicine OC) anti-IL1 treatment was started in the patients who were resistant despite receiving the OC. The FMF50 response of the resistant FMF patients under colchicine or other treatment modalities for at least 6 months has been determinates retrospectively by using data from the patients’ records. Complete clinical and laboratory remission has been evaluated for all resistant FMF patients. Results: A total 74 FMF patients has been considered colchicine resistant FMF(Cr FMF).FMF 50 response has been obtained in only 5/74(6.7%) patients under colchicine treatment. Among patients under other treatment options, FMF 50 response has been obtained in 23/48(48%) patients under OC, in 13/18(72.2%) patients treated with anakinra and in 26/28(93%) patients under canakinumab treatment. There was a statistically significant difference in FMF50 response according to treatment modality (p<0,05). Clinical remission has been achieved in 18/48(37,7%) patients under Opacalcium colchicine, 12/18(66,7%) patients under anakinra and in 25/28(89,3%) patients under Canakinumab. There was a statistically significant difference between patients treated with OC and those with anti-IL-1 according to complete clinical remission (p<0,05). Laboratory remission has been obtained in 19/48(39.6%), 11/18(61,1%) and 23/28(82,1%) patients treated with OC, anakinra and canakinumab, respectively. The laboratory remission was significantly different between patients treated with OC and anti-IL-1 agents (p<0,001). In patients treated with maximum dosage of colchicine (1,7±0,4 mg/day, max 2 mg/day), the most seen adverse effect was diarrhea in 18/74(24,3%), transaminase elevation in 4/74(5.4%) patients and leukopenia only in 2 patient. Diarrhea was seen in 10/48(20.8%) patients under opacalcium colchicine treatment, which is lower comparing to previously mentioned colchicine form. There was no serious adverse event with Anti- IL 1 treated patients. Conclusion: Despite the regular and maximum dose of colchicine use, Cr FMF cases are seen at a substantial rate. In these CrFMF cases, FMF50 response was higher in both canakinumab and anakinra, and clinical and laboratory remission found more than other colchicine form. In this study found that, the both of anti IL-1 agents were safe and effective in colchicine resistant FMFŞener 1 , E. Arslanoglu Aydin 2 , E. Aliyev 1 , I. Bagrul 2 , S. Türkmen 3 , O. Akgün 4 , Z. Balık 1 , A. Tanatar 4 , Y. Bayındır 1 , Z. Kızıldağ 5 , R. Torun 5 , A. Günalp 6 , T. Coskuner 3 , R. İşgüder 5 , T. Aydın 5 , F. Haşlak 6 , M. Kasap Cuceoglu 1 , E. Esen 7 , U. Akçay 3 , Ö. Başaran 1 , A. Pac Kisaarslan 7 , F. Akal 8 , S. Özdel 2 , M. Bülbül 2 , Y. Bilginer 1 , N. Aktay Ayaz 4 , B. Sözeri 3 , O. Kasapcopur 6 , E. Unsal 5 , S. Ozen 1 1 Hacettepe University Faculty of Medicine, 2 Dr. Sami Ulus Maternity and Child Health and Diseases Research and Training Hospital, Ankara, 3 Umraniye Research and Training Hospital, 4 Istanbul University Faculty of Medicine, Istanbul, 5 Dokuz Eylül University Faculty of Medicine, Izmir, 6 Istanbul University Cerrahpasa Faculty of Medicine, Istanbul, 7 Erciyes University Faculty of Medicine, Kayseri, 8 Department of Computer Engineering, Hacettepe University, Ankara, Turkey Correspondence: E. D. Batu Introduction: Familial Mediterranean fever (FMF) is the most common monogenic systemic autoinflammatory disease. Colchicine forms the mainstay of FMF treatment. Around 5-10% of FMF patients are colchicine resistant and require anti-interleukin 1 (IL-1) drugs. Objectives: To compare the characteristics of colchicine resistant and colchicine responsive patients and to develop a score for predicting colchicine resistance at the time of FMF diagnosis. Methods: FMF patients enrolled in the TURPAID (Turkish Pediatric Autoinflammatory Diseases) registry were included. All patients met the Eurofever FMF criteria and had confirmatory MEFV genotype. Colchicine resistance was defined as having one or more attacks per month or presence of subclinical inflammation despite receiving maximum tolerated dose of colchicine. The predictive score for colchicine resistance in FMF was developed by using univariate/multivariate regression and ROC analyses. Results: A total of 2944 FMF patients (297 colchicine resistant and 2687 colchicine responsive) were included (F/M=1.02, mean age at diagnosis 78.3±47.7 months). In colchicine resistant patients, the age of symptom onset and diagnosis was younger (39.1±33.9 vs. 55.4±43.8; p<0.001 and 64.2±43.1 vs. 79.7±48.0 months; p<0.001, respectively) the mean duration of attacks (2.8±1.0 vs. 2.6±1.0 days, p=0.009) and the number of attacks in the one year before diagnosis (16.7±12.9 vs. 11.3±10.4, p<0.001) were higher than colchicine responsive patients. Considering the clinical findings, fever (91.4% vs. 82%), malaise (5.4% vs. 2.8%), erysipelas-like erythema (12.5% vs. 5.7%), arthralgia (50.8% vs. 44.3%), arthritis (34.9% vs. 18.1%), myalgia (16.1% vs. 10.1%), abdominal pain (91.4% vs. 84.2%), diarrhea (10.1% vs. 5.6%), and chest pain (31.9% vs. 16.3%) were significantly more prevalent among colchicine resistant patients compared to colchicine responsive patients (p<0.05). Also, comorbidity (28.4% vs. 14.5%), parental consanguinity (24.9% vs. 15.4%), and homozygosity or compound heterozygosity for exon 10 MEFV mutations (90.3% vs. 62.3%) were more frequent in colchicine resistant than responsive patients (p<0.05). With univariate and multivariate regression and ROC analyses, a score for predicting colchicine resistance in FMF was developed (Table 1). The cut-off value that discriminated best between colchicine resistant and colchicine responsive FMF patients was ≥6. Its sensitivity was 78% while its specificity was 60%. Conclusion: The strongest predictors of colchicine resistance at the time of FMF diagnosis were the presence of arthritis, chest pain, homozygosity/compound heterozygosity for exon 10 mutations, and ≥1 attack/month. This predictive score could help us to identify FMF patients with a higher risk of severe disease. Funding: This study was supported by the Science Academy’s Young Scientist Awards Program (BAGEP) of Turkey. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
E. D. Batu, Y. Bayındır, Z. Balık, S. Sener, E. Aliyev, M. Kasap Cuceoglu, U. Kaya Akca, O. Basaran, Y. Bilginer, S. Ozen Turkey Correspondence: E. D. Batu Introduction: Familial Mediterranean fever is a monogenic autoinflammatory disease characterized by febrile episodes of serositis. In some FMF patients, attacks are triggered by menstruation. There are no studies analyzing peri-menstrual FMF attacks focused on adolescent girls with FMF. Objectives: To analyze the characteristics and treatment in adolescent FMF patients with menstruation-triggered attacks compared to FMF patients who did not have any menstruation-triggered FMF attacks. Methods: Post-pubertal girls (12-18 years of age) with FMF were included. All patients met the Eurofever criteria for FMF and had confirmatory MEFV genotypes. Results: A total of 153 adolescent girls with FMF were included. FMF attacks were triggered by menstruation in 37 (24.2%). In these patients, the median number of attacks within one year increased from 4 to 10 after menarche (p=0.005). The median age at disease onset and diagnosis were younger and both pre-menarche and post-menarche attack frequency were higher in FMF patients with menstruation-triggered attacks compared to the rest of the group (Table). Dismenorrhea was more common among these patients, as well (Table). Regarding the characteristics of non-menstruation associated FMF attacks, the features were similar between two groups (Table). Also, the frequency of patients with exon 10/exon 10 MEFV mutations was similar among two groups. 17 out of 37 patients did not receive any treatment addressing their menstruation-triggered attacks. Colchicine dose was increased or a different preparation of colchicine was initiated in 14 patients and 11 of them benefit from this change. On demand anakinra or corticosteroid use was prescribed to two and one patients. Two patients were receiving NSAIDs during the first 3 days of their menstruation which prevented menstruation-triggered attacks. Conclusion: Menstruation-triggered FMF attacks are probably more common among adolescents. These patients often have earlier disease onset and higher attack frequency in the pre-menarche period, as well. Raising awareness about these patients would help us to improve effective managementY. Bayındır 1 , E. D. Batu 1 , O. Bölük 2 , C. Koşukcu 3 , E. Sağ 2 , A. Özcan 2 , M. Kasap Cüceoğlu 1 , Z. Balık 1 , S. Şener 1 , E. Aliyev 1 , H. Ö. Başaran 1 , Y. Bilginer 1 , S. Özen 1 1 Hacettepe University Faculty of Medicine, 2 Ankara Research and Training Hospital, 3 Hacettepe University, Department of Bioinformatics, Institute of Health Sciences, Ankara, Turkey Correspondence: Y. Bayındır Introduction: Systemic autoinflammatory diseases (AIDs) are usually associated with disturbances of innate immune system. Although clinical phenotypes may suggest a specific SAID, most of these diseases are diagnosed with genetic tests. Objectives: To determine the diagnostic yield of our autoinflammatory gene panel (mainly covering inflammasomopathies) in a cohort of AID patients and analyze and compare the characteristics of gene panel positive versus negative patients. Methods: Patients with features of AIDs were included in this study followed in the Department of Pediatric Rheumatology at Hacettepe University, all initially screened for the common MEFV mutations. They were screened with a gene panel including IL1RN, ADA2, IL10RA, IL10RB, NOD2, PSMB8, LPIN2, MEFV, MVK, NLRP3, PSTPIP1, TNFRSF1A, NLRP12, NLRP7, CARD14, TNFRSF11A, ELANE genes. Patients with a definite or probable disease-causing variant were classified as being ‘panel positive’. ROC analysis was used to determine best performing cut-off value for C-reactive protein (CRP). Results: In our cohort, we enrolled 122 (40.2% female) patients suspected of AID. The median age at symptom onset was 23 months (range 0.1-180). Gene panel provided a definite or probable disease-causing variant in 26 of 122 patients (21.3%). These patients were diagnosed with: FMF (n=12), HIDS (n=8), CAPS (n=1), TRAPS (n=1), Blau syndrome (n=1), PAPA (n=1), DADA-2 (n=1), NLRC3-related disease (n=1). Of the panel negative patients, 14 were fulfilling Eurofever criteria for autoinflammatory recurrent fevers. Male gender (76.9% vs. 55.2%; p=0.046) and diarrhea (38.5% vs. 19.8%; p=0.048) were more prevalent and the median CRP levels during disease flares were higher (12.8 vs. 6.39 mg/dl; p=0.009) among panel positive patients than panel negative patients (Table 1). The cut-off CRP value that discriminated best between panel positive and panel negative patients was >6 mg/dl (sensitivity 72%; specificity 50%). Conclusion: In our cohort, the aforementioned panel offered diagnosis in 21.3% of the patients. Urticarial rash and arthralgia/arthritis were more frequent among panel positive patients and a higher CRP predicted gene panel positivity. The reason for this could be due to the fact that the gene panel used was mainly directed to genes associated with IL-1.M. Birk-Bachar 1 , H. Cohen 2 , E. Sofrin-Dr 1,2 , N. Kropach-Gilad 2,3 , N. Orenstein 1 , L. Kornreich 2,3 , R. Tal 1,2 , G. Amarilyo 2,3 , Y. Levinsky 2,3 , M. Sokolov 2,3 , E. Raveh 1,2 , M. Gerlic 2 , L. Harel 1,2 1 Schneider Children’s Medical Center of Israel, Petach Tikvah, 2 Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, 3 Schneider Children’s Medical Center of Israel, Petach Tikva, Israel Correspondence: M. Birk-Bachar Introduction: Gain-of-function mutations in the NLRP3 gene, lead to hyperactivation of the NLRP3 inflammasome, resulting in the inappropriate release of inflammatory cytokines including IL-1β, and cause a spectrum of autosomal-dominant systemic autoinflammatory diseases called cryopyrin-associated periodic syndromes (CAPS). Many of these patients also develop progressive sensorineural hearing loss (SNL) due cochlear autoinflammation, which in rare cases may be the primary finding. (1-4) A Jewish Ashkenazi family, presented at our clinic with: autosomal dominant progressive sensorineural hearing loss, without clinical features consistent with typical CAPS, and a novel missense variant in the NLRP3 gene (NM 001079821:c.1790G>A, p.Ser597Asn). Objectives: The combination of this unique clinical presentation spanning 3 generations, alongside the novel variant in NLRP3, led us to explore whether carriers of the novel variant indeed show evidence of NLRP3 inflammasome hyper-activity. Methods: We conducted a prospective study in 15 family members (10 known carriers, 5 age-group matched non-carriers), which included clinical and hearing assessment along with functional measurement of the NLRP3 inflammasome activity. Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood. IL-1 levels secreted by PBMCS were measured under 3 main conditions: basal state, exposure to IL-1 stimulant (lipopolysaccharide, LPS) and exposure to LPS + MCC950 (specific inflammasome inhibitor). Quantitative in-vitro measurement of secreted IL-1 in supernatants of carriers and controls was conducted using ELISA (Enzyme-Linked Immunosorbent Assay). Results: Of 10 known carriers, 6 family members from 3 generations, suffered from progressive SNL, varying from isolated high frequency impairment to severe hearing loss and cochlear implant. All family members with hearing impairment were variant carriers, and carriers who had normal hearing function were all under the age of 13. 6 carriers reported occasional episodes of fever/arthralgia, 3 of which reported past episodes of monoarthritis. Functional assessment of the inflammasome in carriers vs. non carriers (controls) revealed that although basal levels of secreted IL-1 were not significantly different in both groups, the fold of change (FC) of secreted IL-1 in response to LPS stimulation was at least 2 times higher among carrier vs. control group (2 way Anova, P-value < 0.05, sidak multiple comparison). Addition of MCC950 inhibited IL-1β secretion after stimulation with LPS+CaCl2, in both groups. Conclusion: The suggestive clinical presentation, genotype-phenotype correlation, and evidence of inflammasome hyperactivation, as seen in functional inflammasome stimulation tests, are proof that the novel variant 001079821:c.1790G>A p.Ser597Asn is a pathogenic gain of function mutation. This hyperactivation state leads to atypical CAPS phenotype, with predominant non-syndromic and syndromic bilateral progressive SNL that initially affects ultra-high frequency ranges. Trial registration identifying number: Study was approved by the local Helsinki Comittee (RMC-0941-20) Patient Consent: Yes, I received consent Disclosure of Interest : None declared
L. Malz 1 , M. A. Lee-Kirsch 1,2 , C. Wolf 1 , B. Mayer 1 , C. Günther 3 , K. Stamos 1 , C. Schütz 1,2 , R. Berner 1,2 , N. Brück 1 1 Department of Pediatrics, 2 UniversitätsCentrum für Seltene Erkrankungen, 3 Department of Dermatology, Medizinische Fakultät Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany Correspondence: N. Brück Introduction: History and main symptoms We report on a family with an unusual clustering of interstitial lung disease as well as acral vasculopathy and polyarthritis. The index patient – a 10-year-old boy - was referred to us with progressive pulmonary deterioration, chilblain lesions and anemia. He presented with severe growth retardation, arterial hypertension and glomerulonephritis. Laboratory investigations showed increased inflammation, strongly positive values for RF, ANA and cANCA. The child’s father had bluish-discolored acral skin lesions and suffered from fibrosing interstitial lung disease since the age of 11 years. Additionally he reported night sweats, pulmonary hypertension, and right ventricular failure. Due to respiratory failure, he had been treated with cyclophosphamide, azathioprine and corticosteroids in adolescence. The family history revealed similar clinical abnormalities in the paternal grandmother and aunt. The grandmother who had suffered from severe rheumatoid arthritis died at the age of 35 years from lung disease. The paternal aunt presented with recurrent pneumonia at the age of 1 year, followed by diagnosis of interstitial pulmonary fibrosis at 11 years, and onset of severe polyarthritis, anemia and accompanying glomerulonephritis at the age of 12. Her pulmonary deterioration was only temporarily controlled with cyclophosphamide and corticosteroids. She died at the age of 20 due to acute respiratory failure. Objectives: Diagnosis In view of the suggestive family history, and high clinical suspicion of STING-associated vasculopathy with onset in infancy (SAVI), genetic testing was initiated revealing the following pathogenic variant in the TMEM173 gene (c.463G>A [p.Val155Met; V155M] heterozygous) within a few days. The child’s father is also carrier of the mutation. SAVI is a rare autoinflammatory disease with autosomal dominant inheritance, caused by gain-of-function variants in the TMEM173 gene (prevalence 1:1 M births). The V155M variant is located in the dimer interface of the STING protein (stimulator of interferon genes), and causes hyperactivation of the type 1 interferon axis. Systemic inflammation and small-vessel-vasculopathy start in infancy. Patients later present with interstitial lung disease and failure to thrive, acral dermatoses, and elevated acute-phase proteins and interferon signature. More rarely, anemia, polyarthritis and chronic glomerulonephritis with nephrotic syndrome are the presenting features. Methods: Case report Results: Therapy and prognosis After confirming the diagnosis, therapy with the Januskinase (JAK) inhibitor Ruxolitinib was initiated, which interrupts the positive interferon feedback loop. Treatment led to normalization of the initially pronounced type 1 interferon activation (score: 8907.36) within 28 days. Clinically, there was a striking improvement in respiratory (FVC 59% to 79%) and renal functions. The index patient’s father is being treated with the JAK inhibitors Baricitinib and Nintedanib Conclusion: Discussion Chilblain lesions and interstitial lung disease in combination with other autoimmune phenomena are suggestive for rare monogenic diseases such as SAVI. Genetic testing is essential for rapid initiation of targeted therapies. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
I. Burgos Berjillos 1 , M. Gonzalez Fernandez 2 , B. Lopez Montesinos 1 , L. Lacruz Pérez 1 , M. Martí Masanet 1,2 , I. Calvo Penadés 1 1 Pediatric Rheumatology Unit, Hospital Universitario y Politécnico La Fe, Valencia, 2 Pediatric Rheumatology Unit, Medical Research Institute Hospital La Fe, Valencia, Spain Correspondence: I. Burgos Berjillos Introduction: IL-1 plays a pivotal role in the pathogenesis of some monogenic autoinflammatory diseases (AID) in which IL-1 inhibitors have been shown to be effective. Objectives: To describe the experience with IL-1 inhibitors in patients with a monogenic AID in a pediatric rheumatology unit in a tertiary hospital. Methods: Clinical and demographical data, efficacy, dose and adverse events were collected from patients with a monogenic AID who received treatment with IL-1 inhibitors (anakinra and/or canakinumab), followed in our pediatric rheumatology unit from January 2007 until December 2021. Results: 31 patients were identified with a monogenic AID diagnosis and treatment with IL-1 blockers (table 1), 16 anakinra treatment courses (3 FMF, 7 HIDS, 5 TRAPS, 1 APLAID) and 26 canakinumab treatment courses (9 FMF, 9 HIDS, 4 TRAPS, 2 CAPS, 2 PAPA). Median (Q1;Q3) treatment time with anakinra and canakinumab was: 1,98 (1,04 ; 3,70) and 5,61 (2,30 ; 7,16) years, respectively. We assessed treatment response: 81,3% of patients treated with anakinra achieved disease control, 25% complete response (3 FMF, 1 HIDS) and 56,3% partial response (6 HIDS, 2 TRAPS, 1 APLAID); for canakinumab courses, 92,3% of patients responded, 73,1% with complete response (6 FMF, 6 HIDS, 3 TRAPS, 2 CAPS, 2 PAPA) and 19,2% with partial response (3 FMF, 2 HIDS). Related to dose management, 4/7 HIDS patients and 2/5 TRAPS patients needed anakinra dose increase (>2mg/kg/day or >100mg/day). 8/9 HIDS patients, 2/4 TRAPS and 1/2 CAPS patients needed canakinumab dose increase (4mg/kg/4 week or 300mg/4 week). During the follow-up, it was possible to decrease the dose (interval increase) in 12/26 (46,2%) patients treated with canakinumab (5 FMF, 3 TRAPS, 2 HIDS and 2 PAPA). Reported severe or of interest adverse events were 2 pneumonia in 2 patients and 2 generalized skin reaction in 2 patients while on anakinra and 5 pneumonia in 4 patients, 2 appendicitis, soft tissue infections in 2 patients and 2 varicella-zoster virus primoinfection while on canakinumab. One patient with HIDS developed hidradenitis during the follow-up. No death was reported. Conclusion: We describe our experience with IL-1 inhibitors in a cohort of patients with monogenic AID. About one third (39%) of our patients with a monogenic AID were treated with IL-1 blockers, leading them to a better disease control. Globally, 67,7% of patients achieved remission. In our cohort, 89% of HIDS patients, and around 50% of TRAPS and CAPS patients needed higher dose of IL-1 inhibitors to achieve disease control. These findings highlight the importance of IL-1 blockade, with an accurate dose adjustment, in monogenic AID. Disclosure of Interest : I. Burgos Berjillos: None declared, M. Gonzalez Fernandez: None declared, B. Lopez Montesinos: None declared, L. Lacruz Pérez: None declared, M. Martí Masanet: None declared, I. Calvo Penadés Consultant with: Novartis, Speaker Bureau with: Novartis, Sobi
A. Calin, C. Scurtu, D. Sfrijan, R. Vidlescu Pediatrics, M.S. Curie Hospital, Bucuresti, Romania Correspondence: A. Calin Introduction: In most cases, the cause of Sweet’s syndrome isn’t known. Acute febrile neutrophilic dermatosis is an uncommon skin condition characterised by fever and inflamed or blistered skin and mucosal lesions. Objectives: Sweet’s syndrome can present in several clinical settings: classical (or idiopathic) Sweet’s syndrome, malignancy-associated Sweet’s syndrome (leukemia or solid tumors, such as breast or colon cancer) and drug-induced Sweet’s syndrome (most commonly a type of drug that boosts production of white blood cells). Classical Sweet’s syndrome usually presents in women between the age of 30 to 50 years, it is often preceded by an upper respiratory tract infection. We present ca case of a 9 years old boy with malignancy-associated SS. Methods: The authors present a case of a 9 years old boy that was admitted to our oncology departement for acute leukemia. He developed a painful skin rash (plaques and nodules), fever resistent to antibiotics, arthralgia, elevated ESR and CRP. Clinical, laboratory and histopatological (oedema of the dermis and a diffuse infiltrate of numerous neutrophils with leukocytoclasis without vasculitis in the superficial and the deep dermis) findings established the diagnostis. Diagnostic criteria for classic acute febrile neutrophilic dermatosis have been proposed. Results: Sweet syndrome, although rare, it can be present in children. This is why pediatricians with rheumatology concerns and beyond, need to know about it and to take it into consideration when needed. Conclusion: Early recognition and treatment of malignancy-associated Sweet syndrome is imperative to limit patient morbidity and provide anti-cancer therapy. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
V. Dang 1 , E. Landy 2 , J. Varghese 1 , L. Van Der Kraak 2 , L. Huang 1 , A. Frank-Kamenetskii 1United States Correspondence: S. Canna Introduction: Interleukin 18 (IL-18) is an inflammasome-activated, IL-1 family cytokine that canonically induces interferon-gamma (IFNg). IL-18 activity is potently inhibited by a soluble, IFNg-inducible antagonist, IL-18 Binding Protein (IL-18BP). IFNg appears central to the pathogenesis of rare, hyperinflammatory states like Hemophagocytic Lymphohistiocytosis (HLH) and Macrophage Activation Syndrome (MAS), and IL-18 is both a biomarker of MAS and may be central to its pathogenesis. In some patients, MAS may compete with IFNg-independent features like arthritis, and IL-18 may contribute to both. More recently, patients with PSTPIP1 mutations causing neutrophilic skin rashes and arthritis were found to have chronic elevation of IL-18. These observations reinforce abundant evidence from model systems that IL-18 is also capable of amplifying Type 2 and 17 inflammatory responses. Objectives: We hypothesized that excess IL-18 will amplify the dominant inflammatory T-cell paradigm and exaggerate models of mixed inflammation regardless of their prevailing type of inflammation (e.g. Type 1, Type 2, Type 17, etc.). Methods: C57BL/6 WT and transgenic mice underwent experimental autoimmune encephalomyelitis (EAE) and inhaled house dust mite (HDM), which are models Type 1/17 and Type 1/2 competition, respectively. Results: Without stimulation, Il18bp -/- mice show no baseline skew towards Type1 responses except in intestinal epithelium. By contrast, Il18tg mice show a mild increase in serum IFNg and IFNg-producing cells in multiple tissues, most notably liver and spleen. Upon induction of EAE, Il18bp -/- mice were profoundly protected from weight loss, clinical score, and CNS infiltration of T-cells. This protection was lost upon systemic neutralization of IFNg, suggesting unopposed IL-18 was protective via induction of IFNg. Intestinal immune responses have been shown to affect EAE. However, mice with an Nlrc4 inflammasome mutation causing Th1-expansion and excess IL-18 restricted to the intestines were not protected from EAE. Notably, CD4 T-cell infiltration into spinal cords was not significantly different during EAE in WT and Il18bp -/- mice, but only Il18bp -/- mice showed significant infiltration of CD8 T-cells. Excess IL-18 protected against airway eosinophilia and Th2 differentiation in HDM in Il18tg but not Il18bp -/- mice, possibly due to minimal induction of IL-18 in the latter. Conclusion: Despite IL-18’s ability to amplify Type 2 and 17 responses in some circumstances, in models where T-cell differentiation states actively compete with each other, excess IL-18 prevents against non Type 1 immunopathology. This likely occurs indirectly, via the products of preferential-enhancement of type 1 responses. This may be relevant to therapeutics aimed at blocking pathogenic Th2 or 17 responses or in predicting the consequences of IL-18 blockade in complex inflammatory states. Patient Consent: Not applicable (there are no patient data) Disclosure of Interest : V. Dang: None declared, E. Landy: None declared, J. Varghese: None declared, L. Van Der Kraak Employee with: Bluesphere Bio, L. Huang: None declared, A. Frank-Kamenetskii: None declared, S. Canna Grant / Research Support with: AB2Bio, Novartis, SOBI, IMMvention Therapeutix, Consultant with: Simcha Therapeutics, Paid Instructor with: Clinical Viewpoints
E. Carlsson 1 , A. Charras 1 , G. Duffy 1 , M. W. Beresford 1 , H. J. Girschick 2 , H. Morbach 3 , C. M. Hedrich 1 1 Department of Women’s and Children’s Health, University of Liverpool, Liverpool, United Kingdom, 2 Department of Paediatrics, Vivantes Klinikum im Friedrichshain, Berlin, 3 Pediatric Immunology and Rheumatology, Children’s Hospital, University of Würzburg, Würzburg, Germany Correspondence: E. Carlsson Introduction: Chronic non-bacterial osteomyelitis (CNO), and its severe form chronic recurrent multifocal osteomyelitis (CRMO), is an autoinflammatory bone disease typically affecting children and adolescents. While the underlying pathophysiological molecular mechanisms are not well understood, dysregulation of monocyte-derived pro- and anti-inflammatory cytokines has been reported in previous studies. Objectives: This study aimed at identifying CNO/CRMO disease-specific gene expression and DNA methylation signatures in monocytes that may be used as biomarkers or therapeutic targets. Methods: Peripheral blood mononuclear cells (PBMCs) from healthy controls (n=12) and CNO/CRMO patients (n=12) were collected at diagnosis and 6-12 months after treatment initiation with naproxen. Cells were immunostained against CD14 and CD16 to identify and characterise monocytes, which were isolated via FACS. Monocyte RNA and DNA were then extracted using a Qiagen AllPrep DNA/RNA Kit. Isolated RNA was subjected to rRNA depletion and RNAseq analysis to identify differentially expressed genes, while isolated DNA was analysed by Illumina Infinium Methylation EPIC Arrays to identify DNA methylation signatures. Gene Ontology (GO) and KEGG pathway analyses was performed for differentially expressed genes and genes presenting at least one promoter differentially methylated position (TSS1500, TSS200, 5’UTR). Results: CNO/CRMO patients exhibited an increased proportion of classical monocytes compared to age- and sex-matched healthy controls (98% vs 93%, p<0.05), which did not significantly change following treatment with naproxen (98% vs 98%, ns). This was associated with altered gene expression and DNA methylation profiles. A total of 54 differentially expressed genes were identified when comparing CNO/CRMO patients at baseline with healthy controls, including IL17RC. Furthermore, 6 genes were found to be differentially expressed when comparing CNO/CRMO patients at baseline with patients at 6-12 months after treatment with naproxen, including PTGES and CXCR5. Differentially methylated positions (n=1176) were identified in promoter regions comparing monocytes from CNO/CRMO patients with healthy controls. These included genomic regions associated with genes encoding pro- or anti-inflammatory cytokines, or their receptors, including IL10RA and IL10RB, and enrichment of genes associated with TGF-beta activated receptor activity. No DMPs were identified comparing CNO/CRMO patients at baseline with follow-up. Conclusion: Gene expression and DNA methylation signatures in CD14+ monocytes differentiate CNO/CRMO patients from matched healthy controls. As molecular signatures largely do not change in response to treatment with naproxen, they may represent early events in the pathophysiology rather than effects secondary to ongoing inflammation. The exact clinical relevance, and whether these may be exploited for therapeutic purposes need to be validated in further studies. Disclosure of Interest : None declared
S. Y. Cetin 1 , O. Kaya Kara 1 , D. S. Kara 2 , S. Yardım 2 , E. Comak 3 , S. Akman 3 , M. Koyun 3 , G. Kaya Aksoy 3 1 Faculty of Health Sciences, Department of Physiotherapy and Rehabilitation, 2 Instin autoinflammatory disease that typically begins in childhood and more common among Mediterranean populations. The primary symptoms experienced by the patients are usually fever and pain with inflammatory attacks, which may cause obstacles in the individual’s role and functions in daily living activities. The Canadian Occupational Performance Scale (COPM) is an individualized measure designed to detect a person’s self-reported activity performance in daily life and can be used among children at least aged 8 years. Practicing COPM helps children to identify their own ideas about the supports required in their daily living activities. The activity and performance of children with FMF has not been questioned in the literature. Objectives: The aim of this study was to determine the activity and performance of children with FMF by using COPM. Methods: The study was included 42 children (22 girls, 20 boys between the ages of 8-17 years) with a mean age of 11.14±3.62 years. COPM was used to determine children’s activity and performance. The assessment was carried out face-to-face with the children by a physiotherapist. During this interview, the children identified up to five key activities they would like to tackle first. Then, the children determine the activities they want to do, should do or are expected to do; however, they determined the activities that they could not do or had difficulty and were not satisfied with because of their rheumatic diseases. Children identified activities for 3 different areas of COPM: self-care, productivity, leisure. For each of these activities, they gave performance and satisfaction scores between 1-10 (1 point indicates that he/she cannot do/’not at all satisfied; 10 points indicates that he/she is able/extremely satisfied). Results: Children with FMF determined 20 different activities that they have difficulty in daily living activities. Children with FMF had most difficulties in three activities including running (64.1%), writing (41%), and climbing stairs (33.3%). Conclusion: According to the results of our study, it was observed that children with FMF had difficulties with a wide variety of activities. It can be thought that COPM may be a useful tool to identify activity performance problems, can be used by clinicians in routine evaluation, and may also be useful in determining goals of rehabilitation programs for children with FMF. References 1- Verkerk GJQ, van der Molen-Meulmeester L, Alsem MW. How children and their parents value using the Canadian Occupational Performance Measure (COPM) with children themselves. J Pediatr Rehabil Med 2021;14 (1):7-17 2- Ben-Chetrit E, Touitou I. Familial Mediterranean fever in the world . Arthritis Care Res 2009;61(10):1447–1453 Patient Consent: Yes, I received consent Disclosure of Interest : None declared
O. Kaya Kara 1 , S. Y. Cetin 1 , S. Karademirıtue of Health Sciences, Department of Physiotherapy and Rehabilitation, 3 Faculty of Medicine, Department of Child Health and Diseases, Akdeniz University, Antalya, Turkey Correspondence: S. Y. Cetin Introduction: Familial Mediterranean Fever (FMF) is a genetic disease characterized by recurrent episodes of fever accompanied by pain in the abdomen or joints. It is most common in individuals of Mediterranean and Middle Eastern descent, and the first attacks typically begin in childhood. In addition, musculoskeletal findings such as arthritis, arthralgia or myalgia can often be seen in patients younger than 18 years of age in children with FMF. The quality of life and psychosocial status of these children may also be adversely affected. These symptoms and negative experiences by children can adversely affect children’s participation of activities in home, school and community settings. The participation of these children has not been evaluated so far and there is no relevant evidence in the literature. Participation and Environment Measure - Children and Youth (PEM-CY) is the most comprehensive measurement based on International Classification of Functioning (ICF) components to evaluate participation of children and adolescents with or without disabilities at aged of 5 to 17 years-old, in home, school, and community settings, alongside environmental factors. Objectives: This aim of this study was determine the participation of children with FMF and compared them with aged matched healthy peers. Methods: The study was included 94 children (57 children with FMF and 37 healthy children between the ages of 7-17) with a mean age of 10.76 ±2.82 years. PEMC-Y was used to assess children’s participation according to ICF. The questions in the scale were answered by the families of children in face-to-face interviews. Student t test and chi-square test was used to compare the groups. Results: When the groups compared in terms of participation at home, a significant difference was found in favor of the healthy group in the involvement, barriers, helpfullness and overall support scores (p:0.00). At school, a significant difference was found in favor of the healthy group in the participation frequency, involvement, barriers, helpfullness and overall support scores (p:0.00). In the community, a significant difference was found in favor of the healthy group in the all of subdomain scores (p:0.00-0.03). Conclusion: According to our study, the participation of children with FMF in home, school and community settings was found to be lower than healthy children. Especially, it was observed that the participation of activities in community setting was lower in every field. The participation of these children should also be taken into account when planning rehabilitation programs and goals, and it should be target to ensure maximum participation at home, school and community settings. References 1-Kaya Kara O, Turker D, Kara K, Yardimci-Lokmanoglu B. Psychometric properties of the Turkish version of Participation and Environment Measure for Children and Youth. Child Care Health Dev. 2020;46:711–722R. Hofmann 2 , A. Cox 3 , F. Schulze 2 , S. Russ 2 , H. Hartmann 4 , S. Haldenby 5 , X. Liu 5 , H. Morbach 6 , H. Wittkowski 7 , P. J. Ferguson 3 , C. Hedrich 1,8, 2 Klinik und Poliklinik für Kinder- und Jugendmedizin, Universitätsklinikum Carl Gustav Carus, TU Dresden, Dresden, Germany, 3 Stead Family Department of Pediatrics, Carver College of Medicine, University of Iowa, Iowa City, United States, 4 Centre for Molecular and Cellular Bioengineering, TU Dresden, Dresden, Germany, 5 Centre for Genomic Research, Institute of Infection, Veterinary & Ecological Sciences, University of Liverpool, Liverpool, United Kingdom, 6 Pediatric Immunology and Rheumatology, Children’s Hospital, University of Würzburg, Würzburg, 7 Klinik für Pädiatrische Rheumatologie und Immunologie, Universitätsklinikum Münster, Münster, Germany, 8 Pediatric Immunology and Rheumatology, Children’s Hospital, University of Würzburg, Liverpool, United Kingdom Correspondence: A. Charras Introduction: Chronic Nonbacterial Osteomyelitis (CNO) is an autoinflammatory bone disease primarily affecting children. It can cause pain, hyperostosis and fractures, thereby affecting quality-of-life and psychomotor development. While increased inflammasome activation has been reported, the exact molecular pathophysiology of CNO is unknown. Objectives: To identify disease mechanisms in CNO to allow patient stratification and individualized treatment. Methods: Whole exome sequencing in families with CNO, and target sequencing of P2X7R in a large German CNO cohort. Findings were integrated with demographic and clinical datasets. Genetically modified THP-1 cells were used to investigate altered potassium flux, inflammasome assembly (ASC specks), cytokine release and pyroptosis. Results: In 8 families with a history of CNO in 2 generations, damaging mutations in P2X7R , a regulator of inflammasome assembly, were identified. Targeted sequencing in 196 unrelated patients identified rare damaging heterozygous variants in 5. In the remaining cohort, common variants were over-represented when compared to the healthy population. Patients with rare variants were younger, more frequently exhibited multifocal disease, and required more aggressive treatment with 2 nd -line agents when compared to the remaining cohort. THP-1 cells expressing variant P2X7 exhibited altered potassium flux, inflammasome assembly, IL-1 and IL-18 release, and pyroptosis. Conclusion: Rare damaging variants in P2X7R account for approximately 2.5% of CNO cases. Common P2X7R variants were present in all remaining (97.5%) patients and may represent a risk allele underscoring the key role of inflammasome dysregulation in CNO. Observations will aid in future patient stratification and individualized care. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
ilona, R. Asaro, C. Martorana, C. Alizzi, M. C. Maggio, G. Corsello A.O.U.P. Paolo Giaccone- Università degli studi di Palermo, Palermo, Italy ilona Introduction: Interferonopathies are a new class of Mendelian inherited disorders, belonging to the group of systemic autoinflammatory diseases(SADs); they are characterized by a constitutive yet anomalous activation of the type I interferon pathway (IFN I). They are clinically heterogeneous; the first identified interferonopathy, the Aicardi-Goutieres syndrome (AGS) , mainly determines neurological and cutaneous involvement. At the beginning, AGS was originally defined as pseudo-TORCH syndrome, identifying a group of serologically negative disorders that mimic congenital TORCH infections, suggesting a similar pathogenetic mechanism. The underlying genetic mutations induce an accumulation of DNA and RNA fragments, generated during the genomic repair process, which act as a trigger for the production of IFN I. Other pathologies, such as systemic lupus erythematosus (SLE) have in common with the interferonopathies the overproduction of IFN and skin involvement. It has been suggested that the accumulation of nucleic acids may lead to the same pathways involved in interferonopathies, with over-production of IFN, suggesting an overlap between interferonopathies and SLE. Recent evidence suggests that interferonopathies should be considered in differential diagnosis in cases of early onset or atypical presentation of rheumatological diseases in the pediatric setting, especially if lipodystrophy, lupus pernio, vascular disease and arthralgia are found among the signs of the disease. Objectives: Our objective is to report a case which shows a possible correlation between a specific mutation and a well known autoimmune pathology such as the Aicardi-Goutieres. Methods: F., 16 years old, comes to our observation complaining of arthralgia, nodular dermatitis affecting lower limbs and Her laboratory evaluation showed positivity of ANA, hypocomplementemia and proteinuria. Adaptive SLE was initially suspected, and the patient was treated accordingly with hydroxychloroquine and corticosteroids, and a rheumatological follow-up was initiated.Results: Despite therapy, there was no regression of the vasculitic lesions, nor normalization of the values of complementemia and proteinuria. The patient was therefore subjected genetic investigation with NGS technique for Interferonopathies. Conclusion: The variant c.2893G> A, found in the patient and in her father, is currently classified as a variant of uncertain significance (VUS). However, considering that literature reports cases of interferonopathies with vasculitis as only clinical presentation, and that genetic correlation is subject to periodic review, to this day an univocal diagnosis has not been made yet the patient. We hope that further observations may help us to define her diagnosis more precisely. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
F. Corona 1 , C. Matucci-Cerinic 2 , S. Pastore 3 , P. Bocca 4 , A. Ravelli 4 , R. Caorsi 4,5 , M. Gattorno 4,5 , S. Volpi 2,4,5 , A. Tommasini 1,3 1 Università degli Studi di Trieste, Trieste, 2 DINOGMI, Università degli Studi di Genova, Genova, 3 IRCCS Institute Burlo Garofolo, Trieste, 4 Center for Autoinflammatory diseases, IRCCS Institute Gaslini, 5 Clinica pediatrica e Reumatologia, IRCCS Istituto Gaslini, Genova, Italy Correspondence: C. Matucci-Cerinic Introduction: COPA syndrome is a rare primary immunodeficiency, characterized by features of autoinflammation, immune dysregulation and autoimmunity caused by heterozygous mutations with incomplete penetrance of the COPA gene. Clinically, the disease is characterized by lung, kidney and joint involvement. No therapeutic guidelines exist for the treatment of COPA syndrome, even if the use of JAK-inhibitors has been reported in some patients. Objectives: to evaluate the efficacy of the JAK 1/2 inhibitor baricitinib in controlling and preventing disease progression in paediatric COPA patients. Methods: the data of 3 COPA patients treated with Baricitinib were retrospectively reviewed. Clinical, serological, immunological and radiological data were collected. Results: all patients were females and had a disease onset before 3 years of age. One patient (P1) carried the p.Arg233His mutation, while the 2 other unrelated patients (P2 and P3) carried the p.Arg281Trp mutation. P1 and P2 mother’s were asymptomatic carriers. P2 has a brother and a sister affected: the brother has an articular and pulmonary involvement, the sister died at 23 years of heart failure. P3’s mother is actually diagnosed with Still disease but no COPA mutation was found in the family. Clinically, all patients presented with arthritis, which was deforming in 2 cases (P1 and P2), and with pulmonary involvement, characterized by interstitial lung disease and by multiple pulmonary cysts on the CT scan. P1 and P3 presented also ground glass opacities and bronchiectasis. No pulmonary haemorrhages were reported. All patients have a normal renal function. P3 has congenital bilateral renal dysplasia. At diagnosis, spirometry in P1 showed a severe reduction of the FVC and of the DLCO, the 6minute walking test (6MWT) was pathologic with a minimal SaO2 of 85%. Spirometry and 6MWT were not performed in the other 2 patients because of the young age. All the patients presented a positive IFN signature at disease onset and were ANA and rheumatoid factor positive. P1 had also positive anti-citrullinated peptide antibodies. P1 had a disease onset 7 years before the first description of COPA disease and underwent several immunosuppressive and biologic therapies before the diagnosis (oral and intraarticular steroids, abatacept, methotrexate, mofetil mycophenolate MMF, rituximab). P2 was diagnosed one year after the disease onset and was previously treated with steroids, MMF and hydroxychloroquine. P3 was started on baricitinib at disease onset. After baricitinib was started, in all the patients a reduction of the inflammatory biomarkers was evident and steroids could be stopped in P1 and P2 (while never used in P3). In P1 a dramatic improvement of the 6MWT was achieved (minimal SaO2 during the test at last f-up 96%), CT scan and spirometry showed no disease progression over the years, and no articular injections were needed in the last 36 months (1-2 intra-articular injections/year before baricitinib start). Both P1 and P2 have a current follow-up of 36 months, P3 of 3 months on baricitinib and didn’t experience any disease relapse since the start of the therapy. Conclusion: baricitinib seems to block lung disease progression in medium term follow-up, controlling arthritis and reducing systemic inflammation in COPA disease. Larger cohorts of patients are needed to confirm these results. Disclosure of Interest : None declared
A. De Matteis 1 , M. F. Natale 1 , M. Chiriaco 2 , C. Cifaldi 3 , S. Di Cesare 2,3 , C. Passarelli 4 , A. Novelli 4 , G. Di Matteo 2,3 , A. Finocchi 2,3 , F. De Benedetti 5 , A. Insalaco 5 1 Division of Rheumatology, ERN-RITA center, IRCCS Ospedale Pediatrico Bambino Gesù, Roma, Italy, 2 Department of Systems Medicine, University of Rome Tor Vergata, 3 Academic Department of Paediatrics, Immune- Infectivology Unit, 4 Translational Cytogenomics Research Unit, 5M. F. Natale Introduction: Mutations in EROS/CYBC1 gene cause a rare form of autosomal recessive (AR) chronic granulomatous disease (CGD), characterized by severe life-threatening infections, hyperinflammation and immune-dysregulation. Objectives: We described an AR-CGD patient with a novel homozygous deletion in CYBC1 gene leading to absent EROS/CYBC1 protein and presenting with a clinical phenotype almost exclusively characterized by inflammatory manifestations. Methods: Whole Exome Sequencing (WES), Flow Cytometry Studies (phenotype, DHR assay, protein expression), Western Blot (protein expression Results: The clinical history of a previously well-being two years old female child was predominantly characterized by autoinflammatory phenotype. At onset she presented with recurrent episodes of fever of unknown origin responsive to glucocorticoids. Subsequently she developed inflammatory bowel disease treated with mesalazine. NBT test was negative. At the age of 9 years old she presented with pancytopenia, hepatosplenomegaly, bowel inflammation and granulomatous lung disease. Laboratory tests showedincreased levels of chitotriosidase (> 10 times normal value) and angiotensin converting enzyme (> 2 times normal value). In the suspicion of sarcoidosis immunosuppressive treatment with high dose of glucocorticoids, methotrexate and adalimumab was started. WES identified a novel homozygous deletion c. 8_7del CTCTCGGGATGTACC in the CYBC1 gene leading to absent CYBC1/EROS protein in PBMC and EBVB cells. Patient’s parents, heterozygous for the mutation, expressed about half of the protein. The gp91phox protein expression/function was impaired in patient’s neutrophils and monocytes (about 50%), but severely compromised in B cells (gp91phox<15%; DHR+ < 4%). Conclusion: This patient’s case emphasize the importance to consider a diagnosis of CGD even in absence of classical clinical presentation and negative NBT test. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
F. G. Demİrkan 1 , Ö. Akgün 2 , V. Guliyeva 2 , N. Aktay Ayaz 1 1 Pediatric Rheumatology, 2 İstanbul School of Medicine, İstanbul, Turkey Correspondence: F. G. Demİrkan Introduction: Interleukin-1 inhibitors are proven treatment options for autoinflammatory disorders. Anakinra and canakinumab are the most commonly used anti-interleukin 1 agents in the field of pediatric rheumatology. Objectives: The aim of this study was to evaluate the broader effectiveness and safety of anakinra and canakinumab in a ‘real world’ pediatric population. Methods: Patients with colchicine-resistant familial Mediterranean fever (crFMF), systemic juvenile idiopathic arthritis (sJIA), or polyarticular JIA treated with anakinra and canakinumab in any order were identified using the database of İstanbul Faculty of Medicine between January 2020-May 2022.Background characteristics of the patients, reason for switching to IL-1 inhibitor, and the side effects observed during the treatment were extracted form the patient files recorded at every 3 month visits. Results: Forty-eight pediatric patients(65%female) were enrolled in this study. The median age was 11 years (4-17 years), and the median disease duration was 37 months (14-59 months). The median (IQR) duration of treatment with anti-IL-1 agents was 16 (12-36) months. Forty-three patients were treated with canakinumab (150 mg/4 week) and 48 patients with anakinra (100 mg/day).Six (12.5%) patients receiving anakinra were diagnosed with sJIA, 1(2%) with polyarticular JIA and 41(85.4%) with crFMF in, and . In the canakinumab group diagnoses were sJIA in 2 (4.6%) and crFMF in 41 (95.3%) patients. The patients with crFMF were first treated with colchicine alone for a median duration of 12.5(8-38) months. Canakinumab was prescribed for crFMF after previous anakinra treatment, whereas no patients who switched treatment from canakinumab to anakinra were identified. According to the current guidelines for the treatment of FMF, colchicine was continued along with the IL-1 inhibitors. The autoinflammatory diseases activity and attacks decreased with both anakinra and canakinumab. However, 18/41(43.9%) patients with crFMF discontinued anakinra due to inadequate response (4 of them with secondary failure after a good initial response) and switched to canakinumab. In 9(21.9%) children anakinra was switched to canakinumab due to patient preference regarding refuse of daily injection. Frequency of flares and the median duration of flares were significantly decreased following switching to canakinumab from anakinra treatment (p<0.01). Anakinra was effective in decreasing proteinuria and canakinumab was also successful in decreasing proteinuria in anakinra unresponsive patients. The modified FMF score was achieved in 76.2% of anakinra and 88.9% of canakinumab group. Anakinra could be stopped due to complete remission in 4 children with sJIA. However, in 2/6 patients, anakinra was switched to canakinumab due to resistant disease course. Injection site reactions (ISRs, n:13) was the most common reason for the discontinuation of anakinra and most of ISRs developed in the first month of treatment. Two severe skin rashes and one significant elevation of transaminases were observed with anakinra. No severe side effects or side effect-related discontinuation of canakinumab was observed. No serious infections were detected in both anakinra and canakinumab group. Conclusion: While canakinumab, a human monoclonal anti-IL-1 beta antibody, for the convenience of its use, became the preferred IL-1 blocker in FMF, anakinra has its own benefits in certain circumstances of FMF. Canakinumab had a favorable safety/tolerability profile. Anakinra is also generally safe with side effects that may be observed in the short and long-term use should be taken into account. Large series and extended follow-up studies are required for establishing the long-term effects of these treatments. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
N. Aktay Ayaz 1 , F. G. Demİrkan 1 , T. Coşkuner 2 , F. Demir 3 , A. Tanatar 1 , M. Çakan 2 , S. G. Karadağ 4 , G. Otar Yener 5 , K. Öztürk 6 , E. Bağlan 7 , F. Çakmak 1 , S. Çağlayan 2 , S. Özdel 7 , K. Ulu 2 , B. Sözeri 2 , H. E. Sönmez 8 1 Pediatric Rheumatology, İstanbul School of Medicine, 2 Pediatric Rheumatology, Umraniye Research and Training Hospital, 3 Pediatric Rheumatology, Acıbadem Healthcare Group, 4 Pediatric Rheumatology, Çam ve Sakura City Hospital, İstanbul, 5 Pediatric Rheumatology, Sanliurfa Mehmet Akif Inan Training and Research Hospital, Sanlıurfa, 6 Pediatric Rheumatology, Istanbul Medeniyet University, School of Medicine, Goztepe Research and Training Hospital, İstanbul, 7 Pediatric Rheumatology, Dr. Sami Ulus Obstetrics and Gynecology, Pediatric Health and Disease Training and Research Hospital, Ankara, 8 Pediatric Rheumatology, Kocaeli School of Medicine, Kocaeli, Turkey Correspondence: F. G. Demİrkan Introduction: As none of the previous scoring systems have a power of estimating the refractory disease course at the initiation of the colchicine treatment in familial Mediterranean fever (FMF) cases, a predictive model appear to be beneficial for achieving early control of disease in colchicine resistant FMF (cr-FMF) patients. Objectives: We intended to develop a novel scoring system based on the initial clinical features and laboratory findings for predicting colchicine resistance in FMF, thus providing a reliable and easy tool for pediatric rheumatologists while evaluating patients at diagnosis. Methods: The medical records including baseline clinical and laboratory findings of patients prior to initiation of colchicine were analyzed. After generating a predictive score in the initial cohort, it was applied to an independent cohort for external validation of effectiveness and reliability. Results: Between June 2020 and December 2021, 1464 patients were admitted to the pediatric rheumatology outpatient clinic with the diagnosis of FMF. Forty-six patients were excluded from the analysis due to short follow-up period (less than 6 months). Among 1418 patients with FMF, 56 (3.9%) were colchicine resistant (cr) and 1312 (96.1%) were colchicine responsive. According to the logistic regression analysis, recurrent arthritis (4-points), protracted febrile myalgia (8-points), presence of ELE (2-points), exertional leg pain (2-points), and carrying M694V homozygous mutation (4-points) were determined as the scoring parameters for predicting patients with cr-FMF. Scores were assigned according to β coefficients in the final model. (Table 1) Conclusion: Intolerance/resistance in a definite portion of patients with FMF is posing a dilemma for pediatric rheumatologists and uncontrolled inflammation resulting in amyloid deposition in organ systems is detrimental for the patients as well. By constructing this novel reliable predictor tool, we enunciate that predicting colchicine resistance in children with FMF at the initiation of the disease and interfering timely before the emergence of complications during the disease course will be possible. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
A. Doležalová, P. Šeferna, A. Zahornadský, Š. Fingerhutová, P. Doležalová Inherited Metabolic Disorders, General University Hospital in Prague, Prague, Czech Republic Doležalová Introduction: Diagnosis of Periodic Fever, Aphtae, Pharyngitis and Adenitis syndrome (PFAPA) is based on its typical clinical picture and exclusion of other conditions. It has been postulated that it is a self-limited disease with spontaneous remission occurring by puberty in majority of patients. Objectives: We aimed at supporting the presumption that diagnosis of PFAPA based on the combination of systematic clinical evaluation using modified diagnostic criteria (1) and standardized follow-up is ultimately confirmed only by its complete resolution. Methods: A structured phone call interview was carried out during May 2022 with patients born between 1999-2008 from our PFAPA cohort described in 2013 (2) that have not been seen at our fever clinic for more than 5 years. Questions covered current health status including existence of any unexplained periodic symptoms, date of the last typical PFAPA episode, tonsillectomy (TE) date. In combination with the hospital electronic record the disease characteristics were derived. Results: During the period of 2004-2011 diagnosis of PFAPA syndrome was confirmed in 125 children (all Czech origin Caucasians) with the median age at onset of 23 months (2). In 60 patients who answered the call the current age was 16.1 (SD 2.3) years (response rate 48%). Mean follow-up (F/U) duration (from the 1 st visit until the call) was 12.7 (SD 1.7) years. In 3/60 (5%) patients recurrence of typical PFAPA was reported after the prolonged (>1year) asymptomatic interval before they entered sustained remission. Only 4/60 (6.7%) patients reported ongoing periodic problems different from the original PFAPA episodes. In 56 patients in remission (93.3%) the total disease duration (from the first to the last reported typical PFAPA episode) was 4.1(SD 2.8) years with the mean age of the last PFAPA episode of 5.9(SD 3) years. Mean asymptomatic interval (from the last reported episode until the interview) was 9.5 years (SD 2.9). 16/60(26.7%) patients reported TE at mean age of 4.8 (SD 2.1) years that was followed by full resolution of PFAPA in 14 cases (87.5%). In one patient there was a short recurrence of typical episodes followed by remission, in the other one typical PFAPA ended post-TE but short febrile episodes with no other symptoms have been recuring until now in 3-monthly intervals. Conclusion: Using a diagnostic approach described previously (2) the original diagnosis of PFAPA was confirmed by its longstanding remission (>9 years) in the majority (93.3%) of cases with symptom resolution occurring during the early school years. TE was a potent remission inductor in almost 90% of cases. Recurrence of symptoms after prolonged afebrile interval was uncommon. Persistence of periodic complaints in 4 patients discovered by the interview will lead to the clinical and genetic re-evaluation of their diagnosis. References: 1. Hofer M, Cochard M, Anton J et al .: PFAPA (periodic fever, oral aphtae, pharyngitis and cervical adenitis) syndrome: a new consensus on diagnostic criteria. Ann Rheum Dis 2009; 68 (Suppl.3): 705 2. Król P, Böhm M, Sula V, et al .: PFAPA syndrome: clinical characteristics and treatment outcomes in a large single-centre cohort. Clin Exp Rheumatol 2013;31(6):980-987F. Dressler 1 , J. Henes 2 , N. Blank 3 , T. Krickau 4 , T. Kallinich 5 , G. Horneff 6 , F. M. Meier 7,8 , I. Foeldvari 9 , F. Weller-Heinemann 10 , B. Kortus-Goetze 11 , M. Hufnagel 12 , J. Rech 13 , P. T. Oommen 14 , J. Weber-Arden 15 , J. B. Kuemmerle-Deschner 16 1 Division of Pediatric Pneumology, Allergology and Neonatology, Hannover Medical School, Hannover, 2 Center of Interdisciplinary Rheumatology, Immunology and autoimmune diseases (INDIRA), University Hospital Tuebingen, Tuebingen, 3 Rheumatology, University Hospital Heidelberg, Heidelberg, 4 Pediatrics, Friedrich-Alexander Universität Erlangen-Nürnberg (FAU), Erlangen, 56 Asklepios Clinic Sankt Augustin, Sankt Augustin, 7 Project Group Translational Medicine and Pharmacology TMP, Fraunhofer Institute for Molecular Biology and Applied Ecology IME, 8 Division of Rheumatology, University Hospital Frankfurt, Goethe University, Frankfurt am Main, 9 Centre for Pediatric and Adolescence Rheumatology Hamburg, Hamburg, 10 Prof. Hess Kinderklinik, Klinikum Bremen-Mitte, Bremen, 11 Division of Nephrology, University of Marburg, Marburg, 12 Division of Pediatric Infectious Diseases and Rheumatology, Department of Pediatrics and Adolescent Medicine, University Hospital Medical Center Freiburg, Medical Faculty, University of Freiburg, Freiburg, 13Erlangen, 14 Clinic of Pediatric Hematology, Oncology and Clinical Immunology, Heinrich-Heine-University Duesseldorf, Duesseldorf, 15 NOVARTIS, Nuernberg, 16F. Dressler Introduction: Familial Mediterranean fever (FMF) is a chronic disease characterized by recurrent episodes of fever and serositis, with a risk of severe complications (e. g. amyloidosis). Treatment of FMF according to EULAR recommendations aims to control acute relapses and subclinical inflammation and improve patients’ quality of life. Objectives: The present study investigates the long-term efficacy and safety of CAN in routine clinical practice in pediatric (age ≥2 years) and adult FMF patients. Methods: RELIANCE is a prospective, non-interventional, multicenter observational study in Germany with a follow-up period of up to seven years. Patients with a clinically confirmed diagnosis of FMF who routinely receive CAN will be enrolled in the study. Disease parameters will be recorded at the time of study inclusion and six-month intervals. [1]AIDAI: Auto-Inflammatory Diseases Activity Index Results: This interim analysis of FMF patients (N=74) enrolled through December 2021 includes data from baseline through the 24-month visit. The mean age in this cohort was 25 years (2-61 years). The proportion of female patients was 51% (N=38). At baseline, the median duration of prior CAN treatment was 1.0 years (0-6 years). At month 24, approximately 63% of patients were in disease remission by physician assessment, and 67% of patients documented inactive disease in the AIDAI[1] score (Table 1). Mutations were documented in a total of N=57 patients, including M694V (9 homozygous, 10 compound heterozygous, 12 heterozygous), V726A (1 hom., 6 comp. het., 1 het.), and M680I (1 hom., 6 comp. het., 1 het.). At baseline, N=16 FMF patients were CAN-naive and received a starting dose of median 150 mg CAN (40 mg/kg - 150 mg every 4 weeks). In this subgroup, a total of 12 dose adjustments were made over time (9 dose increases, 3 dose reductions). A total of 18 SAE[2] were reported, of which 2 (tonsillectomy and tachycardia) were classified as drug-related. [1]AIDAI: Auto-Inflammatory Diseases Activity Index [2]SAE: Serious Adverse Event [1]SAE: Serious Adverse Event Conclusion: Interim data from FMF patients in the RELIANCE study, the longest running canakinumab registry, confirm the efficacy and safety of long-term treatment with canakinumab. Disclosure of Interest : F. Dressler Grant / Research Support with: Novartis, Consultant with: Abbvie, Mylan, Novartis, PfizerT. Kallinich Speaker Bureau with: RocheF. M. Meier Speaker Bureau with: Novartis, I. Foeldvari Consultant with: Novartis, F. Weller-Heinemann: None declared, B. Kortus-Goetze Consultant with: Novartis, M. Hufnagel Grant / Research SupporP. T. Oommen Grant / Research Support with: Novartis, J. Weber-Arden Employee with: Novartis, J. B. Kuemmerle-Deschner Grant / Research Support with: Novartis, AbbVie, Sobi, Consultant with: Novartis, AbbVie, Sobi
S. Ertem, Z. B. Ozcakar, F. Aydin, N. Cakar, F. F. Yalcinkaya School of Medicine, Ankara, Turkey Correspondence: S. Ertem Introduction: Familial Mediterranean fever (FMF) is the most common systemic autoinflammatory disease characterised by recurrent, self-limiting attacks of fever and serositis. The pandemic caused by novel coronavirus-2 (SARS-CoV-2), known as coronavirus disease 2019 (COVID-19), is a global public health problem leading to significant mortality and morbidity worldwide. To prevent the spread of COVID-19, state and local governments enacted numerous restrictions on human movement and physical interactions. The effect of COVID-19 pandemic and the implemented restrictions on the frequency of attacks and the course of pediatric FMF remains unknown. Objectives: The aim of this study was to investigate the impact of COVID-19 restrictions on clinical status of FMF patients. Methods: We have enrolled patients with FMF, diagnosed according to Turkish pediatric FMF criteria, who were admitted to outpatient clinic of pediatric rheumatology department between June 2021 and December 2021. Medical records of the patients were evaluated retrospectively. Demographic data, family history, genetic results, clinical findings before and after FMF diagnosis, and the treatment modalities were recorded. Patients were also questioned about the pandemic days, information about school attendance, clinical findings of FMF, frequency of respiratory infections, colchicine dosages were noted. The clinical and laboratory findings of the patients before and during the pandemic were compared. The schools were closed for three semesters and children received online education during this period. Parameters were expressed as mean ± standard deviation (SD), median [interquartile range (IQR)], and number (percentage). The Cochran Q test was used to compare categorical variables between dependent groups. McNemar test was used to determine which of the categorical variables caused the difference between the three groups (post-hoc analysis), and Bonferroni corrected alpha values were presented. A p<0.05 was considered to be significant. Results: A total of 201 patients ( 97 male, 48% ) with a median age of 13 years ( 2-19 years ) and median follow up of 6 years (3-8.5 years) were included. Major clinical findings of FMF including abdominal pain, fever, arthritis, artralgia, leg pain, heel pain, and frequency of FMF attacks were significantly reduced in pandemic era compared with prepandemic era (Table 1). Moreover, the frequency of upper respiratory tract infections were also reduced during pandemic period. Attack free white blood cell (WBC) count and sedimentation rate levels were significantly decreased in pandemic era, compared to prepandemic era (p<0.05). Conclusion: Attacks of FMF can be triggered by various factors including infections, lack of sleep or stress. During COVID-19 restrictions, patients with FMF had reduced attack frequency in childhood period. Together with online education; the rate of respiratory tract infections decreased, possibly resulting in decreased disease activity. This showed us once again the role of environmental factors in autoinflammatoryE. S. Fedorov 1 , S. S. Salugina 1 , M. Kaleda 1 , M. Cherkasova 2 , Z. Kolkhidova 1 1 Pediatrics, 2 1 Immunology and Molecular Biology of Rheumatic Diseases, V.A. Nasonova Research Institute of Rheumatology, Moscow, Russian Federation Correspondence: E. S. Fedorov Introduction: Interleukin1 plays a leading role in the pathogenesis of autoinflammatory diseases (AIDs), to which systemic juvenile arthritis (sJA) also belongs. Objectives: IL 18 is a member of the interleukin-1 superfamily with a range of special properties; that is why it is of interest to estimate its level in patients with monogenic and polygenic AIDs. Methods: 147 patients with sJA and monogenic AIDs (FMF, CAPS, TRAPS) participated in the study. The diagnosis in all patients with monogenic AIDs was confirmed basing on detection of pathogenic alleles of corresponding genes. IL18 was detected in blood serum through ELISA method using Invitrogen kits (Bender MedSystems GmbH, Austria): reference parameter 0-732.7 ng/ml. Statistical processing was performed using the program “Jamovi”. The reliability of differences between the FMF, CAPS, TRAPS and JA groups was assessed under the Mann-Whitney criterion. Results: The study including 61 patients with sJA: male/female 23/38; age of inclusion into the study 3-19 years; of them ≥ 18 years - 2 (3%), FMF 40 patients; male/female 22/18; age 3-37 years, of them ≥ 18 years - 8 (20%), CAPS 30 patients; male/female 19/11; age 1-51 years, of which ≥ 18 years - 13 (43%), TRAPS 16 patients; age 4-38 years, of which ≥ 18 years - 4 (25%). In the group of patients with sJA, the Median (Me) of IL18 concentration amounted to 4 245.51 ng/ml, 1st quartile 1 136.94 ng/ml, 3rd quartile 4 324.6 ng/ml, interquartile range (IQR) 3,187.6 ng/ml. In patients with FMF Me 657.08 ng/ml, 1st quartile 180.96 ng/ml, 3rd quartile 1 815.51 ng/ml, IQR 1 634.55 ng/ml. In patients with CAPS Me 207.6 ng/ml, 1st quartile 89.0 ng/ml, 3rd quartile 291.0 ng/ml, IQR 202.0 ng/ml. In patients with TRAPS Me 338.6 ng/ml, 1st quartile 159.04 ng/ml, 3rd quartile 1 924.08 ng/ml, IQR 1 765.4 ng/ml. Differences between patients with sJA and patients with FMF, CAPS and TRAPS were reliable: sJA - FMF, sJA - CAPS p<0.001; sJA – TRAPS p<0.009. Conclusion: The maximum concentrations of IL18 are observed in patients with multifactorial AID - sJA, which exceed the concentrations in patients with monogenic AIDs (FMF, CAPS, TRAPS). This fact may explain the greater predisposition to development of macrophage activation syndrome in patients with sJA compared to the above-stated monogenic AIDs. Among patients with monogenic AIDs, the maximum concentrations of IL18 are in patients with FMF, and the minimum concentrations - in patients with CAPŠ. Fingerhutová 1 , H. Lachmann 2 , E. Papadopoulou-Alataki 3 , J. Frenkel 4 , L. Cantarini 5 , L. Obici 6 , G. Fabio 7 , G. Fabio 7 , I. Koné-Paut 8 , G. Amaryan 9 , W. Armbrust 10 , E. Hoppenreijs 11 , J. Kuemmerle-Deschner 12 , E. Moreno 13 , M. Alessio 14 , N. Ruperto 15 , M. Gattorno 16 , P. Dolezalova 1 on behalf of for the Paediatric Rheumatology International Trials Organisation (PRINTO) and the Eurofever Registry 1 General University Hospital in Prague, Centre for Paediatric Rheumatology and Autoinflammatory Diseases, Prague, Czech Republic, 2 Royal Free Campus, National Amyloidosis Centre, London, United Kingdom, 3 Papageorgiou General Hospital, Fourth Department of Pediatrics, Aristotle University of Thessaloniki, School of Medicine, Faculty of Health Sciences , Thessaloniki, Greece, 4 Wilhelmina Kinderziekenhuis, Department of Pediatric Immunology and Rheumatology, Utrecht, Netherlands, 5 University of Siena, Department of Medical Sciences, Surgery and Neurosciences, Rheumatology Unit, Siena, 6 Fondazione IRCCS Policlinico San Matteo, Centro per lo Studio e la Cura delle Amiloidosi Sistemiche, Pavia, 7 Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Dipartimento di Medicina Interna, UOS Malattie Rare, Milano, Italy, 8 National Referral Centre of Auto-Inflammatory Diseases and inflammatory amyloidosis, CEREMAIA, CHU de Biĉetre, APHP, University of Paris Sud, Department of Pediatric Rheumatology, Le Kremlin Biĉetre, France, 9 Arabkir Medical Complex - Institute of Child and Adolescent Health, National Pediatric Centre for Familial Mediterranean Fever; Yerevan State Medical University, Yerevan, Armenia, 10 Beatrix Kinderkliniek, University Medical Center, Department of Pediatric Rheumatology, Groningen, 11 Radboud UMC, Paediatric Rheumatology , CUKZ 435, Nijmegen, Netherlands, 12 University Hospital Tuebingen, Department of Pediatrics, Division of Pediatric Rheumatology and Autoinflammation Reference Center, Tuebingen, Germany, 13 University Hospital Valle de Hebron, Rheumatology Unit, Barcelona, Spain, 14 Universita’ di Napoli Federico II, Dipartimento di Pediatria, Napoli, 15 IRCCS Istituto Giannina Gaslini, UOSID Centro Trial, PRINTO, 16 IRCCS Istituto Giannina Gaslini, UOSD Centro Malattie Autoinfiammatorie e Immunodeficienze, Genoa, Italy Correspondence: Š. Fingerhutová Introduction: Despite estimates of the rate of AA amyloidosis complicating autoinflammatory diseases (AID), its true incidence especially in children is not known. It is anticipated that kidney involvement manifesting as proteinuria can be the first detectable marker of organ amyloid A deposition. Objectives: To assess the frequency of renal pathology in a large cohort of patients with AID reported to the Eurofever registry. Methods: Clinical and genetic data were extracted from the Eurofever registry based on optional responses. Data from the registration form submitted until July 2021 were analysed. Results: From the total of 6628 patients enrolled in the Eurofever registry to date, in 229 (3.46%; 114 females) abnormal value of proteinuria (PU) and/or microalbuminuria (miALBU) was reported. Mean age at the time of registration was 23.1 years (SD 17.1), 123 (53.7%, 62 females) were children. Disease duration from symptom onset was 14.9 (SD 14.1) years. The most common diagnosis was Familial Mediterranean Fever (FMF) (n=89, 38.9%), undefined AID (uAID) (n=35, 15.3%), Syndrome of Undifferentiated Recurrent Fever (SURF) (n=26, 11.4%), cryopyrinopathy (CAPS) (n=26, 11.4%), Mevalonate Kinase Deficiency (MKD) (n=14, 6.1%) and Tumor Necrosis Factor Receptor-Associated Periodic Syndrome (TRAPS) (n=13, 5.7%). Chronic nonbacterial osteomyelitis (CNO), DADA2, PFAPA, Behcet disease, PAPA syndrome, Blau syndrome, CANDLE and TNFAIP3-associated autoinflammatory syndrome were less common (2.6%, 2.2%, 2.2%, 1.8%, 0.9%, 0.9%, 0.4% and 0.4%) respectively. Presence of amyloidosis was confirmed in 47/6628 patients (0.7%; 25 females, mean age 39.8 years, SD 17.2), 6 of them were children (12.8%, 4 girls, mean age 11 years, SD 4.1). Majority of patients had TRAPS (n=19, 40.4%) and FMF (n=16, 34%) followed by CAPS (n=6, 12.8%), MKD (n=4, 8.5%), PAPA and Blau syndrome (n=2, 4.3%). Amyloidosis in paediatric patients was associated with FMF (4 cases), TRAPS and MKD (1 case each). Conclusion: The data suggest that despite low numbers children are at risk of developing amyloidosis. High proportion of children with proteinuria warrants further analysis of follow-up data. As these have been available for minority of patients only this should alert physicians to keep submitting these data into the registry. This study was supported by the Czech Health Research Council (AZV CR) grant NU21-05-00522 Disclosure of Interest : None declared
R. Galindo Zavala 1 , L. G. Martín-Pedraz 1 , G. Díaz-Cordovés Rego 2 , E. Núñez-Cuadros 1 1 Pediatrics, 2 Rheumatology, Hospital Regional Universitario de Málaga, Malaga, Spain Correspondence: R. Galindo Zavala Introduction: Pamidronate showed to be an effective and secure treatment for chronic recurrent multifocal osteomyelitis (CMRO). Nevertheless, there are very few reports about other bisphosphonates. Objectives: To compare pamidronate vs zoledronate’s effectivity and safety in children suffering from CMRO. Methods: Prospective, descriptive, and analytical research on children younger than 16 suffering from CMRO (diagnosed according to Jansson criteria) and treated with bisphosphonates between January 2013 and December 2020. Flares treated with pamidronate (0,5 mg/kg day 1; 1 mg/kg day 2; 1 mg/kg day 3 followed by 1 mg/kg/month) were compared to those treated with zoledronate (0,025 mg/kg/3 months). Results: We identified 16 bisphosphonates treated flares, 6 with pamidronate and 10 with zoledronate, in 12 patients. Average length of pamidronate and zoledronate courses were 3,67 and 12,3 months (p < 0,01), respectively. Epidemiological and clinical data are shown in table 1. There were no significative differences in sex, age or clinical features in patients treated with pamidronate vs zoledronate. 50% of patients treated with pamidronate received corticosteroids simultaneously vs 40% of patients treated with zoledronate (p =0,696). There were no differences in the highest corticosteroid dose (0,33 vs 0,72mg/kg/day; p=0,095) or the corticosteroids’ treatment length (1,67 vs 1,85 months; p=0,879) 66,7% of pamidronate treated patients reached full response vs 80% of patients who were treated with zoledronate (p=0,474). There were no differences in the clinical response time (2 months vs 0 months; p =0,524), the remission time after discontinuing bisphosphonate (9,25 vs 10 months; p=0,877) or the recurrences rate while on treatment (1 vs 0; p=0,298). 33,3% of patients on pamidronate and 40% of patients on zoledronate needed switching to anti-TNFa (p=0,790). Flu-like syndrome was the only reported adverse event. It was recorded in 16,7% of patients treated with pamidronate, and 40% of patients who received zoledronate (p=0,588). Conclusion: Pamidronate’s and zoledronate’s effectivity and security in children suffering from CMRO seem to be similar. Zoledronate permits a more comfortable dosage schedule and reduces hospital stay, improving the quality of life of these patients. Therefore, it may be selected as treatment of choice in children suffering from CMRO resistant to NSAIDs. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
A. Gunalp, M. Deveci, F. Haslak, M. Yildiz, A. Aliyeva, O. Koker Turan, S. Sahin, A. Adrovic, K. Barut, O. Kasapcopur stanbul University-Cerrahpasa, Cerrahpasa Medical Faculty, Istanbul, Turkey Gunalp Introduction: Cryopyrin-Associated Periodic Syndrome (CAPS) is a rare inherited autoinflammatory disease with uncontrolled inflammatory symptoms due to excessive secretion of IL-1β caused by a mutation in the NLRP3 gene. Objectives: The aim of this study is to evaluate the efficacy and safety of canakunimab in a large patient cohort. Methods: 24 patients who were diagnosed with CAPS according to the Eurofever/PReS diagnostic criteria were included. The frequency of attacks and acute phase reactants before and after canakinumab treatment in 2 year follow up were evaluated. Results: A total of 24 (%54 female, %46 male) patients with 5.18 mean age (11-13) were included. 19 of patients were diagnosed with FCAS, 1 Muckle Wells Syndrome and 1 was CINCA. Pathogenic mutation was found in NLRP3 gene in 21 of the patients. 3 of patients had no mutation. The clinical symptoms of our patients were; all of them had urticeria-like skin rash, 21 had fever, 13 had arthritis, 9 had abdominal pain, same 6 patients had oculer findings and convulsions, 4 patients had hearing loss, 1 had bone deformity and 1 had myalgia. 4 of the patients had central nervous system involvement with pathological MRI findings. The main reason to iniate canakinumab treatment was unresponsiveness and non-compliance to the current treatment (anakinra). The frequency of the attacks in the prior year of canakinumab treatment was median 17. 3 (5-30) and it decreased to median 1.17 (0-5) following 1 year use of canakinumab (p<0.01). Statistically significant difference was found between repeated measures of ESR levels when compared prior and following 1, 6, 12, 24 months of canakinumab treatment (p:0.01, p:0.013, p: 0.21, respectively). Mean canakinumab treatment duration was 4.2 (2-8,7) years. In the follow up one of our patients had MAS attacks and SLE-like autoimmune disease developed in the 5th year of canakinumab treatment in the same patient. One of the patient with CINCA syndrome died in the 15th month of treatment at the age of 39 months. No significant side effects were observed in our patients. Conclusion: Cryopryrinoyrin-Associated Periodic Syndrome is a rare childhood disease with urticaria-like rash, fever and arthritis. Canakinumab is a safe and effective agent to control the inflammation of the disease. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
W. J. Jang 1 , J. W. Rhim 2 , S. Y. Lee 3 , H. Y. Choi 4 , S. J. Lee 5 , M. Kim 6 , D.-C. Jeong 1 1 Pediatrics, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul, 2 Pediatrics, Daejeon St. Mary;s Hospital, College of Medicine, The Catholic University of Korea, Daejeon, 3 Pediatrics, Bucheon St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Bucehon, 4 Pediatrics, Seoul St. Marys’s Hospital, College of Medicine, The Catholic University of Korea, Seoul, 5 Pediatrics, International St. Mary’s Hospital, Catholic Kwandong University , Incheon, 6 Laboratory Medicine, Catholic Genetic Center, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul, Korea, Republic Of Correspondence: W. J. Jang Introduction: Haploinsufficiency of A20 (HA20) is a newly described autoinflammatory disease caused by mutations in tumor necrosis factor-α-induced protein 3 (TNFAIP3) gene. Patients present wide spectrum of manifestations with early-onset systemic inflammation like Behcet’s disease or other autoimmune features. Objectives: To investigate the clinical course and immunologic characterization in HA20 patient. Methods: We report the case of HA20 initially diagnosed with systemic juvenile idiopathic arthritis (sJIA). An 11-year-old girl has presented recurrent episodes of fever with vomiting, diarrhea since 11-month of age. She was diagnosed with sJIA presenting fever with skin rash, lymphadenopathy and arthritis with pericardial effusion at 27-month-old. She improved after high dose steroid, but developed fever with gastrointestinal symptoms including abdominal pain and vomiting about five times per year in spite of disease-modifying anti-rheumatic drugs therapy. Acute phase reactants were markedly increased during the period of her symptoms and decreased by conservative treatment with hydration, but still showed elevated levels when she was well-being state. She was shown as growth retardation, and microcytic hypochromic anemia in spite of iron supplement. Results: At the age of 11, she admitted due to the recurrent gastrointestinal symptoms with high acute phase reactants. Colonoscopy showed multiple ulcers on intestinal mucosa compatible with Crohn’s disease. The next generation sequencing identified the pathogenic heterozygous mutation (c.427C>T) at TNFAIP3 gene, leading to HA20. This genetic mutation presents to fail to the negative feedback mechanism for nuclear factor kappa B (NF-κB) activation, developing autoinflammatory features such as fever and systemic inflammation. Immunologic study showed high expression of IL-17 and IFN-γ in CD4 and CD8 T cells independent on zinc, and elevated level of regulatory T cells. Also, IL-1β and TNF-α in plasma were shown high level than healthy control. Conclusion: HA20 should be considered in the patient with recurrent inflammatory disease that presented with fever and gastrointestinal symptoms which might be regarded as respective episodes of acute infections. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
H. D. Karakas, Z. B. Ozcakar, F. Aydin, N. Cakar, F. Yalcinkaya nkara University, Ankara, Turkey Correspondence: H. D. Karakas Introduction: Familial Mediterranean fever (FMF) is the most prevelant hereditary autoinflammatory disease among children, manifesting with recurrent attacks of serositis accompanied by fever, usually lasting 12-72 hours. Abdominal pain and various gastrointestinal system (GIS) manifestations may arise directly from FMF or from other causes independent of primary disease. Objectives: The aim of this study was to evaluate gastrointestinal complaints other than classical peritonitis attacks in patients with FMF and to interpret laboratory, endoscopic and histopathological findings of GIS manifestations. Methods: The medical records of the cases with FMF, who attended to the Ankara University Pediatric Rheumatology outpatient clinic, were evaluated retrospectively from December 2011 to December 2021. Demographic data, anthropometric measures, main clinical symptoms of the episodes, treatment modalities, genetic mutations, family history, gastrointestinal system complaints, endoscopic and histologic data, serum aminotransferase levels (alanine aminotransferase [ALT], aspartate aminotransferase [AST]) were recorded. Descriptive statistics were used. All statistical analyses were carried out using SPSS 25.0. Results: A total of 576 pediatric patients (female 301, 52.3%), diagnosed with FMF since 2011 to 2021, were included. Majority of the patients (n=416, 72.2%) were found to have at least one exon 10 mutations. All of the patients were treated with colchicine, and almost 90.3% uses colchicine regularly. Functional gastrointestinal symptoms were reported by 106 patients (18.4%). Among these, abdominal pain (65.1%) and diarrhea (42.5%) were the most common complaints, followed by dyspepsia (33%), nausea and vomiting (27.4%). Malnutrition was observed in 42 patients (7.3%). Appendectomy was notified in 40 patients (6.9%). High serum aminotransferase levels were detected in 105 FMF patients (18.2%) at any visits, only 38 patient had hypertransaminasemia (AST x 3 unv; ALT x 3 unv). The most common cause of hypertransaminasemia was viral infections (31.6%). Only three patients’ elevated enzymes were associated with colchicine usage and stopped only the longest period of a month. Gastroenterology referral was done in 144 patients (25%) during follow-up. A total of 174 patients was monitored by abdominal ultrasonography for any reason. Upper GIS endoscopy was performed in 61 patients (10.6%), and colonoscopy in 30 (5.2%) patients. Accompanying GIS diseases were detected in 78 patients (13.5%). Upper gastrointestinal tract diseases (gastroesophageal reflux disease, gastritis, duodenitis, peptic ulcer) (6.1%), inflammatory bowel disease (2.6%), and irritable bowel syndrome (1%) were the most common coexisting GIS diseases. Conclusion: Patients with FMF could have frequent GIS complaints other than classical abdominal attacks. Taking a good medical history, clinical evaluation and gastroenterology consultation will result in early diagnosis and treatment of coexisting or associated diseases in these patientU. Kaya Akca 1 , B. Aydın 2 , E. Sag 2,3 , Y. Bayındır 1 , Y. Bilginer 1 , S. Ozen 1,2 1 Pediatric Rheumatology, 2 Pediatric Rheumatology Unit, Translational Medicine Laboratories, Hacettepe University, 3 Pediatric Rheumatology Clinic, Ankara Training and Research Hospital, Ankara, Turkey Correspondence: U. Kaya Akca Introduction: Varying levels of immune checkpoint molecules have been reported in inflammatory and autoimmune diseases. Chronic nonbacterial osteomyelitis (CNO) is a rare autoinflammatory bone disease characterized by sterile bone inflammation. Objectives: This study aimed to investigate the levels of checkpoint molecules in pediatric patients with CNO. Methods: Plasma samples were collected from CNO patients at diagnosis or during treatment with a biologic agent. Plasma levels of PD-1 (programmed cell death protein 1) and TIM3 (T cell immunoglobulin and mucin domain-containing protein 3), which are immune checkpoint molecules, were measured using the sandwich enzyme-linked immunosorbent assay (ELISA) method. Plasma samples of healthy controls were used as the control group. Results: Plasma samples were obtained from 18 CNO patients at the time of diagnosis and from nine patients after receiving biologic treatment. A total of 27 CNO patients (51.8% male) and six healthy controls (50.0% male) were included in the study. The median age of the patient and control groups was 14.5 years and 13.5 years, respectively (p=0.762). Plasma TIM3 levels were within the normal range in both the patient and control groups, and no significant difference was found between the two groups (p=0.981). Median plasma PD-1 levels were significantly lower in the total CNO group (treated and at diagnosis) compared to healthy controls (998.7 vs 8263.1, p=0.011). Plasma PD-1 levels of CNO patients at diagnosis were also lower compared to the healthy controls (p=0.023). There was no difference in plasma TIM3 and PD-1 levels between diagnosis and after treatment with a biologic agent (p=0.136 and p=0.735, respectively). Also, the plasma TIM3 and PD-1 levels of CNO patients were not different between those with and without spinal lesions (p=0.072 and p=0.621, respectively). Conclusion: Plasma PD-1 levels were significantly lower in CNO patients compared to healthy controls. This may reflect the low contribution of adaptive immunity in the pathogenesis of the disease since CNO is an autoinflammatory disease. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
Y. Y. Choi 1 , S. H. Kim 2 1 Pediatrics, 2 Department of Pediatrics, Seoul National University Children’s Hospital, Seoul, Korea, Republic Of Correspondence: S. H. Kim Introduction: In recent years, the phenotype of PSTPIP1 associated autoinflammatory syndrome has expanded. Classic phenotype is PAPA syndrome (Pyogenic Arthritis, Pyoderma gangrenosum, and severe nodulocytic Acne) and a newly identified PSTPIP1-associated myeloid-related proteinemia inflammatory (PAMI) syndrome has some unique features including very early onset severe chronic systemic inflammation, lymphadenopathy, hepatosplenomegaly and pancytopenia. Objectives: I would like to share our experience on familial PAMI syndrome, one of the familial autoinflammatory syndromes. Methods: We described our first 1 st case of familial PAMI syndrome in Korea. Results: A 14-month-old male patient presented with fever for 11 days, diarrhea, cervical lymphadenitis, and cyclic rash. Despite prolonged antibiotics treatment, fever continued for 12 days, and subsided spontaneously from 13th day. Fever usually spiked at night, but sometimes presented during daytime. CSF study confirmed sterile. Abdominal CT-scan showed mild hepatomegaly and several mesenteric lymph node enlargement. Peripheral blood smear showed mild leukopenia with a few plasmacytoid lymphocytes (2%). Laboratory work up showed LDH(Lactate dehydrogenase), CRP(C-reactive protein), ESR(erythrocyte sediment rate) elevation. At his 15-month of age, he experienced prolonged fever for 10 days again and subsided spontaneously. At his 16th month of age, he visited again with rhinorrhea, voice change, cervical lymphadenitis, erythematous rash, and fever. Laboratory work up showed elevated LDH, CRP, ESR and mild leukopenia. Immunoglobulin profile (IgG,A,M,D,E) showed mild IgG elevation, and ANA appeared positive (1:80). Echocardiogram revealed no cardiac anomaly. From medical history of unexplained recurrent fever with persistently increased inflammatory markers, we suspected autoinflammatory syndrome and performed targeted exome sequencing, which revealed pathogenic known mutation on his PSTPIP1 gene, which caused protein change (E275K). The same point mutation was found in his father as well and detailed history taking revealed his father had similar symptom throughout his life. The patient’s father was treated for lymphadenitis when he was four years of age, and he has been treated for periodic fever and JIA from his high school period since now. Recently, he is experiencing recurrent abdominal pain, diarrhea and oral ulcers which is thought to be symptoms of this disease. Conclusion: Here, we present the 1 st case of familial PAMI syndrome in Korea. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
J. B. Kuemmerle-Deschner 1 , B. Kortus-Goetze 2 , P. T. Oommen 3 , A. Janda 4 , J. Rech 5 , C. Schuetz 6 , T. Kallinich 7 , F. Weller-Heinemann 8 , G. Horneff 9 , I. Foeldvari 10 , F. M. Meier 11,12 , M. Borte 13 , T. Krickau 14 , J. Weber-Arden 15 , N. Blank 16 1 Department of Pediatrics, Division of Pediatric Rheumatology, University Hospital Tuebingen, Tuebingen, 2 Division of Nephrology, University of Marburg, Marburg, 34 Department of Pediatrics, University Hospital Ulm, Ulm, 56 Pediatrics, Medizinische Fakultaet Carl Gustav Carus, Technische Universitaet Dresden, Dresden, 78 Prof. Hess Kinderklinik, Klinikum Bremen-Mitte, Bremen, 9 Asklepios Clinic Sankt Augustin, Sankt Augustin, 10 Centre for Pediatric and Adolescence Rheumatology Hamburg, Hamburg, 11 Division of Rheumatology, University Hospital Frankfurt, Goethe University, Frankfurt am Main, 12Frankfurt am Main, 13 ImmunoDeficiencyCenter Leipzig (IDCL), Hospital St. Georg gGmbH Leipzig, Leipzig, 14 Pediatrics, Friedrich-Alexander Universität Erlangen-Nürnberg (FAU), Erlangen, 15 Novartis, Nuernberg, 16 Rheumatology, University Hospital Heidelberg, Heidelberg, Germany Correspondence: A. Janda Introduction: Cryopyrin-associated periodic syndromes (CAPS) are monogenic autoinflammatory diseases with severe systemic inflammation. The IL-1β inhibitor canakinumab (CAN) leads to a rapid remission of CAPS symptoms in clinical trials as well as in practice. Objectives: The RELIANCE registry investigates the long-term safety and efficacy of CAN under routine clinical conditions in pediatric (≥2 years) and adult patients with CAPS, including MWS, FCAS, and NOMID/CINCA[1]. [1]CINCA: chronic infantile neurologic cutaneous articular syndrome, FCAS: familial cold-induced autoinflammatory syndrome, MWS: muckle-wells syndrome, NOMID: neonatal multisystem inflammatory syndrome Methods: This prospective, non-interventional, observational study enrolls patients with a clinically confirmed diagnosis of CAPS who routinely receive CAN. Clinical data, physician assessments, and patient-reported outcomes will be collected at baseline and at 6-monthly visits. Results: 98 CAPS patients (52% female; median age 20 years) were enrolled through December 2021. At the 36-month visit, both physicians and patients of all ages rated current disease activity as absent or mild/moderate (Table 1). The proportion of patients without disease activity was highest in <12-year-old patients (78%). More pediatric compared to adult CAPS patients received higher than standard CAN (<12 years: 76%, 12-17 years: 43%, ≥18 years: 35%). Proportionate, more AE, SAE, and presumably drug-related SAE occurred in the pediatric cohort. Pathogenic mutations were documented for a total of N=38 patients, including R260W: N=15, A439V: N=9, T348M: N=9, D303N: N=3, and E627G, G755R, and G569R: N=1 each. N=27 patients with pathogenic mutations received standard dose CAN and N=9 patients received higher dose. Conclusion: The 36-month interim analysis of the RELIANCE study shows that long-term treatment with CAN is safe and effective in patients with CAPS regardless of the underlying mutation. Pediatric patients tend to have a higher infection rate with a better response rate. Patient Consent: Yes, I received consent Disclosure of Interest : J. B. Kuemmerle-Deschner: None declared, B. Kortus-Goetze Consultant with: Novartis, P. T. Oommen Grant / Research Support with: Novartis, A. Janda: None declared, J. Rech Grant / Research Support with: Novartis, Sobi, Consultant with: AbbVie, Biogen, BMS, Chugai, GSK, Janssen, Lilly, MSD, Mylan, Novartis, Roche, Sanofi, Sobi, UCB, Speaker Bureau with: AbbVie, Biogen, BMS, Chugai, GSK, Janssen, Lilly, MSD; Mylan, Novartis, Roche, Sanofi, Sobi, UCB, C. Schuetz: None declared, T. Kallinich Speaker Bureau with: RocheI. Foeldvari Consultant with: Novartis, F. M. Meier Speaker Bureau with: Novartis, M. Borte Grant / Research Support with: Pfizer, ShirJ. Weber-Arden Grant / Research Support with: Novartis, AbbVie, Sobi, Consultant with: Novartis, AbbVie, Sobi, N. Blank Grant / Research Support with: Novartis, Sobi, Consultant with: Novartis, Sobi, Lilly, Pfizer, Abbvie, BMS, MSD, Actelion, UCB, Boehringer-Ingelheim, Roche
R. Kumrah, R. Rikhi, D. Suri, A. Rawat, S. Singh Advanced Pediatrics Centre, PGIMER, Chandigarh, India Correspondence: R. Kumrah Introduction: Blau syndrome (BS) is a rare monogenic form of autoinflammatory disease caused by gain-offunction mutation in NOD2 gene and is characterized by granulomatous arthritis, dermatitis, and uveitis since early childhood. Objectives: To perform gene expression analysis of pro-inflammatory cytokines and associated transcription factors in patients with Blau syndrome Methods: Confirmation of genetic diagnosis in patients suspected with Blau syndrome was carried ouGenes for pro-inflammatory cytokines and associated transcription factors were selected. Comparison of fold change [2^(-ΔΔCT) method] between patients and controls were performed. Results: BS was genetically confirmed in 6 patients (3 males, 3 females from 5 families). Missense heterozygous mutation involving amino acid change from arginine to tryptophan at position 334 (hotspot) was identified in the nucleotide-binding domain of NOD2 gene in all. All the patients were on treatment in clinic with methotrexate, corticosteroids and adalimumab. Real-time PCR analysis revealed reduced NOD2 gene expression in patients as compared to control. Expression of Eomes, IL-1B, FOXP3 was also found to be less in patients with Blau syndrome. Reduced expression of transcription regulators of inflammation (NFĸβ1 and NFĸβ2) was noted in patients as compared to healthy control. Elevated expression of pro-inflammatory (TNF- ) and master regulator for T cells (T-bet) was noted in patients. Other genes (ROR-γT, GATA3, IL-6, IL18) were comparable in patients and healthy controls. Conclusion: R334W variant was identified in all patients with clinically suspected BS. We found altered expression of various cytokines and transcription factors in patients with Blau syndrome. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
L. Levantino 1 , S. Pastore 2 , F. Biscaro 3 , M. Girardelli 2 , A. Tesser 2 , S. Martelossi 3 , A. Zabotti 4 , A. Tommasini 1,2 1 Department of Medical, Surgical and Health Sciences, University of Trieste, 2 Department of Pediatrics, IRCCS Burlo Garofolo, Trieste, 3 Department of Pediatrics, Santa Maria di Ca’ Foncello Hospital, Treviso, 4 Department of Rheumatology, AOU Santa Maria della Misericordia, Udine, Italy Correspondence: L. Levantino Introduction: Haploinsufficiency of A20 (HA20) is an immune dysregulation disease due to heterozygous loss-of-function mutations in TNFAIP3 , encoding A20 protein, a crucial negative regulator of the NF-kB/TRAF6 pathway. HA20 leads to an imbalance in both innate and adaptive immunity, with high penetrance but variable expressivity. As a result, the phenotype of HA20 may feature both autoinflammatory and autoimmunity disorders. Objectives: To describe phenotypic features, response to treatments, and natural history of HA20 in children and adults. Methods: We performed a retrospective analysis of a case series with HA20, currently cared for at three rheumatologic centres in North-eastern Italy. Results: Overall seven patients from three unrelated Italian families with a genetic diagnosis of HA20 were included in the study. Four were female (57%) and the median age was 10 years (range 1-62). All patients were symptomatic, showing clinical manifestations with an early onset and a relapsing-remitting course. Previous diagnoses included PFAPA, Behçet’s disease, Hashimoto’s disease, celiac disease, autoimmune hepatitis, IBD, psoriatic arthritis, rheumatoid arthritis, Sjogren syndrome, and SLE. In all cases, a history of recurrent aphthous was present since infancy. Other clinical features were recurrent fever (4/7), recurrent upper respiratory infections (4/7), gastrointestinal symptoms (3/7), arthritis/arthralgia (4/7), skin involvement (2/7), psychiatric complaints (4/7), and autoimmune disorders (3/7, including celiac disease, thyroiditis, hepatitis). About laboratory findings, acute-phase reactants were elevated in 100% of patients and ANA/ENA autoantibodies were positive in one; IFN-score was high in 85% of cases and in two cases it increased during anti-TNF treatment. All patients had been received corticosteroids; six of them needed other immunosuppressive treatments. Conclusion: HA20 is an heterogenous immune disorder characterized by both autoinflammation, tending to develop in early childhood, and autoimmunity, usually appearing from late childhood/adolescence. The “amplifier” role of A20 deficiency on distinct pathways in the lifetime may challenge therapeutic and preventive approacheY. Levinsky, L. Harel, R. Tal, G. Amarylio Schneider children medical center of Israel, Petah Tikva, Israel Correspondence: Y. Levinsky. Despite the progress research since the discovery of the MEFV gene, colchicine treatment is still considered a main treatment for FMF patients. It reduces the frequency of attacks and effectively prevents the complication of secondary amyloidosis. Therefore, it is currently recommended to continue colchicine prophylaxis during the treatment with IL-1 inhibitors, despite clinical remission. Objectives: Our aims where to evaluate the rate of adherence to colchicine prophylaxis among patients with FMF under IL-1 Inhibitors treatment and to examine different risk factors for low adherence among that group. Methods: The databases of Maccabi Health Services (MHS), a 2.6 million member state-mandated health provider in Israel was searched for patients with FMF diagnosis. Patients under treatment with IL-1 inhibitors were matched to themselves – before and after starting the treatment. A sub-analysis was done among patients without amyloidosis or chronic renal failure. In the second phase, patients treated with IL-1 inhibitors were matched in a ratio of 1:4 to patients on colchicine only. Medication possession ratio (MPR) was used as main outcome measure. Results: The final cohort included 4526 patients. Among them, 108 patients were treated with IL-1 inhibitors. The MPR was higher after starting IL-inhibitors among the total population (19.7±13.1 before versus 32.2±14.5 after, p<0.01), and among patients without amyloidosis or chronic renal failure (21.6±15.07 before versus 32.8±15.3 after, p<0.01). In the second phase, patients treated with IL-1 inhibitors were matched in 1:4 ratio to 432 “colchicine only” patients. The total MPR in each groups was similar (78.9 ±41.4 versus 82.5 ± 80.6, P=0.5). In the sub-analyses, females treated with IL-1 inhibitors had less MPR than in the matched group (81.4 ± 33.2 versus 85.1 ± 91, P=0.03). Conclusion: Contrary to initial concerns, the adherence to colchicine increases among the same patients after starting IL-1 inhibitors, which may reflect the improvement in disease perception following the need to start the biological treatment. Moreover, the adherence is similar to the general FMF population after matching. Physicians who take care of FMF patients can consider treatment with IL-1 inhibitors when indicated without fear of decline in adherence to colchicine. Education regarding the importance of colchicine treatment is important among all FMF patients. Disclosure of Interest : None declared
R. Livny 1 , Y. Bitterman 2 , Y. Butbul Aviel 2 1 Pediatric Rheumatology, Schneider’s childrens hospital, Petach Tikva, 2 Rambam medical center, Haifa, Israel Correspondence: R. Livny Introduction: Familial Mediterranean fever (FMF) is an autosomal recessive, auto-inflammatory disease, presenting with recurrent bouts of fever and polyserositis. FMF has been associated with central nervous system (CNS) manifestations and recurrent aseptic meningitis (RAM) is a rare described one. There are only few case reports of aseptic meningitis due to FMF. Objectives: In this case-based review, we present a pediatric patient with FMF, who suffered from proven episodes of RAM while on colchicine and responded dramatically to treatment with anakinra. We compare this patient to previously described patients with FMF who contracted aseptic meningitis. Methods: A systematic search of the literature was performed retrieving English-language original case reports, case series, case-based reviews, and review articles on aseptic meningitis in FMF patients, up to may 2022. Articles involving patients with FMF suffering from aspetic meningitis, recurrent or single episode – were included. Results: In addition to our case, we identified seven cases describing aseptic meningitis in patients with underlying proven FMF; 6 of 7 cases were described in the adult population, with patient age ranging from 32 to 64 years of age, two (including our case) were in the pediatric population (13 and 14 years old). All cases in the literature responded well to colchicine treatment and aseptic meningitis episodes were diminished. Our case report is the first to document a resistance to colchicine and complete response to anti IL-1 treatment of RAM due to FMF. Conclusion: Central nervous system (CNS) manifestations of FMF are rare and under debate. Primary headaches are common among FMF patients, and may develop due to the autoinflammation. There are several reports of headaches due to recurrent episodes of RAM presumed to be related to FMF, mainly in adults. In most cases, events subsided or resolved following colchicine treatment. however, reports are scarce and vary significantly in criteria for FMF diagnosis (with or without genetic proof). According to Capron et al. Current diagnostic criteria for RAM related to FMF are: (a) RAM episodes due to FMF should be accompanied by other clinical or biological features of FMF attacks; (b) Colchicine prevents or lessens episodes; and (c) other classical causes of RAM (drugs, HSV2 infection, systemic diseases and cystic brain lesions) should be excluded. Our patient fulfilled both criteria for FMF and at least two of the criteria for RAM related to FMF. Only seven confirmed cases investigating association between FMF and RAM were found in the systemic review. Colchicine is the main therapeutic modality for reducing inflammatory attacks and preventing amyloidosis due to FMF. Nevertheless, 30-40 percent of FMF patients continue to suffer from recurrent attacks despite therapy, and 5-10 percent are considered resistant to colchicine. Until recently there was no other known effective treatment for FMF. Based on the role of pyrin in the regulation of interleukin (IL)-1β activation, the efficacy of IL-1 inhibitors has been assessed and well established in FMF patients who were resistant or intolerant of colchicine. Our study is the first report of RAM due to FMF that is responsive to anakinra. The immediate response strongly suggests there was a relationship between FMF and RAM in this patient. Our experience also offers a novel therapeutic option for colchicine resistant RAM and maybe even other CNS manifestations of FMFC. Maggio 1 , C. Scalisi 1 , F. Benfratello 2 , S. Amato 3 , G. Corsello 1 1 University Department PROMISE “G. D’Alessandro”, 2 University of Palermo, University of Palermo, 3 Children Hospital “G. Di Cristina”, Arnas Palermo, Palermo, Italy Correspondence: M. C. Maggio Introduction: Noise is an environmental factor that can influence human health status. Any sound that causes stress, irritability or disorder on the normal course life can be defined as noise. The WHO, in 2011 defined noise pollution as the most important environmental public health risk factor after air pollution. On the contrary, music is universally recognized as an anti-stressor method, and can be used as a therapeutic strategy. Children with autoinflammatory diseases (AIDs), especially with CAPS, need to wait in the hospital during the days of day hospital for blood sampling and biological drugs parenteral administration, sometimes for many hours, waiting the results of blood sampling. Many patients refer malaise and fatigue the afternoon and the evening of the day hospital, as possible effect of tiredness and stress. Objectives: To evaluate the music impact on wellness of patients with AIDs during the days of day hospital for blood sampling and biological drugs parenteral administration. To empower the nurse’s role in the global care of patients with AIDs, as main actor of alternative strategies to improve the treatment compliance. Methods: We engaged a musician nurse for the care of our patients with AIDs, especially with CAPS. The nurse played piano, involving patients to contribute with other instruments. In fact, in the following meetings, some patients chose to play music with the nurse in the waiting room in front of other patients. Results: We collected the records of the effects of music therapy on their wellness perception, both during the hours of day hospital and after coming back home. Most of the patients referred a positive impact of music on the personal patient journey. Conclusion: Patients with CAPS and generally with AIDs experience stress as a trigger for further attacks. Programs that improve care and support children and their families, through the complex way of AIDs, significantly improve the quality of life of patients with AIDs and their families. Healthcare providers should be aware of the impact of the long diagnostic journey on families and must work to create an environment of trust and collaboration facing a prolonged and difficult diagnostic process. Music can be considered as a winning strategy for supportive care, especially in children with CAPS or other AIDs, who experience stress as a trigger of disease attacks. Disclosure of Interest : None declared
, M. Liuzzo Scorpo, R. Panzeca, G. Corsello Palermo, Italy Correspondence: M. C. Maggio Introduction: Familial Mediterranean Fever (FMF) is an inherited auto-inflammatory disorder and is still extremely underdiagnosed in the Mediterranean area. Objectives: We collected a retrospective case series of children with recurrent febrile or inflammatory episodes and referred to the Children Hospital of Palermo. Methods: We divided all the patients with a diagnosis of FMF of our centre in 2 groups. Group A: 16 families with 24 children (15 M; 9 F) with the polymorphism R202Q (heterozygous in 14), (homozygous in 2), that show elevated inflammatory markers during the attacks, do not fulfil the PFAPA criteria, but respond to colchicine. They show the classical symptoms of FMF, fulfilling the Eurofever/PRINTO FMF classification criteria (fever, arthralgia, abdominal pain, rash, aphthous stomatitis, thoracic pain, with attacks induced by triggers as stress, fatigue, menses, etc). The children were treated with colchicine, 4 stopped colchicine after 4 years or more for the remission of clinical presentation and are still in remission. 2 children (8%) showed a partial response to colchicine, with the reduction but not the complete resolution of recurrent attacks. Parents were frequently symptomatic in childhood and in the 13% of the families, symptoms persisted in adulthood. Group B: 16 families with 36 children (19 M; 17 F) carrying MEFV gene mutations (M694V: heterozygous in 2, heterozygous associated with R202Q in 2, homozygous in 1; A289V: heterozygous in 3; A744S: heterozygous in 2, heterozygous associated with P369S, R408Q in 1; V796T: heterozygous in 2; I591T, P369S, R408Q: heterozygous in 1; F479D, E167D, R202Q: heterozygous in 3; R348H, R202Q: heterozygous in 3; E148Q: heterozygous in 10; R408Q, P369S: heterozygous in 2; R716H: heterozygous in 1; P369S, R408Q, R202Q: heterozygous in 1; R716H: heterozygous in 2) were included. Results: In group A, tIn group B, 7 children were asymptomatic; 29 were symptomatic before treatment, and treated with colchicine (27) or colchicine plus anakinra (1), or colchicine plus canakinumab (2). 4 patients stopped colchicine for the resolution of symptoms and a good control of the disease without treatment. They are still in follow-up. Their parents showed a different clinical presentation, and, in all the cases, the diagnosis was done after the genetic study of their children. In the 16 families, all the parents, except 1, did not show fever. Most of them, however, had anamnestic records of recurrent fever in childhood. Most of the parents had recurrent abdominal pain, arthralgia at the limbs, headache, fatigue. In children, the clinical presentation was typically characterized by recurrent attacks of , with no significant differences with the children of group A. Conclusion: We can speculate that children in group A, carrying R202Q mutation, are patients with SURF (Syndrome of undifferentiated recurrent fever, as proposed by M. Gattorno and coll.). 92% responded to colchicine. The clinical presentation of the parents diverged from A to group B: most of the patients of group A do not refer a family history of recurrent fever and/or symptoms of SURF. Most of the patients of group B have a family history of recurrent fever in siblings and parents, and actual records of abdominal pain, arthralgia at the limbs, headache, fatigue. Disclosure of Interest : None declared
C. Matucci Cerinic 1,2 , G. Viglizzo 3 , R. Caorsi 2,4 , S. Volpi 1,2 , C. Malattia 1,4 , M. Gattorno 2,4 1 DINOGMI, Università degli studi di Genova, 2 Centro malattie autoinfiammatorie e immunodeficienze, 3 UO Dermatologia, 4 Clinica pediatrica e reumatologia, Istituto Giannina Gaslini, Genova, Italy Correspondence: C. Matucci Cerinic Introduction: SAPHO is a heterogeneous autoinflammatory disease, characterized by bone and joint involvement and by a wide variety of dermatologic manifestations, including palmo-plantar pustulosis (PPP), acne, hidradenitis suppurativa (HS), pyoderma gangrenosum (PG), psoriasis, Snedd-Wilkinson disease and Sweet syndrome. The SAPHO treatment still today is a challenge, and no therapeutic guidelines are available in children: NSAIDs are frequently used as first line treatment for CRMO lesions, followed by bisphosphonates, methotrexate and biologic therapies. In the literature there are many case reports but only a few case series, of which the present one is the largest in Europe (29 cases reported in China). Objectives: to report the clinical and radiological features and the response to therapy of a cohort of SAPHO children Methods: the clinical data (serological, imaging and therapy) of 13 SAPHO patients, followed between 2001 and 2021 at the Unit for Autoinflammatory diseases at the Gaslini Hospital were reviewed. Patients were divided into 2 groups according to their cutaneous manifestations in an acne-HS and in a PPP group. Results: 8 patients presented an acne-HS while 5 patients had a PPP. In the former group, 7/8 patients were male, while in the latter group 5/5 patients were females. At disease onset, in the acne-HS group, skin manifestations were the first symptom, while in the PPP group were concomitant or subsequent to bone manifestations. In the PPP group, the cutaneous involvement was mild and responded well to topical treatments, while the bone involvement required a therapy with methotrexate or salazopirin, that induced remission in 4/5 patients. 1 PPP patient showed a refractory bone disease and underwent several biological therapies. The acne-HS skin involvement was characterized as follows: 7 patients presented with severe acneiform eruptions (pustular, nodular, cystic, comedonic and ulcerative lesions), of these, the 3 patients with comedonic acne presented also HS, and 1 PG. In the acne-HS group, the osteoarticular involvement responded well to conventional DMARDS, but the skin manifestations required a therapeutic upgrade on biologics. Etanercept obtained only a partial result in 5 patients, Adalimumab had an optimal response in 6 patients, in 1 obtained a partial response even if requiring an increased dose (80 mg per week) and 1 was swapped to dapsone. Anakinra (3), ustekinumab and secukinumab (2) were not efficacious. The 3 patients with comedonic acne were refractory to all treatments and needed therapy cycling, which achieved a partial remission only. When comparing the 2 groups, sternal and axial involvement were more frequent in the acne-HS group (75% and 87,5% versus 40 and 60% respectively) while clavicular and mandibular involvement were more frequent in the PPP group (60% and 40% vs 25% and 0% respectively). An involvement of the metaphysis of the long bones was present in both groups, however in PPP a major involvement of the tibia and fibula was present (100 and 80% vs 62,5 and 50% respectively) Conclusion: All our patients were characterized by well-known SAPHO clinical features. For what concerns skin involvement, all patients with comedonic acne were refractory to treatments while PPP and nodulo-cystic acne achieved remission. Interestingly, a difference in the bone manifestations was found in the 2 groups. Our observation may suggest that in SAPHO two different phenotypes exist. In fact, skin involvement may have a different response to the treatment, and comedonic acne seems to belong to a separate cluster of patients that poorly respond to treatmN. Ruperto 13 , M. Gattorno 14,15 , I. Kone-Paut 16 1 Center for Autoinflammatory Diseases, IRCCS Istituto Giannina Gaslini, 2 DINOGMI, Università degli studi di Genova, Genova, Italy, 3 Pediatric rheumatology and CEREMAIA, Bicêtre hospital, APHP, Paris, France, 4 Department of Pediatrics, King Faisal Specialist Hospital and Research Center; College of Medicine, Alfaisal University, Riyadh, Saudi Arabia,Foundation Trust, London, United Kingdom, 6 Rheumatology Unit, Department of Medical Sciences, Surgery and Neurosciences, University of Siena, Siena, Italy, 7 Division of Rheumatology, Department of Internal Medicine, Istanbul Faculty of Medicine, Istanbul University, 8 Cerrahpasa Medical School, Istanbul University-Cerrahpasa Turkey, Istanbul, Turkey, 9 Division of Pediatric Rheumatology, Department of Pediatrics and autoinflammation reference center Tuebingen, University Hospital Tuebingen, Tuebingen, Germany, 10 Department of Pediatric Rheumatology, Hacettepe University, Ankara, Turkey, 11 Sorbonne Universités, Department of Internal Medicine and Clinical Immunology, CEREMAIA, AP-HP, Groupe Hospitalier Pitié-Salpêtrière., Paris, France, 12 Rheumatology Research Center, Shariati Hospital, Tehran University of Medical Sciences, Tehran, Iran, Islamic Republic Of, 13 UOSID Centro Trial, IRCCS Istituto Giannina Gaslini , 14 Center for Autoinflammatory Diseases, IRCCS Istituto Giannina Gaslini, 15 Pediatrics and Rheumatology Clinic, IRCCS Istituto Giannina Gaslini, Genova, Italy, 16 Pediatric rheumatology and CEREMAIA, Bicêtre hospital, APHP; University of Paris Saclay, Paris, France Correspondence: C. Matucci-Cerinic Introduction: Behçet disease (BD) is an autoinflammatory disease characterized by a variable vessel vasculitis. In children, the first BD symptoms may start early in life, mimicking other autoinflammatory diseases and making the diagnosis a real challenge in the paediatric population. While many criteria exist for the diagnosis or classification of BD in adults in children only classification criteria exist, the PEDBD criteria (2015), created by international Expert consensus. Objectives: to perform an external validation of the PEDBD criteria and attempt to improve their sensitivity and specificity with respect to other autoinflammatory diseases. Methods: 210 patients were randomly selected from the Eurofever Registry: patients were included if enrolled after 2010 with a diagnosis of BD, PFAPA, FMF, MKD, TRAPS, SURF and undefined autoinflammatory diseases (UND); patients were excluded if they participated in the first PEDBD study. A set of 11 Experts blinded to the original diagnosis, were chosen to evaluate the patients, and reach a consensus defined as > 80% for the following diagnosis: BD, PFAPA, FMF, MKD, TRAPS, SURF, UND. In the 1 st round only clinical and serological data were shown; in the 2 nd round genetic data were added; and in the 3 rd round the other Experts’ votes and comments were shared with all experts. Using the expert consensus as gold standard, the PEDBD, the ISG and the ICBD criteria were applied to BD patients and to the confounding diseases in order to define the sensitivity and specificity. Results: In the 1 st round a consensus was reached in 45/210 (21%) of patients (22/70 BD, 5/35 FMF, 2/26 MKD, 11/40 PFAPA, 2/22 TRAPS, 2/12 SURF, 1/5 UND). While in the 2 nd round a consensus was reached in another 58/163 (35%) of patients, for a total consensus on 103/210 patients (49%): 31/70 BD, 17/35 FMF, 21/26 MKD, 15/40 PFAPA, 14/22 TRAPS, 3/12 SURF, 2/5 UND. The 3 rd round is now ongoing, and the data are not yet available. The next step will consist on the application of the PEDBD, the ISG and the ICBD criteria to the cohort of classified BD, and to the other diseases in order to see their performance on BD and confounding diseases. Conclusion: the classification of Behçet disease showed a wide range of opinions among Experts. The heterogeneity of the disease is a challenge but a more robust classification can at least allow homogeneous groups of patients for research purpouro 1 , E. Bizzi 2 , A. M. Pisacreta 2 , V. Ansuini 1 , C. Carollo 2 , A. Pedroli 2 , F. Casini 3 , F. Savoia 4 , A. Brucato 5 , L. Bernardo 6 1 Pediatric Rheumatology Unit, 2 Department of Medicin, Rheumatology Unit, ASST Fatebenefratelli-Sacco, 3 Department of pediatrics, Vittore Buzzi Children’s Hospital, Milan, 4 Childhood Cancer registra of campania, Santobono-Pausilipon Children’s Hospital, Naples, 5 Department of Medicin, 6 Department of Pediatrics, ASST Fatebenefratelli Sacco, Milan, Italy Correspondence: A. Mauro Introduction: Pericarditis is a clinical syndrome characterized by inflammation of the pericardium with or without pericardial effusion. The course can be acute, recurrent or chronic. Recurrent pericarditis (PR) is defined as the recurrence of pericarditis after the first attack, with a symptom-free interval greater than 4-6 weeks. Etiopathogenesis of RP is idiopathic in 70% of pediatric cases,s. PR can occur isolated or in the context of a polyserositis with characteristics of systemic inflammation (fever, leukocytosis, pleural effusion and more rarely peritoneal). Objectives: The aim of the study is to assess the differences in laboratory parameters between two groups of patient affected respectively by systemic recurrent pericarditis (PRs) and isolated recurrent pericarditis (PRi). Methods: Patients diagnosed with PRs and PRi aged <18 years were included. Inclusion criterion was the presence of fever and pleural effusion, in patients with PRs. Demographics, clinical characteristics, and blood tests were collected and compared. Results: We prospectively enrolled 42 patients (14 RPs and 28 RPi). The distribution by gender and age was similar in the two groups (M / F 1: 1; age of onset: 15 years (IQR 13-17). The PRs compared to the PRi had significantly higher levels of leukocytes, neutrophils and CRP (p < 0.01). In addition, patients with PRs performed pericardiocentesis more frequently (p <0.01) Conclusion: Our study showed that in patients with PRs mechanisms of autoinflammation, probably mediated by IL1, which manifest themselves with elevated levels of CRP and neutrophilia, seem to play a fundamental pathogenetic role, compared to the PRi forms. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
A. Mauro 1 , E. Bizzi 2 , L. Trotta 2 , M. Pancrazi 2 , F. Casarin 2 , V. Ansuini 2 , S. lassainato 3 , A. Brucato 4 , L. Bernardo 5 1 Departments of Pediatrics, Rheumatology Unit, 2 Departments of Medicin, Rheumatology Unit, ASST Fatebenefratelli-Sacco, 3 Departments of Pediatrics, Vittore Buzzi Children’s Hospital, 4 Department of Medicin, 5 Department of Pediatrics, ASST Fatebenefratelli sacco, Milan, Italy Correspondence: A. Mauro Introduction: Chronic pericardial effusion of unknown etiology is a very rare event in pediatric age. The autoinflammatory etiology of acute and recurrent pericarditis often causes acute pericarditis and simultaneous pericardial effusion as an inflammatory epiphenomenon. Idiopathic pericardial effusions not associated with acute pericarditis in children is not reported in literature. Objectives: We describe a case of pediatric patients with an Idiopathic, chronic and large pericardial effusion Methods: Male patient, 9 years old, hospitalized for persistent fever and pharyngotonsillitis. On chest x-ray he presented cardiomegaly then confirmed by echocardiography which showed abundant circumferential pericardial effusion, with heart oscillating in the pericardial cavity, without signs of cardiac tamponade and normal inferior vena cava (VCI). He had no symptoms due to pericardial effusion. Results: Pericardiocentesis was carried out with the withdrawal of 650 ml of citrine liquid. In suspicion of acute pericarditis, prednisone therapy (25 mg / day) was initiated without any benefit. Blood tests showed: PCR 147 mg / L, GB 20,000 / mm3, anti-Mycoplasma IgM positive. Throat swab for SBEGA, autoimmunity, thyroid functions, troponin and Mantoux intradermal reaction were negative. Discharged in good general conditions, he began therapy with indomethacin (50 mg / day). After 7 days, for the finding of pericardial effusion of 1 cm, he combined therapy with Prednisone (37.5 mg / day) with little benefit on the extent of the effusion. For this reason he added Colchicine (1 mg / day), and made a slow decalage of the steroid. Over the years he performed numerous echocardiographic exam that showed a slow increase in effusion of about 0.5-1 cm per year with non-dilated and normocollassing VCI, well tolerated. At the last visit, about 7 years after onset, the patient reported full well-being. On physical examination, PA 100/70 mmHg, HR 60 bpm. Non turgor of the jugular, cardio-thoracic and abdominal examination were negative. Not organomegaly. Echocardiography showed slightly dilated, normocollassing VCI; circumferential massive pericardial effusion, with the heart oscillating in the pericardial cavity. At the moment he does not perform any therapy. Conclusion: It was not possible to clarify whether patient presented an effusion secondary to acute pericarditis, possibly autoinflammatory and / or due to mycoplasma infection, or an occasional finding of idiopathic pericardial effusion in the course of acute pharyngotonsillitis. Generally, acute pericarditis and related effusion respond to anti-inflammatory therapy, while occasional pericardial effusion does not respond to anti-inflammatory therapy and is often well tolerated. To our knowledge, this is the first case of idiopathic, asymptomatic, pericardial effusions not associated with acute pericarditis in pediatric ageA. Mauro 1 , S. Lassainato 2 , F. Casini 2 , E. Chittano Congedo 2 , V. Ansuini 1 , C. Carollo 3 , A. Pedroli 3 , F. Casarin 3 , A. Brucato 3 , L. Bernardo 4 1 Department of Pediatrics, Rheumatology Unit, ASST Fatebenefratelli-Sacco, 2 Department of Pediatrics, , Vittore Buzzi Childrem Hospital, 3 Department of Medicin, 4 Department of Pediatrics, ASST Fatebenefratelli-Sacco, Milan, Italy Correspondence: A. Mauro Introduction: Recurrent pericarditis (RPVery often RP is idiopathic in pediatric age and more rarely, it can be secondary to autoinflammatory, autoimmune diseases, tumors, metabolic diseases or infection. Objectives: We describe a case of Bardet-Biedl syndrome associate to RP Methods: A. is a 9-year-old boy with Bardet-Biedl syndrome characterized by retinitis pigmentosa, polydactyly of the left foot, kidney cysts and learning difficulties. He presented the first episode of pericarditis at the age of 12, successfully treated with colchicine. He also presented three further recurrences of pericarditis at a distance of 1 year each other. During the last episode, due to the presence of large pericardial effusion, he performed pericardiocentesis with evacuation of 900 ml of serum-hematic liquid. Culture and cytological examinations of the pericardial fluid were negative. The patient came to our attention at age 16 for retrosternal pain. At the visit he showed BP 130/90 mmHg, cardio-thoracic and abdominal physical examination negative. He performed blood tests (complete blood count, ESR, PCR, troponin, liver and kidney function, electrolytes) which were in normal range. Echocardiography showed the presence of a minimal effusion of the free pericardium wall (5 mm) with the presence of fibrin. Colchicine therapy (1 mg / day) was started. Results: After two months he returned to our department with chest pain which worsened with movement. On physical examination the patient was in fairly good general conditions, BP 150/90 mmHg, HR 110 beats/minute. Negative cardio-thoracic and abdominal physical examination. Blood tests were carried out and showed an increase of inflammation index (CRP 170 mg / L, ESR 80 mmHg) and white blood cells count (WBC) 10750 / mm3, Neutrophils 7920 / mm3. Anti-nucleus antibodies, ENA antibodies, anti-phospholipid antibodies and Mantoux test were performed and all were negative. Chest X-ray and electrocardiogram were negative and Echocardiography revealed the presence of pericardial effusion (20 mm). He started therapy with Indomethacin (50 mg / day), Prednisone (25 mg / day), Anakinra (100 mg / day). Progressively, the patient showed an improvement of the clinical, laboratory and instrumental conditions and, gradually reduced steroid treatment. Conclusion: To our Knowledge, this is the first case reported the association between Bardet-Biedl Syndrome and recurrent pericarditis. Given the brilliant response to therapy with Anakinra, an autoinflammatory etiology has been hypothesized for pericarditis. Are Pericarditis there a new manifestation of Bardet-Biedl syndrome or a form of autoinflammatory pericarditis associated with it? Patient Consent: Yes, I received consent Disclosure of Interest : None declared
M. Mohanna 1 , E. Roberts 2 , L. Whitty 1 , J. Gritzfeld 1 , A. Theos 3 , P. Ferguson 4 , J. Preston 1 , C. Hedrich 1,2 1 University of Liverpool, 2 Alder Hey Children’s Hospital, Liverpool, United Kingdom, 3 Georgetown University, Washington, DC, 4 University of Iowa Stead Family Children’s Hospital, Iowa City, United States Correspondence: M. Mohanna Introduction: Chronic non-bacterial osteomyelitis (CNO) is a rare autoinflammatory bone disease that mainly affects children and young people. It is characterised by bone pain and reduced function (1). The pathophysiology of CNO is incompletely understood and there are no licensed treatments, diagnostic criteria, or outcome measures (2). Objectives: The aim of this study was to explore and compare the experience of patients, their families, and clinicians with CNO, its diagnosis and treatment. Furthermore, opinions on research priorities were explored. Methods: Questionnaires were designed in 2020 to capture patient (20 items) and clinician (24 items) experience with the diagnosis and treatment of CNO and to explore research priorities. Questionnaires were circulated internationally in 2021 (Google Form) among patients and families through the CNO Facebook group, shared by other patient groups on twitter including fight_CRMO, and mailing list from the CRMO Foundation, and among clinicians historically involved in CNO research and participating in international working groups on diagnostic and therapeutic approaches. Results: We received 93 responses to the patient questionnaire ; 23% were from young people affected by CNO, and 77% were from parents or carers on behalf of the young person they care for. There were 21 responses (27 contacted) from clinicians. The median time taken for the patient group to receive a diagnosis was 6 months (mean: 14 months). This matched the clinician perspective that estimated a diagnosis after 6 months. 56% of patients/families reported to have received alternative diagnoses before they received a CNO or CRMO diagnosis, most commonly infection (29%). The most common medications taken for CNO amongst the patient group were NSAIDs (34%), followed by bisphosphonates (28%). The most frequently prescribed by clinicians were NSAIDs, followed by bisphosphonates. Research priorities showed strong alignment between patients and clinicians. Pathophysiology was the most commonly mentioned research priority in both patient (45%) and clinician (34%) groups, followed by medication trials (18%, 33% respectively). Based on this survey, four topics were identified for further discussion between clinicians and patients/families at the inaugural “ International conference on CNO and autoinflammatory bone disease ”, held in Liverpool, UK in May 2022: pathophysiology, nomenclature, clinical trials, and psychosocial aspects and psychological support. Conclusion: This survey compared clinician and patient/family experience with CNO to identify unmet need and research priorities. Results will inform roundtable discussions to guide future research and provide ongoing support and information for patients and their families. References: 1.Vol. 4, Journal of Translational Autoimmunity. 2021. 2. Hedrich CM, Morbach H, Reiser C, Girschick HJ. New Insights into Adult and Paediatric Chronic Non-bacterial Osteomyelitis CNO. Curr Rheumatol Rep. 2020. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
Nashawi 1 , A. Sabo 2 , F. Blankenburg 2 , A. Heinkele 3 , P. Müller-Abt 4 , T. Hospach 3 1 Pediatric Rheumatology, King Abdulaziz University, Jeddah, Saudi Arabia, 2 Klinikum Stuttgart, Stuttgart, Germany, 3 Pediatric Rheumatology, 4 Pediatric Radiology, Klinikum Stuttgart, Stuttgart, Germany Correspondence: M. Nashawi Introduction: Growing pains (GP) are frequent in children. They usually encompass mostly bilateral, nocturnal pains, with changing localization, especially in the lower extremities, which respond well to symptomatic therapy and resolve until the next day. It is however a diagnosis of exclusion, and selected cases warrant further investigation. Objectives: Our objective is to point out that growing pain is a diagnosis of exclusion by presenting a case that had been falsely diagnosed as a growing pain in the beginning. Methods: we present a case report. Results: We present a 3-yo girl with recurrent pain of the lower extremities which was initially diagnosed with growing pains. Upon further investigation, we found severe bilateral sacroiliitis. Considering the mediterranean origin of the family we sought to investigate the MEFV Gene and found a homozygous single nucleotide mutation strongly associated with familial mediterranean fever (FMF). Conclusion: Especially in the pediatric population, sacroiliitis (SI) is a rare and uncommon localization for FMF-associated arthritis. In this case, the patient was very young and nocturnal pains were the only manifestation of this disorder. In the population with a susceptible genetic background, we recommend further investigation of limb and lower back pains, to rule out FMF-associated arthritis. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
M. F. Natale 1 , C. Celani 2 , S. Federici 2 , F. De Benedetti 2 , A. Insalaco 2 1 Rheumatology, Bambino Gesu Children’s Hospital, 2 Rheumatology, Bambino Gesù Children’s Hospital, Rome, Italy Correspondence: M. F. Natale Introduction: Deficiency of adenosine deaminase type 2 (DADA2) is an autosomal recessive disease caused by loss-of-function mutations of the ADA2/CECR1 gene which encodes adenosine deaminase type 2 (ADA2). The disease is associated with a highly variable clinical presentation, such as vasculitis of small- and medium-sized arteries, systemic inflammation, skin involvement, hematologic manifestations, immunodysregulation and neurological ischemic or hemorrhagic stroke. It is important to arrive at an early diagnosis in order to start an effective therapy to control clinical manifestations and reduce risk of complications. Objectives: We described a 16 years old male patient with a genetically confirmed DADA2 characterized by a complex clinical phenotype. Methods: Next generation sequencing Results: The patient was first seen at our center at age 16 with a clinical history, started about 1 year earlier, characterized by intermittent fever (mainly serotin), persistent increase of inflammatory markers, splenomegaly (14cm) and diffuse lymph-node enlargement. At home, empiric antibiotic and glucocorticoid therapy was attempted without benefit. In family history, an older brother died when he was 12 years old with an undefined hyperinflammatory syndrome. In our unit we performed extended microbiological tests resulted negative. Circulating autoantibodies were absent. Radiological exams confirmed splenomegaly and enlarged lymph-nodes in supra and subdiaphragmatic areas with 18F-FDG enhancement at PET-TC. In the suspicion of lymphoproliferative disease a lymphonode biopsy was performed (resulting in reactive lymphadenopathy) and bone marrow aspiration did not reveal atypic cells. Immunological exams revealed an immunodisregulation with a low level of circulating immunoglobulins (IgG 861 mg / dl; IgA 54 mg / dl, IgM 30 mg / dl) and a reduction of total count of memory B cells. Given the complex clinical picture and the family history, a large NGS diagnostic panel for autoinflammatory diseases and immunodeficiencies was performed revealing the homozygous c.1358A>G mutation in ADA2 (CECR1) gene described in literature as pathogenetic. We performed a brain MRI to exclude neurological involvement and then started biological therapy with Anti-TNF alfa (Etanercept) with a rapid and brilliant clinical and instrumental response. Conclusion: This case emphasize the importance to consider a diagnosis of DADA2 in complex clinical cases characterized by systemic inflammation and/or immunedysregulation Patient Consent: Yes, I received consent Disclosure of Interest : None declared
Z. Nesterenko 1 , A. Kozlova 1 , V. Burlakov 1 , A. Laberko 1 , Y. Rodina 1 , E. Viktorova 1 , E. Deordieva 1 , A. Moiseeva 1 , A. Mukhina 1 , N. Kuzmenko 1 , D. Bogdanova 1 , A. Khoreva 1 , D. Yukhacheva 1 , V. Bludova 1 , N. Kan 1 , D. Balashov 2 , A. Shcherbina 1 1 Immunology, 2 Hematopoietic Stem Cell Transplantation , Dmitry Rogachev National Medical Research Center Of Pediatric Hematology, Oncology and Immunology, Moscow, Russian Federation Correspondence: Z. Nesterenko Introduction: AIDs are a group of diseases caused by dysregulation of mechanisms controlling innate immune response and characterized by systemic inflammation and a plethora of manifestations, including hematological symptoms. Objectives: To analyze hematological manifestations and their response to treatment in a group of monogenic AIDs. Methods: We retrospectively analyzed data of 31 patients (12- females, 19- males) in whom persistent hematological symptoms were part of their AID phenotype. Results: The forms of AIDs with hematological symptoms included: PSTPIP1-associated syndrome (PAID) - 8, adenosine deaminase 2 deficiency (DADA2) - 8, type I Interferonopathies - 8, mevalonate kinase deficiency (MKD) - 4, A20 haploinsufficiency (HA20) - 1, POMP-related autoinflammation and immune dysregulation (PRAID) – 1, NLRC4-associated autoinflammatory disorder – 1 (AIFEC). Median age of onset of any disease symptoms was 2 months (range 0–16 years). Median age of onset of hematologic manifestations was 2.5 years (range 0–33 years). Hematological manifestations as the first symptom of AID were seen in 23/31 patients. Median age at diagnosis was 8 years (range 0.5-33), with the mean diagnostic delay of 4 years (range 0.5-32). 9/31 patients had three-lineage cytopenia, 12/31 – two-lineage cytopenia and 5/31 - single lineage cytopenia. Also myelodysplastic syndrome was seen in 1/31 and hemophagocytic lymphohistiocytosis in 4/31. Jak-inhibitors monotherapy were effective in 3/11 cases with interferonopathies, in 1/11- with DADA2, and in 1/11 - with HA20. Combination of an IL-6 inhibitor and JAK-inhibitor had effect in 1 patient with DADA2 (Tabl.1). Anti-IL1 therapy was successful in 3 patients with MKD and 1 patient with AIFEC. Immunosuppressive therapy (steroids, high-dose IVIG, mycophenolate mofetil, cyclosporine A, methotrexat) were not effective in any of the patients treated. Hematopoietic stem cell transplantation (HSCT) was performed in 5 patients (3 – with PAID, 2- with MKD), the median follow-up time after HSCT is 2.16 years (1,08–4,58). Four patients are alive, with predominantly donor chimerism, good immune function and complete absence of the disease symptoms. Conclusion: The hematological manifestation of AIDs represent a therapeutic challenge. HSCT might be a viable treatment option. Disclosure of Interest : None declared
P. T. Oommen 1 , T. Kallinich 2 , J. Rech 3 , N. Blank 4 , J. Weber-Arden 5 , J. B. Kuemmerle-Deschner 6 12 Department of Pediatrics, Division of Pulmonology, Immunology and Critical Care Medicine, Charité University Medicine Berlin, Berlin, 4 Rheumatology, University Hospital Heidelberg, Heidelberg, 5 NOVARTIS, Nuernberg, J. Weber-Arden Introduction: Hyper-IgD Syndrome/Mevalonate Kinase Deficiency (HIDS/MKD) is a rare autoinflammatory disease caused by a defect in the gene encoding mevalonate kinase. Treatment with the interleukin-1β inhibitor canakinumab (CAN), which since 2017 has been approved for use in HIDS/MKD patients, resulted in rapid remission in most patients, both in clinical trials and in practice. Objectives: The present study investigates the long-term efficacy and safety of CAN under routine clinical conditions in pediatric (age ≥2 years) and adult HIDS/MKD patients. Methods: RELIANCE is a prospective, non-interventional, multicenter observational study based in Germany with a follow-up period of up to 7 years. Patients with clinically confirmed HIDS/MKD diagnosis who routinely receive CAN will be enrolled in the study and followed at 6-month intervals. Results: The present interim analysis includes baseline data from 8 HIDS/MKD patients enrolled in the study through December 2021 and preliminary 18-month data. Of these patients, N=5 (63%) were female and the median age at study entry was 8 years (2-39 years). The median duration of prior CAN treatment at study entry was 1.5 years (0-5 years). Standard, low-dose, and high-dose CAN treatment was evenly distributed at each interval. Preliminary results indicate stable remission and disease control (as assessed by physicians and patients as well as laboratory parameters; Table 1). Overall, adverse drug reactions occurred in N=4 patients, but none were considered serious. During the study, N=4 patients underwent vaccinations, with one vaccination reaction classified as non-therapeutic occurring after a DTP (diphtheria, tetanus, pertussis) vaccination. According to mutational analysis, pathogenic or likely pathogenic mutations (some homozygous) were present in N=7 patients. In no patient disease activity was considered to be severe. Conclusion: Baseline characteristics and preliminary data from HIDS/MKD patients in the RELIANCE study suggest good disease control. In addition, interim analysis at 18 months revealed no unexpected safety concerns. In relation to vaccinations, vaccination reactions unrelated to CAN therapy occurred in only one case. Disclosure of Interest : P. T. Oommen Grant / Research Support with: Novartis, T. Kallinich Speaker Bureau with: RocheJ. M. Garcia Aznar 1 , N. Palmou Fontana 2 , N. P. Gandeaga 3 , J. G. Ocejo-Viñals 4 , M. A. Gonzalez-gay 2 1 Inmunology, Health in code, A Coruña, 2 Rheumatology, 3 IGENETISTA, 4 Inmunology, Universitary Marques De Valdecilla Hospital, Santander, Spain Correspondence: N. Palmou Fontana Introduction: The monogenic autoinflammatory disease accounts for at least 10% of cases with the suspected autoinflammatory syndrome. inflammasome complex, overproduction of IL-1β and IL-18. Different inflammasomes have been described in which genetic defects have been identified in their protein components, resulting in the development of Pyrin-associated inflammasopathy (MEFV), Cryopyrin (NLRP3), NLRP1, NLRC4, PSTPIP1, WDR1, LPIN2, IL1RN, and IL36RN. Another group is the interferonopathies, which include three types depending on the class of interferon involved in the dysregulation: IFNα, IFNβ, IFNγ, and IFNλ. The genes involved have JAK1/2, TYK2, nucleic acid degradation-associated genes such as TREX1, IFIH1, DNASE1/2/L3, and proteasome genes (POMP, PSMA3, PSMB8/9/10, PSMG2), among others. Genes associated with deregulation of the NF-kB/TNF pathway (NF-kBopathies) also result in autoinflammatory disorders characterized by aberrant activation of NF-kB (NFkbopathies), including TNFAIP3, TNFRSF1A, OTULIN, LUBAC complex genes, NOD2, ADA2, or RIPK1 among others. Additionally, other genetic factors not included in any of the above categories can affect both intrinsic and adaptive immunity genes with an autoinflammatory component (COPA, PLCG2, LRBA, C2). Objectives: Defining and identifying the phenotypic spectrum associated with these diseases is challenging for clinical practice. The clinical manifestations, although heterogeneous, allow defining criteria for the categorization of autoinflammatory disease. Methods: Ninety-nine patients with suspected autoinflammatory diseases were included in the cohort, with a panel containing more than 270 genes. According to the genetic result, the subjects were categorized into five groups: inflammasome disorders, interferon disorders, NFkB/TNF alteration, unfavorable genetics, and other pathologies. A group of patients phenotype divided into eight categories. Results: The study results revealed that 48% of the studies performed with suspected autoinflammatory disease presented relevant genetic findings. The genetic variants identified were distributed in the following genes from higher to lower frequency: MEFV (21%), NOD2 (9%), PSTPIP1 (9%), NLRP12 (9%), TNFRSF1A (6%), TNFAIP3 (6% ), NLRC4 (4%), JAK1 (4%), NLRP3 (4%), TNFRSF1A (2%), ADA2 (2%), OTULIN (2%), TNFRSF11A (2%), BANK1 (2%), C2 (2%), PLCG2 (2%), PSMB8 (2%), LRBA (2%), NLRP1 (2%), POMP (2%), RNF31 (2%). 23% contained genes associated with inflammasome disorders, 4% interferon disorders, 16% NF-kb disorders, and 3% genes from other metabolic pathways. The mean age of disease onset was lower in patients with NF-kb-pathway disorder. Overall was lower in all categories than the studies that did not have relevant genetic variants. Among patients with the autoinflammatory genetic disease, febrile and gastrointestinal manifestations were more frequent in 0-11 years. In contrast, rash and joint manifestations appeared more frequently in patients with 36 years old Conclusion: The genetic study turned out to be a helpful tool for identifying the altered inflammatory pathway, which is an essential element for selecting treatment. Knowledge of the clinical presentation of each age and group of diseases adds value to selecting patients with the suspected autoinflammatory disease. Disclosure of Interest : None declared
D. B. Pandya, M. Agarwal, S. Sawhney Pediatric Rheumatology Department, Sir Gangaram Hospital, New Delhi , India Correspondence: D. B. Pandya Introduction: Deficiency of ADA2(DADA2) is the first molecularly described monogenic vasculitis syndrome which is caused by autosommal recessive loss of function mutations in the ADA2 gene(CERC1). Deficiency of ADA2 has been linked to an imbalance in differentiation of monocytes towards proinflammatory M1 macrophages, neutrophil extracellular traps(NETosis) dysregulation and endothelial dysfunction. DADA2 can present with Vasculitis or Vasculopathy , Bone marrow failure , Lymphoproliferation , Immunodeficiency and Type 1 interferonopathy. 1 Objectives: We aim to unveal Characteristics of DADA2 deficiency in five children at Our Center. Methods: This is a retrospective analysis of five children who were treated at our unit between August 2013 & August 2021 for severe systemic polyarteritis nodosa. In view of resistent disease in these children , ADA2 gene analysis was ordered and returned Positive. Results: Conclusion: Early age of onset, female sex, severe disease activity mainly affecting gastrointestinal system , recurrent and resistant course in polyarteritis nodosa may suggest monogenic vasculitis syndrome - DADA2 deficiency. Trial registration identifying number: 1. J Clin Immunol. 2018, Published online 2018 Jun 27. doi: 10.1007/s10875-018-0525-8 Deficiency of Adenosine Deaminase 2 (DADA2): Updates on the Phenotype, Genetics, Pathogenesis, and Treatment Isabelle Meyts and Ivona AksentijevichPatro 1 , A. Abraham 1 , J. Raghuram 2,3 , A. P. Rao 1,3 1 Pediatric Rheumatology, Manipal Hospital, 2 Pediatric Rheumatology, Aster Whitefield Hospital, 3 Pediatric Rheumatology, Indira Gandhi Institute of Child Health, Bengaluru, India Correspondence: D. Patro Introduction: First described in 1972 by Giedion et al, Chronic nonbacterial osteomyelitis (CNO) is a rare autoinflammatory bone disorder that presents as recurrent bone pain in one or multiple sites. Objectives: 1. To understand the clinical profile of CNO patients from Indian subcontinent 2. To elucidate the clinical outcomes of the children affected with CNO. Methods: We retrospective reviewed 38 cases diagnosed as CNO as per Bristol Diagnostic criteria in the Pediatric rheumatology clinic in Manipal hospital, Bengaluru, India after obtaining ethical approval. Results: The findings are summarized in the table below. Of the 164 multifocal lesions detected by WB- MRI in 33 cases, tibial lesions (26%) were the commonest. LPIN2 deficiency was also reported in two cases. All cases were initiated with naproxen. 27 patients were started on methotrexate of which 2 cases each responded to Bisphosphonates and TNF inhibitor respectively. 4 out of 25 cases required additional TNF inhibitor after pamidronate and methotrexate therapy. 2 cases responded well to pamidronate infusion alone. 8 children had frequent flares. 14 children are in remission on drugs and 12 children are in remission off drugs. 2 cases are lost to follow up. Conclusion: With increasing knowledge and timely referral to a rheumatology center may bridge the gap to delay diagnosis. WB- MRI can detect bone lesions and prevents unnecessary invasive procedures. In a resource-limited setting, methotrexate and pamidronate have helped in achieving remission. TNF inhibitors are helpful in resistant cases with good response. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
A.-M. Ramazan 1 , A. Apostol 2 , S. Mehdi 3 1 Rheumatology Department, 2 Paediatric Onco-Hematology , 3 Dermatology Department, Emergency County Clinical Hospital “Sf Apostol Andrei”, Constanta, Romania Correspondence: A.-M. Ramazan Introduction: Pyoderma gangrenosum (PG) is a rare neutrophilic dermatosis that presents with rapidly developing, painful skin ulcers hallmarked by undermined borders and peripheral erythema. The cause of PG is not well understood, but PG is generally considered an autoinflammatory disorder. Children hospitalized in US for PG were found to have higher odds of thyroid disease, inflammatory bowel disease, hematologic malignancy, and other autoimmune disorders. Objectives: To report a rare autoinflammatory and autoimmune condition with therapeutical difficulties Methods: A 13-year-old girl without any past medical history was sent to our clinic for consultation and treatment of refractory pyoderma gangrenosum (PG) of bilateral lower extremities. Results: Our patient had incomplete therapeutical response with high doses of prednisone indicated by our dermatologist. We tried to add Cyclosporine (CsA), accordingly to the first line of therapy in PG, followed by a significant flare of dermatological and biliary involvements (only hepatic cytolysis), as our best option remained Azathioprine with progressive increasing doses. The abnormal liver tests were obviously under CsA when they had a peak, a clear sign of worsening and an autoimmune involvement. Conclusion: PG could be more than a dermatological disease and the rheumatologist must look for the underlying autoimmune disease, a required diagnosis for the adjustment of therapy. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
C. Reiser 1 , J. Klotsche 2,3 , T. Hospach 4 , G. Heubner 5 , D. Windschall 6,7 , R. Trauzeddel 8 , N. Grösch 2 , M. Niewerth 2 , K. Minden 2,3 , H. Girschick 9 1 Department of Pediatrics, LKH Bregenz, Bregenz, Austria, 2 Deutsches Rheuma-Forschungszentrum Berlin, ein Institut der Leibniz-Gemeinschaft, 3 Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, 4 Department of Pediatrics, Olgahospital, Klinikum Stuttgart, Stuttgart, 5 Städtisches Klinikum Dresden-Neustadt, Klinik für Kinder- und Jugendmedizin, Dresden, 6 Clinic for Pediatric and adolescent Rheumatology, St. Josef-Stift, Sendenhorst, 7 University of Halle –Wittenberg, Halle, 8 Fachambulanz Kinderrheumatologie, Helios Klinikum Berlin-Buch, Klinik für Kinder- und Jugendmedizin, 9 Vivantes Klinikum Friedrichshain, Children’s Hospital, Berlin, Germany Correspondence: C. Reiser Introduction: Chronic non-bacterial osteomyelitis (CNO) is an autoinflammatory bone disease of unknown origin. A 5 year longitudinal analysis of patients in the German National Pediatric Rheumatologic Database (NPRD) with CNO was performed. Objectives: To document the long term course and to assess risk factors for severe disease and items defining remission of patients with CNO. Methods: From 2015-2020 all patients with a confirmed diagnosis of CNO, who were registered in the NPRD during their first year of disease course and at least one follow-up visit, were included in this analysis. Results: Additional to the previously reported cross-sectional analysis of almost 800 CNO patients, a data set of 314 eligible patients including up to 5 years of follow-up visits was analysed. Selected patients’ characteristics are as follows: 64% of the patients were female; the medium age was 11.2 years. 10.8% of patients were HLA-B27 positive. The mean number of clinical lesions was 2.2; using whole-body MRI imaging, the mean number was 2.9 lesions per patient at diagnosis. Co-manifestation of the skin was present in 17%. MRI-scans (whole-body) were performed in 255 patients, and most of them (86%) showed a positive TIRM/STIR signal. The analysis of the location of the lesions showed that the distribution changed over the years. Whereas in the first year of disease course sites like vertebrae or mandibula were inflamed (8% and 2% of all affected sites), these sites completely resolved over time, while other sites like pelvis or tibia (18%/20%) remained affected after 5 years. Initially, patients were mainly treated with non-steroidal anti-rheumatic drugs (NSAIDs). During disease course the proportion of patients treated with conventional or biological disease modifying anti-rheumatic drugs (DMARDs) raised during these five years from 37% to 54%, or 5% to 12.5% respectively. Inflammation of pelvis or femur at baseline posed an elevated risk for long term severe disease (OR 1.51/1.61, each p=0.0001). Other risk factors for severe disease (defined as physician reported disease activity (PRDA) ≥4 (Visual Analogue Scale 0-10)) were raising numbers of lesions, elevated ESR (erythrocyte sedimentation rate) and multifocal disease (defined as more than one lesion). The risk for arthritis increased with the initially higher number of lesions. Items defining remission best were PRDA <1, patient reported pain score and number of radiologically defined inflammatory bone lesions. The composite Childhood health assessment score (C-HAQ) and patient reported overall well-being did not correlate with inactive disease. Conclusion: The NPRD long-term cohort documents a large number of children and adolescents with CNO. Most of the patients were treated effectively with non-steroidal anti-inflammatory drugs, second-line treatment are disease-modifying agents, steroids or bisphosphonates. An improvement of patient-, physician- and imaging-defined disease activity measures was documented, suggesting CNO generally as benigne disease with a modest number of complicated disease course. Remission defining items and risk factors for severe disease course were defined. Patient Consent: Yes, I received consent Disclosure of Interest : C. Reiser Grant / Research Support with: Pfizer, Speaker Bureau with: Novartis, J. Klotsche: None declared, T. Hospach: None declared, G. Heubner: None declared, D. Windschall: None declared, R. Trauzeddel: None declared, N. Grösch: None declared, M. Niewerth: None declared, K. Minden Speaker Bureau with: Pfizer, Novartis, and Medac, H. Girschick: None declared
A. Remy 1 , W. Abou Chahla 2 , G. Boursier 3 , M. Le Coniac 4 , B. Catteau 5 , H. Reumaux 6 1 General pediatrics, 2 Hematology, CHU Lille, Lille, 3 Rare and Autoinflammatory diseases laboratory, Molecular Genetics and cytogenomics , CHU de Montpellier, Montpellier, 4 General pediatrics, CH Valenciennes, Valenciennes, 5 Dermatology, 6 Rheumatology, CHU Lille, Lille, France Correspondence: A. Remy Introduction: Auto-inflammatory diseases have a broad range of symptoms and thus are difficult to diagnose. Objectives: To underline the significance of multispecialty collaboration in the diagnosis of rare inflammatory disease. To describe the phenotype of a patient with undescribed mutations of WDR1 gene. Methods: We report a case of a child where multispecialty collaboration led to a diagnosis of Periodic Fever Immunodeficiency and Thrombocytopenia (PFIT). Results: The girl was born to unrelated Caucasian parents. From infancy, she presented with recurrent purulent conjunctivitis and unexplained severe mouth ulcers. The case was found atypical to her general pediatrician that sought the help of a dermatologist. Indeed, on top of mucous involvement, the patient had recurrent pustulosis on different parts of her body. Rheumatologist help was also sought because of recurrent stereotyped attacks, with or without fever, associated to high blood inflammation in and in between outbreaks (Orosomucoide: 5 g/L). Joints have never been involved and the patient had no hepatomegaly. Immunohematologist joined the discussion when the patient presented with mild diarrhea. The hypothesis of an underlying primary immunodeficiency was confirmed with lymphocyte immunophenotyping revealing B cell lymphopenia (40/mm 3 CD19+ cells) and inadequate postvaccination antitetanus immune response, along with history of recurrent infections. The hematologist point of view also gave a significant breakthrough: the patient had relative thrombopenia with lower platelets than expected regarding inflammation (157-222 × 10 9 /L). When pediatrician, dermatologist, hematologist, rheumatologist met around this case, all agree to say that the atypical symptoms were suggestive of an autoinflammatory disease. Behcet disease was unlikely due to uncommon skin lesions. PFIT was hypothesized and confirmed by molecular analysis revealing compound heterozygosity of WDR1 gene for 2 likely pathogenic variants: NM_017491.5:c.1264G>A p.(Val422Met) and c.1790C>T p.(Ala597Val). Conclusion: PFIT is caused by mutations in the WDR1 gene leading to actin accumulation, pyrin activation and IL-18 release.(1) Impaired cytosqueleton homeostasis impacts hematopoietic cells especially leukocytes (resulting in immunodeficiency) and platelets (resulting in microthrombocytopenia). PFIT is a new entity first described by Standing et al in 2017.(2) Our patient has a milder phenotype than the previously described patients with no pathological scarring or microstomia to date. She had a good clinical response to colchicine, a cytosqueleton targeted therapy that has shown partial efficacy in PFIT. Should the treatment be unsuccessful, IL-18 targeted blockade might be effective, if available. To conclude, routine blood tests and patient history as seen by a multidisciplinary team prevail extended genetics. Diagnosis and understanding of rare auto-inflammatory diseases such as PFIT require a collaboration between specialists. With respect of precision medicine, the functional consequences of those newly described WDR1 mutations need to be clarified to optimize targeted therapy. REFERENCES: (1) Pfajfer L, Mair NK, Jiménez-Heredia R, et al. Mutations affecting the actin regulator WD repeat-containing protein 1 lead to aberrant lymphoid immunity. J Allergy Clin Immunol . 2018;142(5):1589-1604.e11. (2) Standing AS, Malinova D, Hong Y, et al. Autoinflammatory periodic fever, immunodeficiency, and thrombocytopenia (PFIT) caused by mutation in actin-regulatory gene WDR1. J Exp Med . 2017;214(1):59-71. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
J. Riedl 1 , C. Eide 2 , J. Tolar 1 , A. Kwiatkowski 3 , J. Heier 3 , A. Almeida de Jesus 4 , R. Goldbach-Mansky 4 , B. Binstadt 1 1 Medical School, Pediatrics, 2 Medical School, University of Minnesota, Minneapolis, 3 Cell Biology, University of Pittsburgh, Pittsburgh, 4 Translational Autoinflammatory Diseases Section, National Institutes of Health (NIH), Bethesda, United States Correspondence: J. Riedl Introduction: The interferonopathies are a group of monogenic disorders defined by persistent type 1 interferon (IFNα,β,k) signaling and upregulation of interferon-stimulated gene (ISG) expression. They are highly heterogeneous, and approximately 20 genes have been implicated in driving ISG expression and pathogenesis in these autoinflammatory disorders. The full spectrum of clinical presentation and mechanisms of disease are not fully described, and treatment remains challenging. Objectives: We identified an 11-year-old male patient with a high IFN score in skin (1000x normal) and peripheral blood (200x normal). The patient has severe inflammatory skin lesions, conjunctivitis, mucositis and joint contractures: symptoms akin to described monogenic interferonopathies Methods: Whole exome sequencing revealed compound heterozygous mutations in CTNNA3 encoding the protein αT-catenin, which localizes to adherens junctions of a subset of cells, where it links the cadherin complex to filamentous actin, providing cell-cell attachment. Results: The CTNNA3 mutations encoded a premature stop codon (R208*) and an amino acid substitution (R378C). ClinVar suggests that these are pathogenic mutations. The R378C mutation is located in an intramolecular salt bridge that regulates protein conformation. Mechanical force breaks the salt bridge to induce conformational change and reveal a cryptic binding site for the protein vinculin (important for both cadherin- and integrin-mediated adhesion to the actin cytoskeleton). The patient also had mutations in NOD2 , a gene linked to Blau and Yao syndromes, This patient’s NOD2 variants included IVS8 +158 and R702W in linkage disequilibrium; both parents have the same NOD2 mutations and are unaffected. We therefore propose a possible link between CTNNA3 and an inflammatory disease with an interferon signature. Conclusion: There are no prior reported associations of CTNNA3 mutations in inflammatory disorders. It is possible that the combination of the CTNNA3 and NOD2 gene mutations together drives patient’s phenotype. Further study is needed to investigate the potential role of CTNNA3 in in interferon-associatedM. Robert, C. Guillemin, L. Rossi-Semerano, C. Galeotti, I. Koné-Paut, P. Dusser Rhumatologie pédiatrique, Hôpital Bicêtre, Le Kremlin Bicêtre, France Correspondence: M. Robert Introduction: Chronic recurrent multifocal osteomyelitis (CRMO) is a rare auto-inflammatory disease. Clinical manifestations include skeletal symptoms that are well described. Whole body magnetic resonance imaging (MRI) is the gold standard for the diagnosis. Sometimes, skin or digestive manifestations occur. Treatment relies on non-steroidal anti-inflammatory drugs (NSAIDs), bisphosphonates are used as second-line treatments. To date, the focus has been set on bone involvement and few data are available on extra-osseous manifestations in CRMO. Objectives: This study aims to further describe these extra-osseous manifestations in CRMO. Methods: A historical cohort was designed with patients followed at the Pediatric Rheumatology Department in a tertiary university hospital. All patients fulfilled Jansson’s criteria. Skeletal and extra-osseous manifestations were characterized. Treatments used were recorded. Results: Forty-one patients were included, with 31 females (75.6%). The mean age at onset was 79.1 months, with a delay at diagnosis of 6.71 months. Twenty-one patients had a familial history of inflammatory diseases (n=21/41, 51.2%). At diagnosis, pain level was 5.71/10. Eleven patients (42.3%) had blood inflammation. The mean number of bone lesions, assessed thanks to MRI, was 6.65. Twenty-seven patients had extra-osseous symptoms (65.9%), with 7 patients with fever (17.1%), 24 patients had skin manifestations (58.5%) including palmoplantar lesions (n=3, 12.5%), acne (n=6, 25.0%), psoriasis (n=5, 20.8%) and aphthous (n=10, 41.7%). Four patients (9.76%) had gastro-intestinal symptoms and 7 (17.1%) had enthesitis. One patient had uveitis. A patient had a vasculitis which mimicked Behçet’s disease. Almost all patients received NSAIDs (n=39/41, 95.1%), 51.2% (n=21/41) of the cohort was treated with bisphosphonates. Nine patients (22.0%) received biologics. Subgroup analysis between CRMO with and without extra-osseous manifestations revealed significant differences for age at diagnosis (126 vs 20.5 months, p=0.03), blood inflammation (87% vs 46.2%, p=0.018) and the use of biologics (33.3% vs 0%, p=0.017). Conclusion: Extra-osseous manifestations have to be carefully searched in CRMO and integrated in the therapeutic strategS. Salugina 1 , E. Fedorov 1 , M. Kaleda 1 , E. Kamenets 2 , E. Zakharova 2 1 Pediatrics, V.A. Nasonova Research Institute of Rheumatology, 2 Selective Screening, Research Center of Medical Genetics, Moscow, Russian Federation Correspondence: S. Salugina Introduction: TRAPS (TNF-receptor-associated periodic syndrome) is a rare autosomal dominant disease associated with a mutation of the TNFRSF1A gene. It belongs to the group of monogenic autoinflammatory diseases (mAIDs) characterized by repeated prolonged episodes of fever, skin rashes, musculoskeletal, ophthalmological symptoms, and an increase in the level of acute phase markers. The main targeted therapy is IL1 inhibitors (iIL–1). Objectives: to present the Russian cohort of patients (pts) with TRAPS according to the Federal Rheumatology Center Methods: For the period from 2013 to 2021, 28 pts with TRAPS were included in the study, 17 of them female, aged 2.5 to 65 years, Me 9.0 [IQR 7.9;14.5] y.o., 7 adults, 21 children. All pts underwent rheumatological examination. Molecular genetic analysis for mutations in the TNFRSF1A gene was made to all pts. Results: Among mAIDs (162 pts), TRAPS was diagnosed in 28 (17.3%). The age of the onset ranged from 1 month of life to 28 years, Me 4.0 [IQR 1.0;7.1] y.o. The majority of pts started to hurt before the age of 10 (89.3%), of them up to a year - 8 (28.6%), from 1 to 5 years – 25%, older than 18 years – 2 (7.1%), at the age of 21 and 28. At the time of the study the duration of the disease and the delay in diagnosis ranged from 3 months to 59 years. The diagnosis was made within 1 year of the disease in 4 (14.3%), ranging from 1 to 10 years in 18 (64.3%), a delay in diagnosis of more than 10 years was in 8 (28.6%), more than 20 years in 4 (14.3%). The duration of attacks ranged from 10 to 30 days, the intervals between seizures ranged from 2 weeks to 6 months. Clinical manifestations included most often: fever – 96.4% of pts, skin rashes - 71.4% (annular in 9, erythema in 6, urticaria in 2), musculoskeletal symptoms - 64.3% (among them oligoarthritis in 9, polyarthritis in 2), gastrointestinal symptoms - 64.3% (more often abdominal pain), lymphadenopathy of various groups (cervical, intra-thoracic, mesenteric lymph nodes) - 35.7%, ophthalmological manifestations - 28.6% (more often periorbital edema-5 and eye redness-5). Less frequently observed: manifestations from the nervous system (headaches, dizziness, fatigue, convulsions) in 6 pts, pharyngitis - in 4, stomatitis - in 2. An increase in ESR was noted in 82.1% pts, CRP-71.4%, leukocytosis - 39.3%. The diagnosis in all pts was confirmed genetically. 10 pts had a low-penetrant mutation R92Q (p. Arg121Gln). 5 family cases were identified, the total number of sick family members was 12, from 2 to 4 in each family, 3 cases of amyloidosis, all adults. Treatment included: GC-19 (67.9%), cyclosporine A - 1, colchicine-5. IL-1 inhibitor (canakinumab) was prescribed to 10 pts (35.7%), duration of administration was from 1 to 9 years; etanercept-1, adalimumab-1, tocilizumab-3. Conclusion: A cohort of pts with TRAPS in the practice of a rheumatologist is presented. The majority of pts had an early onset of the disease (before the age of 10), the delay in diagnosis was 10 years or more in a third of pts. Most pts had a typical inflammatory phenotype, the diagnosis in all was confirmed genetically. About a third of the pts needed the appointment of biological drugs, mainly iIL-1. There was a complete response to the therapy in most pts and good tolerability of the treatment. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
C. Schuetz 1 , N. Blank 2 , J. Henes 3 , T. Kallinich 4 , P. T. Oommen 5 , M. Borte 6 , M. Hufnagel 7 , A. Janda 8 , J. Weber-Arden 9 , J. B. Kuemmerle-Deschner 10 1 [email protected], Medizinische Fakultaet Carl Gustav Carus, Technische Universitaet Dresden, Dresden, 2 Rheumatology, University Hospital Heidelberg, Heidelberg, 3ImmunoDeficiencyCenter Leipzig (IDCL), Hospital St. Georg gGmbH Leipzig, Leipzig, 8 Department of Pediatrics, University Hospital Ulm, Ulm, 9 NOVARTIS, Nuernberg, 10Germany Correspondence: C. Schuetz Introduction: TRAPS[1] is a rare hereditary autoinflammatory disease characterized by periodic fever and severe systemic and organ inflammation. Successful treatment was achieved with the interleukin-1β inhibitor canakinumab (CAN) in a pivotal phase 3 study, in which 45% of patients achieved clinical remission. CAN has been approved for the treatment of TRAPS patients since 2017. 1TRAPS: Tumor necrosis factor receptor-associated periodic syndromepractice conditions in pediatric (age ≥2 years) and adult TRAPS. Patients with a clinically confirmed diagnosis of TRAPS who routinely receive CAN will be enrolled in the study to evaluate the efficacy and safety of CAN under standard clinical conditions at baseline and at six-month intervals. Results: The interim analysis of TRAPS patients enrolled through December 2021 included baseline data (N=19) and preliminary 24-month data. Of these patients, N=12 (63%) were female and the median age at baseline was 16 years (3-43 years). Mutation information was provided for a total of N=13 patients, all of whom were pathogenic or likely pathogenic (Table 1). N=4 patients had dose adjustments (↑ dose increase, ↓ dose reduction): patient 1: ↑, patient 2: ↑↓, patient 3: ↑↑, patient 4: ↑↑↓↑. Preliminary results at baseline, 12 months and 24 months indicate stable disease control by remission according to physician assessment (47, 73 and 50% of patients), laboratory parameters (CRP 0.2, 0.1, 0.2 mg/dl and Serum Amyloid A (0.5, 0.4, 0.4 mg/dl median) and disease control according to patient assessment (1.5, 1.0, 0.0). Of N=9 serious adverse events, none occurred in association with therapy. A total of N=7 adverse drug events were observed, none of which were considered serious. Conclusion: Baseline and interim data from the RELIANCE trial for TRAPS patients indicate continued good efficacy and safety of various CAN doses. Disclosure of Interest : C. Schuetz: None declaredT. Kallinich Speaker Bureau with: Roche, P. T. Oommen Grant / Research Support with: Novartis, M. Borte Grant / Research Support with: Pfizer, Shire, M. Hufnagel Grant / Research Support with: Novartis, A. Janda: None declaredNazzar 1 , L. Moorthy 2 , S. Lapidus 3 , G. Schulert 4 , J. Tousseau 5 , M. Correia Marques 6,7 , L. Mansfield 8,9 , M. Twilt 10 , M. Gutierrez 11 , S. Angevare 12 , F. Dedeoglu 13 , K. Durrant 5 1 UCLA, Los Angeles, 2 Rutgers-RWJMS, New Brunswick, 3 The Joseph M. Sanzari Children’s Hosp., Hackensack, NJ, 4 Cincinnati Children’s Hosp., Cincinnati, 5 Autoinflammatory Alliance, San Francisco, 6 NIAMS, NIH, Bethesda, 7 Children’s Nat’l Hosp., Washington DC, 8 Hosp. for Special Surgery, 9 Weill Cornell, NY, United States, 10 Alberta Children’s Hospital, Calgary, Canada, 11 Johns Hopkins Hosp., Baltimore, United States, 12 KAISZ, Amsterdam, Netherlands, 13 Boston Children’s Hosp., Boston, United States Correspondence: G. Schulert Introduction: The exaggerated inflammatory responses to SARS-CoV-2 infection and the paucity of data on the risks for patients with Systemic Autoinflammatory Disease (SAID) patients is concerning. Objectives: To assess the outcomes of SAID patients with COVID-19. Methods: The CARRA Autoinflammatory Working Group and the Autoinflammatory Alliance conducted an anonymous online survey that was distributed to SAID patients through social media between January and March of 2021 about their experiences with COVID-19 in 2020. Results: We analyzed data from 593 individuals with SAID including Cryopirin-associated Periodic Syndromes (CAPS) (18%, n=108), Undifferentiated Systemic Autoinflammatory Disease (USAID) (12%, n=70), Periodic Fever Aphthous Stomatitis Pharyngitis and Cervical Adenitis (PFAPA) (11%, n=64) and others. Seventy four percent of the patients were from the United States, US territories and Canada and 26% from other countries. Fifty-two subjects had COVID-19. Of these, 12% (n=6) were asymptomatic, 81% (n=42) had mild or moderate symptoms managed at home and four were hospitalized. Thirty-three percent (n=17) had a flare of their underlying SAID concurrently (n=10) or after recovering from COVID-19 (n=7). Half of the patients who were hospitalized (n=2/4) noted increased frequency of flares during the pandemic, whereas the ones who had asymptomatic infection had decreased or unchanged flare frequency. Twenty-one (40%) of patients reported post-COVID manifestations (Table 1), which were seen more often after moderate illness and hospitalized patients (60%, n=15/25), compared to mild and asymptomatic cases (22%, n=6/27). Thirty three percent (n=7) of the SAID patients who had post-COVID symptoms did not have a specific SAID diagnosis listed, 14% (n=3) had USAID, and 9.5% (n=2) for each diagnosis had CAPS, PFAPA or Familial Mediterranean Fever. Conclusion: These data reflect the largest known global cohort of SAID patients’ experiences with COVID-19. Most of the SAID patients who had COVID-19 reported mild to moderate symptoms managed at home with a third presenting an exacerbation of their baseline SAID and lingering symptoms. The severity of acute COVID-19 was associated with reported changes in the frequency of flares during the pandemic and with the likelihood of post-COVID symptoms. Patient Consent: Not applicable (there are no patient data) Disclosure of Interest : S. Nazzar: None declared, L. Moorthy Grant / Research Support with: Brystol Myers Squibb, S. Lapidus: None declared, G. Schulert Consultant with: Novartis, J. Tousseau: None declared, M. Correia Marques: None declared, L. Mansfield: None declared, M. Twilt: None declared, M. Gutierrez: None declared, S. Angevare: None declared, F. Dedeoglu: None declared, K. Durrant: None declared
E. Krekhova 1 , E. Alexeeva 1 , M. Shingarova 1 , T. Dvoryakovskaya 22 , I. Tsulukia 2 , A. Pushkov 3 , K. Savostyanov 3 1 Pediatric, Sechenov First Moscow State Medical University, Pediatric, Moscow, Russian Federation, 2 Rheumatology, 3 Molecular geneticM. Shingarova Introduction: Cryopyrine-associated periodic syndromes (CAPS) are a group of rare congenital auto-inflammatory diseases (AID) in an autosomal dominate manner and caused by variants in NLRP3 gene. The main difficulties in diagnosing CAPS are the similarity of their clinical manifestations with other rheumatic diseases. Objectives: To reveal genotype and phenotype characteristics in patients with NLRP3 gene variants at the National Medical Research Center of Children’s health, Moscow, Russia. Methods: Retrospective study included 60 patients (37 females, 62%) with NLRP36,3 (interquartile range (IQR) 0,1:13,7) years. The diagnosis of CAPS was founded according Eurofever/PRINTO criteria. Results: 5/60 (8,3%) patients had NLRP3 pathogenic variants: two had c.943A>G variant, and one each with c.2173C>A, c.1991T>C and c.214G>A respectively. Likely pathogenic variants had 5/60 patients (8,3%)/ Among them two had c.1085T>A , two - c.410G>A , and one - c.796C>T ; all heterozygous. Clinical manifestations 10/60 (16,7%): rash in 7 (70%), fever in 7 (70%) patients, hepatomegaly in 9 (90%), splenomegaly in 6 (60%) patients, lymphadenopathy in 8 (80%), and serositis in 4 (40%) patients. Аrthritis was observed in 10/10 (100%) patients, affection eyes in 4/10 (40%), сentral nervous impairment observed in 6(60%), sensorineural hearing loss was in 2 (20%) patients. The criteria of CAPS accorded 9/10 (90%) children. One patients with pathogenic variants was sibling child with CAPS (asymptomatic carrier). Variant with variant with conflicting pathogenicity c.598G>A was observed in 10/60 (16,7%). Clinical manifestations 10/60 (16,7%): rash in 3 (30%), fever in 7 (70%) patients, hepatomegaly in 5 (50%), splenomegaly in 3 (30%) patients, lymphadenopathy in 4 (40%), serositis in 1 (10%) patients, arthritis was observed in 9/10 (90%) patients, affection eyes in 4/10 (40%), сentral nervous impairment observed in 2(20%), and sensorineural hearing loss was in 1 (10%) patients. The criteria of CAPS accorded 4/10 (40%) children, juvenile polyarthritis (RF-) - 4/10 (40%), persistent oligoarthritis – 1/10(10%) and 1/10 with DADA2 . Among the remaining patients, 30/60 (50%) had polymorphism c.2113C>A , 8/60 had VUS c.2861C>T , c.584C>T , c.585G>A, c.459C>G and 2/60 patients - probably benign variants : c.2664-26G>C and c.1050G>A , respectively. Conclusion: The criteria of CAPS accorded 14 patients. The most frequent clinical manifestations were in musculoskeletal involvement 93%, fever 86 % and rash 79 % of children. Central nervous impairment observed in 7/14(60%), sensorineural hearing loss was in 3/14(21,4%) and affection eyes in 4/14(29%). The number of identified causal variants of the NLRP3 gene in these 14 patients showed high diversity, while the high frequency of polymorphism c.2113C>A in the studied sample was consistent with data obtained in other populationsS. Signa 1 , A. Bertoni 1 , G. Del Zotto 2 , M. Bonacini 3 , A. Corcione 1 , M. Bartolucci 4 , M. Bruschi 5 , A. Petretto 4 , R. Bertelli 6 , S. Croci 3 , S. Della Bella 7 , M. Gattorno 1 , F. Schena 1 1 Centro per le malattie autoinfiammatorie e immunodeficienze, 2 Core Facilities Flow Cytometry and Cell imaging Lab, IRCCS, Giannina Gaslini Institute, Genova, 3 Clinical Immunology, Allergy and Advanced Biotechnologies Unit, Azienda Unità Sanitaria Locale-IRCCS di Reggio Emilia, Reggio Emilia, 4 Core Facilities-Clinical Proteomics and Metabolomics, 5 Laboratory of Molecular Nephrology, 6 Laboratory of Human Genetics, IRCCS, Giannina Gaslini Institute, Genova, 7 Lab of Clinical and Experimental Immunology, IRCCS Humanitas Research Hospital, Milano, Italy Correspondence: S. Signa Introduction: Deficiency of Adenosine deaminase 2 (DADA2) is a monogenic autoinflammatory disorder presenting a broad spectrum of clinical manifestations, including vasculitis, immunodeficiency and hematologic disease. Biallelic mutations in ADA2 gene have been associated to an insufficient ADA2 activity and a consequent accumulation of extracellular adenosine. Recently a chronic neutrophil activation and a dysregulation of NETosis triggered by extracellular Adenosine and inducing TNF-α secretion from macrophages, has been implicated in the pathogenesis of DADA2. Objectives: The aim of the project is to analyze NETosis in DADA2 and healthy neutrophils, quantifying suicidal and vital NETosis induced by several stimuli and by analyzing NETs remnants. Moreover, we investigated the mechanisms of NETs removal, evaluating DNAse activity. To determine if NET epitopes can change depending from the inflammatory microenvironment and if protein composition of NETs is disease specific, we used quantitative proteomics approach to characterize DADA2 patients’ NET proteins. To investigate the cross-talk between neutrophils and Dendritic cells (mDCs), we performed DCs immune-phenotype analysis in DADA2 patients and analyzed in vitro moDC maturation and cytokine production in presence of NETs. Methods: We analyzed NETosis by Imaging Flow Citometry. We evaluated NETs remnants and DNAse in the plasma samples by ELISA assay whereas DNAse activity by DNA digestion. We used quantitative proteomics approach to characterize NET proteins. We isolated monocytes from peripheral blood with microbeads and we generated moDC after culture stimulation with LPS/NETs. On the 7th day we analyzed by flow cytometry the phenotype and the markers of differentiation. Quantification of cytokines was performed by flow cytometry bead array. Results: Neutrophils from DADA2 patients show a significant increased suicidal NETosis, and we found an increased vital NETosis. Accordingly, plasmatic levels of circulating nucleosomes were elevated in patients; DNAse levels were normal but its activity was reduced, possibly due to presence of inhibitors. We set up experimental conditions for proteomic analysis of NETs, induced by PMA, Adenosine and TNF-α, testing two patients, two HDs and two patients with non-genetic vasculitis: in total we identified 1770 proteins among which a hundred of proteins were significantly up or down-modulated in DADA2 NETs compared to controls NETs. DC phenotype in DADA2 patients result normal, as well as moDCs cytokine production after LPS stimulation. We observed a stimulatory effect of patients’ NETs towards induction of TNF-α , IL-6 production and IP-10 from moDCs in both HD and DADA2. Conclusion: Our findings confirm a dysregulation in NETosis process in DADA2 patients. An increase of vital NETosis was also identified. Proteomic profile of NETs isolated from DADA2 is different from HD and PAN patients: NETs are qualitatively different between HD and DADA2. NETs in DADA2 may therefore interact differently with innate immunity compartment, stimulating DCs to produce cytokines, contributing to the typical inflammatory phenotype. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
P. Bocca 1 , A. Tesser 2 , D. Cangelosi 3 , M. Ulivi 4 , F. Candotti 5 , A. Tommasini 2,6 , M. Gattorno 1 , S. Volpi 1 Clinica Pediatrica e Reumatologia, IRCCS Istituto G. Gaslini, Genova, 2 Pediatric Department, Institute for Maternal and Child Health - IRCCS “Burlo Garofolo”, Trieste, 3 Unità di Bioinformatica Clinica, IRCCS Istituto G. Gaslini, 4 Centro Biotecnologie Avanzate, TIB Molbiol, Genova, Italy, 5 Laboratory of Inherited Immune Disorders, Division of Immunology and Allergy, Lausanne University Hospital and University of Lausanne, Lausanne, Switzerland, 6 University of Trieste, Trieste, Italy Correspondence: M. Gattorno Introduction: Type I interferon (IFN) signature analysis is extensively used to identify pathological conditions characterized by a type I IFN dysregulation (i.e. monogenic interferonopathies, dermatomyositis, systemic lupus erythematosus), and to direct therapy approaches. IFN signature is used to discriminate patients with IFN-related inflammation from conditions predominantly mediated by other cytokines (i.e. periodic fevers), through the calculation of an IFN score (IS). However, no standardized method is yet available for the clinical practice, and comparing values at different time points or between different centers remains a challenge. Objectives: To implement a standardized method for IFN signature detection with the use of a synthetic control, and to evaluate the concordance of IFN signature results obtained with this method among the laboratories of two Italian Hospitals. Methods: Peripheral blood expression analysis of the six IFN Stimulated Genes (ISGs) IFI27, IFI44L, IFIT1, ISG15, RSAD2 and SIGLEC1 was detected by Real Time PCR. A synthetic control was used to determine copy numbers of the selected genes. The IS for each subject was calculated as the geometric mean of the genes’ copy numbers normalized using an endogenous reference gene. IS cut-off value was obtained as the mean + 2SD of 20 healthy donors’ IS. The method was validated by analyzing the IS of 89 patients with different inflammatory, autoimmune and infectious disorders driven by type I IFN or other inflammatory pathways. To assess inter-laboratory variability, 12 patients with different degrees of type I IFN inflammation were run in parallel in two different centers. Results: As expected, our test confirmed a positive IFN score for inflammatory diseases such as systemic lupus erythematosus, type I interferonopathies, dermatomyositis and Sjögren syndrome. The test demonstrated a high reproducibility as assessed by multiple analyses of the same sample. Furthermore, IS obtained from the samples shared by two different laboratories resulted in comparable values. Conclusion: The proposed method represents a quantitative, standardized and easy to perform assay for the evaluation of type I IFN signature in IFN-driven inflammatory conditions. The reproducibility of the synthetic control minimized the inter-assay and inter-laboratory variability. The application of a standardized method for type I IFN signature detection greatly improves the test interpretation, patient follow up and clinical discussion among centers promoting the use of type I IFN signature in clinical practice. Disclosure of Interest : None declared
Y. Vyzhga 1 , V. Hentgen 2 , R. Papa 3 , H. Wittkowski 4 , N. Ruperto 3 , E. Lainka 5 , K. Theodoropoulou 6 , R. Caorsi 3 , S. Fuehner 4 , D. Foell 4 , M. Gattorno 3 , M. Hofer 6Vinnitsya, Ukraine, 2 Versailles Hospital, Le Chesnay, France, 3 IRCCS Instituto G. Gaslini, Genova, Italy, 4 University Hospital ChildreńPatients with recurrent fever without molecular diagnosis can often be diagnosed as Periodic Fever, Aphthous Stomatitis, Pharyngitis, Adenitis (PFAPA) syndrome, the most common pediatric autoinflammatory fever syndrome, with a highest incidence in children up to 5 years of age. Nevertheless, a number of patients do not fulfil current diagnostic criteria and have to be classified as undefined autoinflammatory diseases (uAID). In recent years the syndrome of undifferentiated recurrent fever (SURF) was proposed as a heterogeneous group of autoinflammatory diseases characterized by the presence of self-limiting episodes of systemic inflammation with multiple clinical presentations, but without confirmed molecular diagnosis. Presence of the similar clinical features makes these 2 conditions to be in a scope of interest from the perspective of the potential overlap and misdiagnosing. Objectives: In a project endorsed by PReS, supported by the EMERGE fellowship program, and performed in line with the Metadata registry for the ERN RITA (MeRITA) project, our objective was to analyse recurrent fevers without molecular diagnosis, the evolution of diagnosis over time and the clinical characteristics in PFAPA, SURF and uAID. Methods: Currently there are two internationalPatients in Germany were recruited to the BMBF-funded AID-Net Registry and the AID-Net Biobank. Patients with PFAPA, uAID or SURF were identified from the registries. Diagnostic (modified Marshall’s criteria) and classification criteria (Eurofever) for PFAPA and the preliminary indications for the suspicion of SURF (Papa et al) were applied to all patients. Results: The overall number of patients with a diagnosis of PFAPA in all three registries was 1230 (Eurofever - 634, AIDnet - 140, JIR cohort - 456). Complete epidemiological and clinical dataset was available in 929 patients (Eurofever - 476, AIDnet - 136, JIR cohort - 317). Marshall’s modified criteria were satisfied in 51.5 to 67.2 % of the PFAPA patients, while 59 to 69.4 % patients fulfilled the Eurofever classification criteria. At the same time, 29.4 to 47.5 % of the patients met both – modified Marshall and Eurofever criteria. Up to 20 % of the patients didn’t fulfil any of the proposed criteria. The overall number of patients with a diagnosis of SURF was n=809 (Eurofever - 429, AID net - 83, JIR cohort - 297). Among them, 561 of them displayed a negative genetic test for hereditary recurrent fevers (Eurofever - 307, AIDnet - 79, JIR cohort - 175). 417 patients satisfied the preliminary indications for the suspicion of SURF (Eurofever - 230, AIDnet - 51, JIR cohort - 136). From 22 to 35 % of SURF patients didn’t fulfil the proposed preliminary indications . From 8 to 17% of PFAPA patients in the three registries fulfilled the preliminary indications for suspicion of SURF. Vice versa, 26 to 37 % SURF patients satisfied at least one PFAPA criteria. Conclusion: Patients with recurrent fevers but without molecular diagnosis are still an heterogenous group of patients regarding clinical manifestations. More accurate classification criteria are needed for both conditions. A better definition of the clinical manifestations associated with the 2 conditions would allow correct grouping of these patients improving clinical management and outcomeY. Vyzhga 1 , V. Hentgen 2 , R. Caorsi 3 , H. Wittkowski 4 , N. Ruperto 3 , E. Lainka 5 , S. Fuehner 4 , K. Theodoropoulou 6 , M. Hofer 6 , D. Foell 4 , M. Gattorno 3 on behalf of AID-NET, Eurofever and JIRcohort registries 1 National Pirogov Memorial Medical University, Vinnytsya, Ukraine, 2 Versailles Hospital , Le Chesnay, France, 3 IRCCS Instituto G. Gaslini, Genova, Italy, 4 University Hospital Childrens Muenster, Muenster, 5 University Hospital Essen, Essen, Germany, 6 Centre Hospitalier Universitaire Vaudois CHUV, Lausanne, Switzerland Correspondence: Y. Vyzhga Introduction: Knowledge about autoinflammatory diseases (AID) has changed the general view of innate immune activation over the recent decades. Based on common pathophysiological mechanisms, autoinflammatory diseases can be divided into groups (IL-1-, interferon type I- and NF-kB-mediated diseases, diseases caused by macrophage activation, and other diseases). Also due to modern sequencing techniques, the number of known entities has grown strongly in recent years, but still their peculiarities in different countries. Access of the patients to diagnostic and treatment options are variable, may affecting disease control and the risk of long term complicationsperform data harmonization and comparative analyses of selected most relevant research questions, e.g. changes in the diagnostic work-up of AID-patients over time, and how newly described disease entities or the emergence of new drugs have changed patient care. This is also meant to improve further work in the registries. Methods: Currently there are two European AID-registries active (Eurofever and JIRcohort) and one German AID-registry, that was active from 2009 to 2018. Cohort design, duration of the observance and patient’s follow-up, main epidemiological, clinical and laboratory information, data collection methods, process of IT-support, etc. were analysed and compared between registries for harmonization of data granularity and further analysis. Results: Together, the 3 registries cover 7825 patients with different AID from participating centers all over the world. The German AID-Net presents a national type of registry involving 36 pediatric rheumatology centers in Germany. JIRcohort and Eurofever are international cohorts, covering about 40 countries in total. The most widely presented AID in all registries were FMF, PFAPA, URF (Undefined Recurrent Fever), CRMO, SoJIA, CAPS, TRAPS, and MKD. Eurofever and JIRcohort additionally covered other exclusively rare AID as PAPA, DADA2, Blau syndrome, HA20, PASH, DIRA, etc. One of the important scopes of the study was evaluation of the diagnostic delay. There was a marked delay between disease onset and diagnosis in all registers, with an obvious trend of its shortening between children. The highest diagnostic delay is more common for classical monogenic AID, as TRAPS, MKD and CAPS, in which the diagnostic delay in adult was mainly related to the time of the identification of the molecular defect in respect to the disease onset. Moreover, the diagnostic delay was also strongly associated with the availability of the genetic testing in different time periods. In all registers capacity of the tested patients exceed 50 % over the whole period of registry work. Detailed data of treatment options shows availability of novel biologic therapy for approximately 20 – 25% of the patients with AID and depends on its availability in the country of patient’s residence. Conclusion: Several important questions arise around clinical features, diagnostic strategy and optimal management of the AID. Analysis of 3 large registries shows still relevant diagnostic delays especially in adult patients and a varying availability of access to diagnostic work up and treatment. Further efforts should be made in data harmonization for future registry studies. This project was supported by PRES-EMERGE and Novartis AG funding for IIR. Disclosure of Interest : None declared
S. Wieland 1 , T. Welzel 2 , S. Fühner 3 , A. Gabrielyan 1 , E. Lainka 4 , C. Kastl 1 , L. Pietzsch 1 , N.-C. Knopf 1 , C. Michler 2 , A. J. Ritter 2 , A. Rösen-Wolff 1 , D. Foell 3 , H. Wittkowski 3 , J. Kuemmerle-Deschner 2 , C. Schuetz 1 1 Department of Paediatrics, Medizinische Fakultät Carl Gustav Carus, Technische Universität Dresden, Dresden, 2 Division of Paediatric Rheumatology, University Hospital Tuebingen, Tübingen, 3 Department of Paediatric Rheumatology and Immunology, Universitätsklinikum Münster, Münster, 4 Division of Pediatric Rheumatology, University Children’s Hospital Essen, Essen, Germany Correspondence: S. Wieland Introduction: Autoinflammatory diseases (AI) like FMF, CAPS or TRAPS lead to a constitutive activation of the inflammasome and secretion of IL-1b with severe long-term consequences like amyloidosis when undiagnosed or insufficiently treated. Currently, clinicians routinely use nonspecific acute phase proteins – most of them synthetized in the liver – to monitor inflammation and treatment efficacy. Objectives: Apoptosis-associated speck-like protein containing a CARD (ASC) is an adaptor protein in different inflammasomes released during pyroptosis. It assembles following activation to a speck-like oligomer in the cytoplasm. Previous studies would detect the ASC- agglomerates intracellularly after time-consuming sample preparation. We have established a rapid and simple method for routine detection of ASC agglomerates in patients’ serum. Methods: We collected 68 serum samples from 52 patients (aged 1 to 30 years) with various AI and at different stages of disease activity. After centrifugation for 10-15 min at 2000g within 2 hours after sampling, the supernatant was removed and cryopreserved at -80°C. For analysis thawed sera were incubated with a PE-linked anti-ASC antibody (Biolegend) diluted 1:4. To detect the ASC agglomerates of different sizes between 1- 3mm, PE-linked microbeads (Polysciences) were used to define our gate. Flow cytometry was performed on a BD LSR II. Clinical data collected via a simplified questionnaire were available for most samples collected at 3 different pediatric centers. Results: In the serum of healthy donors, null to minor amounts of ASC agglomerates were present. In contrast, we measured relevant amounts of ASC agglomerates in serum of patients with FMF, CAPS, TRAPS, sJIA and SURF. During disease flares, the amount of ASC specks was significantly elevated in samples from FMF and CAPS patients constituting over half of the collected specimens. Conclusion: Measurement of ASC agglomerates may be taken as a surrogate for inflammasome activity. The methodology presented allows detection in serum without laborious preanalytics. In clinical practice, monitoring this novel biomarker may be of relevance for diagnosis, monitoring and treatment decisions. We will extend our study by measuring more specimens of patients with TRAPS, SURF/PFAPA and sJIA comparing disease flares with periods of remission. Disclosure of Interest : S. Wieland: None declared, T. Welzel: None declared, S. Fühner: None declared, A. Gabrielyan: None declared, E. Lainka: None declared, C. Kastl: None declared, L. Pietzsch: None declared, N.-C. Knopf: None declared, C. Michler: None declared, A. Ritter: None declared, A. Rösen-Wolff: None declared, D. Foell: None declared, H. Wittkowski: None declared, J. Kuemmerle-Deschner: None declared, C. Schuetz Grant / Research Support with: Novartis
J. Wojtowicz, A. Gazda, B. Kolodziejczyk, I. Szczygielska, M. Szwarc-Bronikowska, I. Witkowska, A. Adamczuk, E. Hernik, P. Gietka National Institute of Geriatrics, Rheumatology and Rehabilitation, Warszawa, Poland Correspondence: J. Wojtowicz Introduction: Chronic non-bacterial osteomyelitis (CNO) or chronic recurrent multifocal osteomyelitis (CRMO) are rare autoinflammatory bone diseases that occur in children. There are diagnosed on the basis of a pooled analysis of clinical data, results of imaging studies and histopathological examinations, if needed. Objectives: The diseases are not widely known. First symptoms, auch as pain, swelling and tenderness can be therefore misinterpreted. Moreover absence of deviations in laboratory assays and initialy correct imaging results (especially x-ray) can extend time from the first symptoms to diagnosis. Methods: The study presents a retrospective analysis of clinical data of 45 patients, diagnosed and treated because of CNO/CRMO at our Department over the past 13 years (2009-2022) with regard to the time from first symptoms to diagnosis. Results: The median time from first symptoms to CNO/CRMO diagnosis in the group was 13 months (minimum 1,5 months; maximum 107 months). The difference in time to diagnosis between the sexes was not statistically significant (girls median 22 months, boys 11 months). There was a weak positive correlation between the age of the patient and the time to diagnosis (r 0,34, p=0,03). The crucial result of the analysis is a strong negative correlation between the year of firts symptoms of CNO/CRMO (2003-2022) and a time to diagnosis (r -0,63, p <0,001). Conclusion: The analysis of the clinical data from our center shows the significant reduction in time to diagnosis of CNO/CRMO in children over the last 13 years. It depends mainly on better and better ability to distinguish symptoms reported by children with joints and bone complaints and ordering targeted diagnostics, including the whole body magnetic resonanse imaging, at our department. This may depend also on greater awareness of the diseases among medical doctors (especjally surgeons, orthopedic surgeons and oncologist) who referre children to the paediatric rheumatology department faster than in the past. However none active educational campain has been organised yetP181. Prevalence, demographic and clinical patterns of uveitis in juvenile idiopathic arthritis at a tertiary care pediatric hospital in Libya P182. Methotrexate therapy associated with lower risk for uveitis-onset in juvenile idiopathic arthritis 83. Non-infectious isolated uveitis in children: clinical features and multimodal imaging of a challenging condition P184. Treatment and prognosis of of chronic non infectious uveitis in rheumatology unit in tripoli children hospital P185. Paediatric uveitis at the royal Victoria infirmary: looking back and forwards 6. Pediatric uveitis: single-center experience for six years P187. Ocular involvement in paediatric Behçet disease: a monocentric experience 8. Subcutaneous tocilizumab in JIA related uveitis: a monocentric experience P189. Juvenile idiopathic arthritis-associated uveitis: a bibliometric and network analyseA. A. Abushhaiwia 1 , S. T. Ashur 2 , M. Zletni 3 , A. Ganbour 4 1 Pediatric Rheumatology, Tripoli Children’s Hospital; Faculty of medicine; University of Tripoli, 2 Family and Community Medicine, Faculty of Medicine, University of Tripoli, Libya, 3 Paediatric Rheumatology , Faculty of Medicine,Tripoli university, Tripoli Children’s Hospital , 4 Paediatric Rheumatology , Tripoli Children’s Hospital , Tripoli , Libya Correspondence: A. A. Abushhaiwia Introduction: Juvenile idiopathic arthritis (JIA) is the most common childhood rheumatic disease. The development of associated uveitis represents a significant risk for serious complications, including permanent loss of vision. Initiation of early treatment is important for controlling JIA-uveitis, but the disease can appear asymptomatically, making frequent screening procedures necessary for patients at risk. It is difficult to assess which JIA patients are at risk of developing uveitis. In the present study, we described the prevalence and clinical profile of JIA-associated uveitis and out come among children in the Libyan clinical settings. Objectives: The present study described the prevalence, demographic and clinical profiles of uveitis in children diagnosed with JIA in a referral pediatric hospital in Libya. The study also compared the demographic and clinical characteristics of children who developed uveitis with those who did not develop uveitis, and examined the association between these characteristics and having uveitis Methods: A total of 90 JIA patients who fulfilled International League of Associations for Rheumatology (ILAR) diagnostic criteria were included in this retrospective study. The data collected were age, gender, age at disease onset and at diagnosis, and follow-up duration. Duration from JIA diagnosis to uveitis diagnosis. Antinuclear antibody (ANA), RF, and human leukocyte antigen B-27 were evaluated for each patient Results: A total of eight uveitis cases were identified among the 90 JIA cases, which gives a prevalence of 8.9%. All cases were females (100.0%), the majority were Libyans (87.5%), and their mean age was 12.3 (SD=4.3) years old. The mean age at JIA onset for this group was 5.3(SD=2.3) years old and that for JIA diagnosis 6.2 (SD=2.6) years old, with an average duration of JIA of 6.8 (SD=3.7) years. The most common JIA subtype in this group was oligoarthritis (50%), followed by poly arthritis (37.5%). The majority of the cases had a negative rheumatoid factor test (75%), and ANA was positive only in 1 of 7 valid cases (14.3%).The mean age at uveitis diagnosis was 8.2 (SD=3.7) years old. The median duration from time at diagnosis of JIA till uveitis diagnosis was 1 (IQR=1-1.7) year. Only 1 out of 7 valid uveitis cases (14.3%) was symptomatic, and it was presented with eye redness. The course of uveitis was acute in 3 out of 6 cases (50.0%). Anterior uveitis constituted 62.5% of all uveitis types, and 66.6% of the cases had unilateral eye involvement. Over half of the patients (62.5%) were on follow-up for uveitis for duration of at least 3 months, while the remaining cases (37.5%) had a follow-up duration shorter than one month. Half of the cases had impaired visual acuity. Concerning complications, cataract and synechiae were the commonest (37.5%), followed by cataract, synechiae and band keratopathy in a quarter of cases. All cases were on a combination of systemic steroids and methotrexate (87.5%).The types of the biological medications varied considerably across those who received biological therapy, however, Etanercept was the most popular one, as it was used either as a single biological treatment (25%), then Infliximab (12.5%), and Adalimumab (12.5%). Conclusion: early recognition of uveitis in JIA is required to improve outcome,the reasons for our lower complication rates and better visual outcome may be the more frequent use of systemic immunosuppressive agents (particularly methotrexate and anti- TNF agents), and close collaboration between our pediatric ophthalmologists and pediatric rheumatologists Patient Consent: Yes, I received consent Disclosure of Interest : None declared
G. Akay 1 , J. W. Straalen 1 , C. V. Kouwenberg 2 , N. M. Wulffraat 1 , S. de Roock 1 , J. H. de Boer 2 , J. F. Swart 1 1 Department of Pediatric Immunology and Rheumatology, Wilhelmina Children’s Hospital, 2 Department of Ophthalmology, University Medical Center Utrecht, Utrecht, Netherlands Correspondence: G. Akay Introduction: Juvenile idiopathic arthritis associated uveitis (JIA-U) is a common complication of juvenile idiopathic arthritis (JIA), with a cumulative incidence of 11.4% (1). It can result in significant visual impairment and blindness if not treated in a timely manner. Risk factors for uveitis-onset include oligoarticular JIA, ANA-positivity, young age at JIA onset and short JIA disease duration (2,3). Objectives: To investigate if the risk for new-onset uveitis in children with JIA is lower when treated with (different doses of) methotrexate (MTX) compared to treatment with other first- and second-line therapy. A second objective is to assess if this risk increases after MTX-discontinuation. Methods: For this case-control study, JIA-U cases from the University Medical Centre Utrecht (UMCU) were matched 1:1 to JIA control patients based on known risk factors for new-onset uveitis. Patient characteristics and medication use during the observation period were compared between JIA-U cases and JIA controls. Multivariable Cox regression analysis adjusted for JIA subtype, ANA-positivity and age at JIA onset was used to evaluate the effect of (different doses of) MTX on new-onset uveitis. MTX therapy was entered into the model as a time-varying covariate. Results: 54 out of 160 identified JIA-U cases were eligible, yielding a total of 108 matched JIA patients with similar uveitis-risk at baseline for cases and controls. The ever-use of drugs did not differ significantly between JIA-U cases and controls, although the ever-use of MTX was higher in JIA controls (n = 36, 66.7%) compared to JIA-U cases (n = 26, 48.1%) (p = 0.05). The number of exposure years for MTX was also higher for JIA controls (median 0.56 years, IQR 0 – 1.62) compared to JIA-U cases (median 0 years, IQR 0 – 1.53) but was not statistically significant (p = 0.18). However, the adjusted hazard ratio (HR) for new-onset uveitis during the observation period as obtained from the survival analysis was significantly decreased for MTX therapy compared to no MTX therapy with a HR of 0.28 (95% CI 0.13-0.58). We observed no MTX dose differences between low (<10 mg/m 2 /wk) and normal dose (≥10 mg/m 2 /wk). Of JIA-U cases, 26/54 (48.1%) were treated with MTX prior to uveitis-onset. Upon occurrence of new-onset uveitis, 34.6% of MTX-users even had ongoing MTX-therapy at that time, while 65% discontinued MTX prior to uveitis-onset. New-onset uveitis occurred most often (82.4%) within the first two years after discontinuation and half of them were during the first 6 months. Conclusion: Our results demonstrate a significantly reduced risk for new-onset uveitis in patients on MTX therapy after adjusting for known risk factors for JIA-U. We therefore suggest earlier initiation of MTX in JIA patients at high risk for new-onset uveitis. Furthermore, we advise more frequent ophthalmologic screenings after MTX-discontinuation since this is in itself a risk factor for new-onset uveitis. References: 1.’s Arch Clin Exp Ophthalmol. 2006 Mar;244(3):281–90. 2. van Straalen JW, Giancane G, Amazrhar Y, Tzaribachev N, Lazar C, Uziel Y, et al . A clinical prediction model for estimating the risk of developing uveitis in patients with juvenile idiopathic arthritis. Rheumatology (Oxford). 2021 Jun 1;60(6):2896–905. 3. Angeles-Han ST, Yeh S, Vogler LB. Updates on the risk markers and outcomes of severe juvenile idiopathic arthritis-associated uveitis. Int J Clin Rheumatol. 2013 Feb 1;8(1):109–21B. Beretta 1 , G. Leone 2 , M. Rossano 1 , E. A. Conti 1 , G. Rogani 1 , F. Baldo 1 , S. Testa 1 , F. Viola 2 , G. Filocamo 1 , F. Minoia 1 , C. Mapelli 2 1 Pediatric Rheumatology, 2 Ophtalmology Unit, FONDAZIONE IRCCS CA’ Granda Ospedale Maggiore Policlinico, Milan, Italy Correspondence: G. B. Beretta Introduction: Although rare, uveitis is the main cause of visual morbidity in children. Juvenile idiopathic arthritis (JIA) is the most frequently associated disease in pediatric uveitis; however, 28–51% of non-infectious uveitis remains idiopathic and still represents a major challenge. So far, there is a lack of evidence about pediatric non-infectious isolated uveitis (NII-U), especially regarding its characterization with multimodal imaging. Objectives: To describe clinical course, ophthalmological features, multimodal imaging and management of a monocentric cohort of pediatric NII-U. Methods: Medical records of pediatric-onset NII-U were retrospectively reviewed regarding systemic and ocular features, treatment and outcome. Ophthalmological evaluation included best corrected visual acuity (BCVA), slit lamp biomicroscopy, intraocular pressure measurement and fundus examination, and it was integrated with fluorescein angiography (FA), indocyanine green angiography (ICGA) and optical coherence tomography (OCT) when needed. NII-U data were compared with the cohort of JIA-associated uveitis (JIA-U) followed up at the same service. Quantitative and qualitative variables were analysed by means of Mann-Whitney U test or chi-square/Fisher exact test, as appropriate. Results: Data from 32 children (53% males) with NII-U were collected. Median age at uveitis diagnosis was 11.3 years (IQR 5.25) and median duration of follow-up was 1.9 years (IQR 3.0). Panuveitis, anterior and intermediate uveitis were diagnosed in 41%, 38% and 22% of cases, respectively; in 12 patients a granulomatous aspect was observed. In 67% of patients (82% of panuveitis) symptoms were present at onset, primarily ocular redness (48%), photophobia (22%), visual loss (26%) and eye pain (15%). Antinuclear antibodies (ANA) were positive only in 10 patients (6 anterior uveitis). In more than half of NII-U ocular damage was observed at diagnosis. FA, ICGA and OCT showed abnormalities in 88% of cases (100% of panuveitis) and major findings are described in Table 1. Methotrexate or cyclosporine were used in 59% and 13% of cases, respectively, with a median interval from diagnosis of 3.7 (IQR 5,6) months. A biological medication was required in 14 children after a median of 13.4 (IQR 21,0) months from diagnosis. At last visit, more than a third of NII-U patients still present active uveitis, with a BCVA < 4/10 reported in 10% of cases. Compared to JIA-U, NII-U was less common in females (47% vs 86%, p 0.0003) with a higher median age at onset (11.3 vs 5.0 years, p <.00001). NII-U patients were more frequently symptomatic (67% vs 0%, p < .00001), with a higher incidence of ocular damage at onset (40% vs 7%, p 0.0007) and a higher frequency of active uveitis at last visit (31% vs 7%, p 0.006). Conclusion: Pediatric NII-U is a challenging condition with a potential impact on visual outcome. Multimodal imaging is crucial in the proper definition of uveitis and should always be included in the diagnostic work-up of NII-U, especially when posterior segment’s involvement is suspected. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
S. Hashad 1 , H. M. Atyari 2 , M. N. Altofeel 3 1 Rheumatology unit, Tripoli children hospital, Tripoli, Libya, 2 Pediatric rheumatology , Tripoli Children Hospital , Tripoli , Lebanon, 3 Pediatric, Rheumatology , Tripoli , Libya Correspondence: S. Hashad Introduction: Noninfectious uveitis is an autoimmune mediated inflammation of the uveal tract, and it is a potentially sight threatening condition with associated consequences on the quality of life of these patients and high costs for the health system. It represents 12% of cases in pediatric rheumatology clinic which is the only clinic caring for non-JIA autoimmune uveitis in all Libya. Pediatric uveitis is a topic of special interest because of its diagnostic and therapeutic challenges. In the last decade, many biomarkers have been identified to help stratify the risk of uveitis and many new treatment modalities were introduced Objectives: • To study the demographic and clinical characteristics of JIA and non-JIA associated autoimmune uveitis. • To Study and compare response to treatment in both categories. Methods: The medical records of the patients with uveitis presented to rheumatology clinic from January 2000 to September 2021 were reviewed and data collected about the demographics (Gender, Date of birth, address) clinical data: date at presentation to ophthalmology, date at presentation to rheumatology, diagnosis, clinical findings at presentation, complications at presentation, treatment and number of flares before presentation, treatment during follow-up, slitlamp examination findings at last visit, complication of systemic treatment Results: A total of 75 cases of uveitis were presented in rheumatology OPD in the research period. Mean age at uveitis onset was 8.3±3.4 the patients years and mean follow-up period of 2.8±2.5 years. JIA associated uveitis represent 32% of cases (62% of them are with various JIA subclasses and 37.5% JIA froste forme) and 68% of them are nonJIA associated. Table 1 summarizes the diagnosis of all cases. Of the JIA cases 54.2% were symptomatic while 100% of the non-JIA uveitis were symptomatic. Anterior uveitis was the most common location in JIA associated uveitis (58.3%) while panuveitis was the most common location in nonJIAassociated uveitis (74.5%). of the cases of JIA cases 62.5% were bilateral and nonJIA associated uveitis 88.2% were bilateral. Methotrexate was the drug used in most cases of JIA and nonJIA associated uveitis (91% of total cases). Oral prednisolone was used in 66.7% of JIA associated cases while it was used in 80.4% of nonJIA cases. Biologics were needed in the treatment of 3 (12.5%) cases of JIA associated uveitis while they were neededin 9 (17.6%) of non-JIA cases. Intraocular injections were not used in JIA associated uveitis while it was used in 8 (15.7%) of the nonJIA associated uveitiOf the JIA cases 2 (8.3%) underwent operations and of nonJIA cases 10 (19.6%) underwent operations. In JIA cases remission was achieved in 75% of the cases with MTX and 95% Of cases with biologics. In nonJIA associated uveitis remission was achieved in 70% of cases with MTX and in 90% of cases with first biologic and in 95% of cases with second biologic. Of JIA cases 58.3% had complications at last visit while 72.5% of nonJIA cases had complications in JIA and nonJIA associated uveitis. Conclusion: Biological and nonbiological DEMARDs are effective in introducing maintaining remission in nonJIA associated uveitis with various diagnosis. Disclosure of Interest : None declared
Jandial, S. Cairns Great North Children’s Hospital, Newcastle, United Kingdom Correspondence: S. Jandial Introduction: Uveitis is an inflammatory eye disorder that if untreated can lead to irreversible damage such as cataract and glaucoma. In children, uveitis is a common association with Juvenile Idiopathic Arthritis (JIA) with a well-established screening guideline for all newly diagnosed JIA patients. Uveitis in non-JIA patients requires careful scrutiny for an underlying diagnosis but many will be labelled as ‘idiopathic uveitis’. Regardless of the underlying diagnosis, management of chronic uveitis requires immunosuppression to prevent persistent inflammation and structural damage to the eye. The immunosuppressive approach is similar regardless of the underlying diagnosis and needs multidisciplinary (MDT) care and careful management. Objectives: In Newcastle (UK), children with uveitis are managed in the Newcastle Eye Centre with rheumatology care provided in the Great North Children’s Hospital. A monthly paediatric uveitis clinic exists but is under considerable strain and is without a uveitis clinical nurse specialist (CNS); we therefore undertook a structured evaluation of the current service to support service development led by a paediatric rheumatology CNS on temporary secondment to support the uveitis service. Methods: Retrospective review of patient data currently attending the paediatric uveitis service at the Newcastle Eye Centre and Great North Children’s Hospital in 2021 with focus on patient number, diagnosis and treatments. Additional information about safeguarding was collected. Patients and carer feedback in a snapshot time period in 2021 was collected via a QR code link to an online survey. Results: A total of 202 patients were known to the paediatric rheumatology team and coming to the uveitis service; 58/202 (29%) for uveitis screening, 5/202 (2%) were shared with renal, 62/202 (31%) had idiopathic uveitis and 77/202 (38%) with JIA-associated uveitis. Of patients on treatment, 56/202 (27%) were not on treatment, 104/202 (51%) were on a single immunosuppressive agent (either adalimumab or methotrexate) and 42/202 (21%) were on multiple immunosuppressive agents (methotrexate, azathioprine, mycophenolate mofetil, adalimumab, infliximab, tozilizumab, abatacept). 67/202 (33%) were ready for transitional care (13yrs and older) which was not routinely planned for. Patient feedback was collected from 16/50 patients seen in June 2021. Satisfaction was high (12/16, 76% very satisfied) and particularly praised the clinical nurse specialist ‘The nursing team have always been very reassuring with myself and my son”. A patient and parent information leaflet ‘All about uveitis’ was developed with patient and clinician input. Conclusion: A significant number of patients attend the Newcastle Eye Centre for care related to paediatric uveitis. Three quarters of these patients are on immunosuppressive treatment, many on complex regimes, requiring multidisciplinary care including counselling, prescribing, blood monitoring and disease progression assessment. This can only be provided in a cross-specialty multidisciplinary service supported by both paediatric rheumatology and ophthalmology, with adult ophthalmology providing input for transitional care. Paediatric uveitis services need to be appropriately funded and must take into account the complex needs of multi-system inflammatoryKasap Cuceoglu 1 , Y. Kapucu 2 , A. Keskin 3 , S. L. Sadigh 2 , S. Sener 1 , Z. Balik 1 , Y. Bayindir 1 , E. Aliyev 1 , O. Basaran 1 , E. D. Batu 1 , S. Kadayıfcilar 2 , S. Ozen 1 , Y. Bilginer 1 1 Pediatric Rheumatology, 2 Ophtalmology, 3 Pediatrics, Hacettepe University, Ankara, Turkey Correspondence: M. Kasap Cuceoglu Introduction: The inflammation of uveal structures (iris, choroid, retina) is called uveitis which is anatomically classified into anterior, intermediate, posterior and panuveitis. 1 At the time of diagnosis, it may present acute or chronic course. Chronic anterior uveitis, particularly in juvenile idiopathic arthritis, might lead to visual impairment and blindness due to its asymptomatic and latent course. 2 Objectives: This study aimed to evaluate the data of pediatric patients with uveitis in our outpatient clinic and statistically exhibit our findings. Methods: 131 patients (<18yo) with uveitis from 553 patients were retrospectively screened using the electronic medical records between January 2015 and January 2021 at Hacettepe University. Demographic and clinical characteristics of patients, drug treatments, frequency of recurrence, and uveitis-related ocular complications were indicated. Results: A total of 190 eyes (68 bilateral, 23 right eyes, 31 left eyes) of 131 patients were assessed, including 70 female (53.4%). The median age at diagnosed for uveitis was 7 (1-17) years. The association of uveitis was recorded in patients of 58 (44.2%) isolated uveitis, 55 (41.9%) juvenile idiopathic arthritis, 11 (8.3%), Behçet’s disease, 2 (1.5%) sarcoidosis, 1 (0.7%) Cogan syndrome. Anterior uveitis was the most prevalent anatomic type (n=101; 77.1%) followed by 14 (10.7%) panuveitis, 10 (7.6%) intermediate uveitis, 6 (4.6%) posterior uveitis. Eighty-seven of the patients were treated with corticosteroid drops, intraocular corticosteroid injection in 12 patients, systemic corticosteroids in 13 patients, synthetic DMARD in 55 patients, and biological DMARD in 43 patients were used. Of biological agents, 34 (75.6%) patients received adalimumab, 7 (15.6%) infliximab, 2(4.4%) tocilizumab, 2 (4.4%) alpha-2a interferons. The ocular complications associated with uveitis were cataracts in 23 patients, posterior synechiae in 12 patients, band keratopathy in 7 patients, glaucoma in 6 patients, macular edema, retinal vasculitis/scar in 3 patients, and vitriol in 2 patients. Intraocular surgery was performed on nine patients who developed uveitis-related ocular complications. Finally, 74 patients had a recurrence of uveitis. Conclusion: Juvenile idiopathic arthritis is the most common rheumatological disease that causes non-infectious chronic uveitis in the childhood. Isolated uveitis is also widely seen. Patients should be closely monitored to prevent possible eye complications, and appropriate treatment strategies should be carefully applied. References 1. Sen. ES, Ramanan A V. Juvenile idiopathic arthritis-associated uveitis. Clin Immunol . 2020;211:108322. doi:10.1016/j.clim.2019.108322 2. Gautam Seth N, Kaur S, Yangzes S, et al. Ophthalmic Complications in Pediatric Uveitis. Ocul Immunol Inflammation. Published online July 2020:1-6. doi:10.1080/09273948.2020.1762897 Patient Consent: Yes, I received consent Disclosure of Interest : None declared
I. Maccora, S. I. Orsini, I. Pagnini, M. V. Mastrolia, E. Marrani, G. Simonini lorence, Florence, Italy Correspondence: Maccora Introduction: Behçet syndrome (BS) is a rare disease in childhood and ocular involvement may lead to several complicationehçet patients with ocular involvement and report the performed treatment. Methods: This is a monocentric retrospective study based on chart review conducted at the Rheumatology Unit of Meyer Children’s Hospital, Florence. The study involved patients with a diagnosis of paediatric BS who fulfilled at least one of the following criteria: International Criteria for Behçet’s Disease Criteria, the International Study Group Criteria for BS, Paediatric BD classification criteria. Demographic, laboratory, and clinical data were collected at onset and during disease course. Ocular characteristics were evaluated, and the different treatment performed were assessed evaluating the outcome according to SUN criteria. Statistical Analysis were performed using SPSS v27. Results: Among 33 paediatric patients with a diagnosis of BS, 9 children (27.3%) showed an ocular involvement. Among these 9 patients, 7 were male (77.8%), 5 had a family history for autoimmunity (55.6%), 3 had ANA positivity (33.3%) and 5 HLA B51 positivity (55.6%). The diagnosis of BS was performed at median age of 12.5 years old (IQR 10.1-15.1) while the diagnosis of uveitis at 12.5 years old (IQR 9-14.2). The median of ESR and CRP at disease onset were respectively 7 mm/h (IQR 2-29) and 0 mg/dl (IQR 0-0). Five patients showed a bilateral ocular involvement (55.6%), 2 showed a panuveitis (22.2%), 4 a posterior involvement (44.4%), 2 an intermediate involvement (22.2%), and 1 an anterior involvement (11.1%). Of these 9 patients, Five patients showed ocular signs and symptoms: 4 hyperaemia, 3 photophobia, 2 ocular pain, 4 blurred vision. One patient showed recurrent fever (11.1%), 8 recurrent oral ulcers (88.9%), 4 recurrent genital ulcers (44.4%), 2 gastrointestinal symptoms (22.2%), 3 cutaneous involvement (33.3%). All the patients received at least one treatment, 4 patients received 2 treatment and one patient 4 treatment to achieve disease control (1 methotrexate, 3 colchicine, 3 Azathioprine, 2 adalimumab and 1 canakinumab). Ocular control was achieved in all patients with the following drugs: 3 Adalimumab, 1 tocilizumab, 1 infliximab, 2 azathioprine, 1 colchicine and 1 systemic corticosteroid. The drug able to achieve ocular control was administered after a median time of disease onset of 2 months (IQR 1-30 months), with a median follow-up of 20 months (IQR 3.5-31.5months) and a median duration of therapy of 16 months (IQR 3-29 months). The median time to achieve a response to therapy was 3 months (IQR 2-3 months), and median time to achieve remission on therapy of 10 months (IQR 6-18 months). One patient treated with adalimumab, and one with azathioprine relapsed respectively after 11 months and 33 months from achievement of remission. The treatment was stopped in 2 patients (1 infliximab and 1 systemic corticosteroid) without relapse. Conclusion: Ocular involvement in paediatric BS is one of the most common sight threatening complications. In our case series, a biologic was required in 5 children in order to achieve the ocular control in BS. Further studies in large cohort of paediatric BS are necessary in order to evaluate the most efficacious treatment of this sight-threatening complMarino 1 , L. Marelli 2 , R. Caporali 1,3 , E. Miserocchi 4 1 Pediatric Rheumatology, ASST Pini-CTO, 2 University of Milan, Milan , Italy, 3 Department of Clinical Sciences and Community Health and Research Center for Pediatric and Adult Rheumatic Diseases (RECAP.RD), University of Milan, 4 Ophtalmology Department, Ospedale San Raffaele, University Vita-Salute, Milan, Italy Correspondence: A. Marino Introduction: Uveitis associated with juvenile idiopathic arthritis (JIA-U) is a cause of ocular morbidity. JIA-U might be difficult to treat since a substantial proportion of children are refractory to methotrexate (MTX) and TNF inhibitors (TNFi). Tocilizumab (TCZ) is a humanized monoclonal antibody to the IL-6 receptor preventing IL-6 from binding to its soluble and membrane-bound receptors; TCZ is approved for the treatment of systemic and polyarticular JIA. Few studies, mainly case series, explored the efficacy of intravenous TCZ in JIA-U, whereas little is known about subcutaneous TCZ (SC-TCZ) for uveitis in patients with JIA. Objectives: To describe the efficacy and safety of SC-TCZ in a cohort of JIA patients with refractory uveitis. Methods: Retrospective observational monocentric study. Patients with JIA-U treated with SC-TCZ were enrolled from the Pediatric Rheumatology Unit of Gaetano Pini Hospital, Milan, Italy from January 2011 to December 2021. Results: Thirteen patients (9 female) were enrolled: 8 patients with oligoarticular JIA and 4 with polyarticular JIA. All patients were ANA positive. The age at articular disease onset was 2.2 ± 2.1 (0.8-6.5) years; the age at uveitis onset was 4.5 ± 2.8 (1.8-10.6) years. The mean follow-up time was 19.3 ± 9.2 (6.8-32.3) years. Ten patients had bilateral uveitis. Uveitis location included anterior uveitis (8 patients and 15 eyes), anterior and posterior (1 patient and 1 eye), intermediate uveitis (1 patient and 2 eyes), posterior uveitis (1 patient and 1 eye), and panuveitis (2 patients and 4 eyes). All patients have been treated with synthetic disease modifying antirheumatic drugs (sDMARDs): all received methotrexate (MTX), 8 of them received cyclosporine too. However, just three patients received MTX in combination with SC-TCZ. The mean number of biological DMARDs (bDMARDs) was 2.4 ± 1.3 (1-5). The bDMARDs which had been used before starting SC-TCZ included adalimumab in 12 patients, infliximab in 7 patients, other TNFi in 10 patients (4 etanercept, 3 certolizumab, 3 golimumab), and 2 subjects received rituximab. Descriptors of subcutaneous tocilizumab treatment in the cohort are reported in Table 1. Two patients started SC-TCZ for articular flare and do not experienced uveitis flare during the anti-IL6 treatment. Overall, SC-TCZ was safe, and no side effects were observed during the treatment period. Three patients discontinued SC-TCZ: 2 patients due to secondary inefficacy (switched to golimumab and rituximab, respectively), 1 subject due to loss of compliance (switched to oral tofacitinib). One patient withdrawn the drug because of pregnancy. Conclusion: Subcutaneous tocilizumab can be effective and safe in patients with JIA and uveitis recalcitrant to several bDMARDs. Further studies, with larger cohorts and prospectively designed, are advisable to verify this opportunity. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
Uveitis is the most common extra-articular manifestation of juvenile idiopathic arthritis (JIA) and may result in sight-threatening complications. Objectives: To explore the current state of publications on JIA-associated uveitis and to characterize it using a bibliometric approach. Methods: An advanced search using the query [(“juvenile rheumatoid arthrit*” AND (uveit* OR iridocyclit*)) OR (“juvenile idiopathic arthrit*” AND (uveit* OR iridocyclit*))] was performed in May 2022 within all titles in the Core Collection of the Clarivate Web of Science database. Only publications until 2021 were includedtilized for scientific production, author, authors collaboration, and citation analyses. Network visualization cluster analysis was conducted in VosViewer (Leiden University, Leiden, The Netherlands). Results: The search yielded 496 items. After 5 items that were published in 2022 and 7 irrelevant items were excluded, 484 papers were analysed. They were published starting in 1970 by 1660 authors in 98 sources, and 97.7% (473 of 484) of them were written in English. Articles (n=225; 46.5%) and meeting abstracts (n=148; 30.6%) comprised the majority of the publications. Singled-authored document were rare (n=22; 4.5%) and were written by 1.0% (n=17) of all authors. The average years from publication was 11.1. The collaboration index was 3.6. There were 0.3 documents per author and 3.4 authors per document and 6.8 co-authors per document. The annual growth rate was 1.6%. The most productive year was 2019 (n=42; 8.7%). Bradford’s law showed that only the following journals formed the core sources: Annals of the Rheumatic Diseases (n=50; 10.3%), Arthritis & Rheumatology (n=35; 7.2%), Journal of Rheumatology (n=33; 6.8%), Ocular Immunology and Inflammation (n=33; 6.8%), Arthritis and Rheumatism (n=29; 6.0%). Top-contributing authors included Heiligenhaus A (n=61; 12.6%), Ramanan AV (n=27; 5.6%), and Minden K (n=26; 5.4%). Heiligenhaus A (h-index=23), Heinz C (h-index=15), and de Boer JH (h-index=14) had the highest local impact. The most prolific institutions included St. Franziskus-Hospital (n=48; 9.9%), Emory University (n=33; 6.8%), Johns Hopkins University (n=31; 6.4%), and the University of Duisburg-Essen (n=31; 6.4%). The h-index of the research field was 54. Totally, there were 9,191 citations with average citations per document of 19.0 and average citations per year per document of 1.8. Each publication had an average of 7.4 references. Shown in Table 1, the United States was the most common corresponding author’s country (106 of 341; 31.1%) and had the highest number of total citations (2,213 of 8,093; 27.3%). Switzerland had the highest average article citations (52.75 citations/article). Network co-authorship analysis showed that 21 countries, each with at least 5 documents, formed 6 clusters; Germany, Switzerland, and the United States had the highest link strength. Conclusion: The current study of publications related to juvenile idiopathic arthritis-associated uveitis identified the major contributors, co-authorship relationship, and article citations. The United States was the most prolific country. Switzerland produced the most influential papers. Germany showed the highest co-author collaboration. St. Franziskus-Hospital was the most productive institu190. A case of 15-years-old girl with parvovirus-arthritis P191. Synovial inflammation in antibiotic-refractory Lyme arthritis is characterized by clonally expanded peripheral T helper cells and TCR convergence P192. Characterization of an ataxia-teleangectasia patient with inflammatory phenotype mimicking a rheumatologic disease P193. Juvenile idiopathic arthritis associated to primary immunodeficiency in pediatric age: a case series I. Pagnini, A. rossi, I. maccora, M. V. mastrolia, E. marrani, G. simonini Aou Meyer, Firenze, Italy Correspondence: I. Pagnini Introduction: Primary Immunodeficiency (PID) are a group of more than 400 monogenic diseases with an estimated overall incidence of 1:10,000. The related immunoregulatory defects may predispose to development of autoimmune diseases, such as inflammatory arthropathies. Juvenile Idiopathic Arthritis (JIA) is the most common chronic rheumatic disease of unknown etiology in childhood. It occurs 50 times more common in DiGeorge syndrome (DGS) than in normal population, and has a prevalence of about 10% in Bruton agammaglobulinemia (BA). Objectives: We describe clinical features, laboratory, imaging data and therapeutic outcomes in children with JIA and PID. Methods: Among 450 JIA children followed at our unit, we enrolled 3 patients, followed in Pediatric Rheumatology Unit at Meyer Children’s Hospital in Florence, affected by JIA, according to ILAR criteria, and fulfilling the diagnosis of PID: 2 patients with DGS and 1 with BA. Results: Case 1: male of 5 years old affected by DGS from 2 years old, who developed oligoarticular JIA. He was treated with NSAID and methotrexate, without benefit. He received two consecutive ankle injections, 6 months apart, in addition to 6 courses of oral glucocorticoids also. Etanercept was then added to methotrexate 8 months after JIA diagnosis. He then achieved remission over 2 months later. Case 2: female of 5 years old affected by DGS, by 3 years and 11 months old of age, who developed oligoarticular JIA. She was treated with NSAID, joint injection and methotrexate, without clinical response after 8 months. Due to the increase of liver tests, methotrexate was then stopped, and Adalimumab started. At 6-month follow-up, she was in clinical remission. Case 3: male of 10 years and 10 moths old affected by BA, since he was 9 years old, later developing polyarticular JIA. He was treated with NSAID, methotrexate, joint injections and three course of oral glucocorticoid with no benefits, plus IVIg at 400 mg/kg/monthly. Due to persistent clinical activity after 5 months, Adalimumab was then added. However, over the following 6 months new multiple joint injections, two course of oral glucocorticoid and increased of IVIg at immunomodulation dose was performed without achieving disease control. Adalimumab was switched to Etanercept and after 4 months he was in clinical remission on medications. Conclusion: The association between autoimmune diseases, including JIA, and immunodeficiency is well described in literature, but scanty evidence is referred to the clinical outcomes according to the standard treatment. In our experience these patients showed severe disease, with recurrent arthritis, although several joint injections, oral glucocorticoids courses and methotrexate treatment. In our cohort, children exhibited no response to the conventional first step treatment with methotrexate, whilst achieved disease remission on medication using anti-TNF agents. Larger, multicenter cohort will be necessary to identify the optimal treatment for these specific patients. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
A. Margaryan WIGMORE CLINIC, Yerevan, Armenia Margaryan Introduction: Acute arthritis is one of the presentations of Parvovirus B19 beside erythema infectiosum and aplastic crisis. Objectives: This is a case report of 15-years-old girl with acute polyarthritis due to parvovirus B19. Methods: This is a case report based on medical chart of the patient. Results: A 15-years-old girl presented to pediatrician on 7 th day of the following complains: pain in upper extremities, difficulty raising arms, fatigue, painful and swollen wrists, rash on palms. On 2 nd day of illness CBC was performed: PLT 91*10 3 /L. Objective examination: general condition was stable, mild hyperemia of cheeks, red-purple papulose rash on palms, swollen wrists, painful and swollen small joints of hands with limited movements. Palpation of the upper extremities from the shoulders to the wrists were painful. No other changes: no changes in gait, no squatting difficulties, no changes in other joints, normal muscle strength, no hepatosplenomegaly. Laboratory findings: CBC’ normal (no anemia, no reticulocytosis, PLT’ 160*10 3 /L), ANA, ALT, AST, LDH, CK’ Normal, Covid-19 total Ab’ positive, Parvovirus B19 IgM’ positive, EBV IgM’ negative. Treatment with NSAID initiated. After two weeks the diagnosis of the parvovirus B19 was proven by seroconversion. Follow-up: after 4 weeks from treatment minor improvement in complains. NSAID had been continued and on 6 th week there were no any complains and examination was normal. Conclusion: Parvovirus B19 associated acute arthritis is mostly self-limited and resolves with symptomatic treatment. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
J. Dirks 1 , J. Klaussner 1 , A. Almamy 1 , G. Haase 1 , U. Fischer 1 , A. Holl-Wieden 1 , C. Hofmann 1 , H. Girschick 2 , H. Morbach 1 1 Children’s University Hospital, Würzburg, 2 Vivantes Klinikum Friedrichshain, Berlin, Germany Correspondence: H. Morbach Introduction: Antibiotic-refractory Lyme arthritis (ARLA) is defined by persistent arthritis despite adequate antibiotic treatment and may develop in up to 10 % of patients with Lyme arthritis. Disease pathogenesis of ARLA is still inadequately understood. In detail, whether chronic inflammation in ARLA is maintained by chronic antigen stimulation (e.g. by persistent borrelial antigens or autoantigens) is not elucidated yetpatients with ARLA. Methods: Flow cytometric analysis of synovial fluid CD4+ T cells from children with ARLA and juvenile idiopathic arthritis (JIA). High-throughput sequencing of the T cell receptor Vβ (TCRVB) repertoire as well as single-cell RNA sequencing (scRNAseq) of sorted CD4+ T cell populations. Results: Multidimensional flow-cytometric analysis revealed a striking expansion of an IL-21 and IFN-γ co-expressing PD-1hiCXCR5-HLA-DR+ CD4+ T cell population resembling peripheral T helper (TPH) cells in the joints of pediatric ARLA patients compared to JIA patients. Indeed, ARLA patients displayed the highest frequencies of TPH cells, which could separate this group of patients from JIA. Accumulating TPH cells exhibited signs of clonal expansion with restricted TCR clonotypes. Those clonotypes showed an overlap between different ARLA patients but not to JIA patients. SF TPH cells were enriched for distinct and overlapping TCRVB motives that showed signs of a convergent immune response and could not be identified in SF CD4+ T PH cells of JIA patients. Conclusion: The inflamed joints of children with ARLA are characterized by a striking expansion of oligoclonal TPH cells with signs of TCRVB convergence that is distinct from other forms of chronic arthritis (e.g. JIA). This peculiar pattern suggests that disease specific immune response may sustain chronic inflammation in ARLA. Current experiments are ongoing to dissect whether this maladaptive immune response targets persisting borrelial antigens or rather autoantigens. Disclosure of Interest : None declared
M. F. Natale 1 , L. De Nardi 2 , C. Celani 3 , F. De Benedetti 3 , A. Insalaco 3 1 Rheumatology, Bambino Gesu Children’s Hospital, Rome, 2 Rheumatology, IRCCS materno infantile Burlo Garofolo, Trieste, 3It is well known that some inflammatory manifestations such as arthritis and lung diseases could be part of both rheumatological and immunological disease, representing a real diagnostic challenge. Ataxia telangiectasia (A-T) is a rare autosomal recessive condition characterized by skin telangiectasia, ataxia and immunodeficiency in which lungs and joints are frequently involved. Objectives: We described an autosomal dominant A-T patient with a novel heterozygous mutation in ATM gene presenting with a clinical phenotype characterized by inflammatory manifestations, articular and lung involvement. Methods: Whole-exome sequencing Results: When the patient was first seen at our center at age 7 years , presented with a clinical picture characterized by poor growth, chronic liver granulomatous inflammation, interstitial lung disease with bilateral bronchiectasis and recurrent episodes of large joint ahrtritis (wrists, knees and ankles). The diagnosis of pediatric sarcoidosis had been done in another center by where a treatment with high dose systemic glucocorticoids and intraarticularar glucocorticoids injection were started with no significant improvement. We performed the combined dosage of angiotensin converting enzyme (ACE) and chitotriosidase, resulted in the normal range excluding the diagnosis of pediatric sarcoidosis. In the suspicion of interferonopathy with pulmonary and joint involvement (COPA syndrome) an interferon signature was performed with a normal value (n.v. <2). Immunological blood exams showed a reduction in total immunoglobulin levels and in the absolute count of T and B lymphocytes, a marked reduction in thymic output (CD4Ra 1.4%), a population B with phenotypic characteristics of “ atypical B Memory“, abnormal NK cells function and very high levels of serum alpha-protein (273ng /ml) Whole exome sequencing identified a novel homozygous mutation in ATM gene causing the ataxia-telangiectasia syndrome Conclusion: This case emphasize the importance to consider a diagnosis of primary immunodeficiency (PID) even in patients with inflammatory lungs and joints involvement mimicking a rheumatologic condition. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
P194. CD8 T-cell expansion may compensate for natural killer cell dysfunction in mice with chronic excess IL-18 95. Liver involvement in systemic juvenile idiopathic arthritis, a multi faced problem P196. Predictors of relapse of macrophage activation syndrome in patients with systemic juvenile idiopathic arthritis: cohort study P197. CD38HIGH/HLA-DR+ CD8+ T cells as diagnostic marker for hlh secondary to visceral leishmaniasis P198. COVID-19 in pediatric patients with rheumatic diseases and the history of macrophage activation syndrome: retrospective single center study P199. CD4 DIM CD8 + T cell frequency distinguishes patients with MAS/SHLH from patients with active SJIA and is associated with MAS/SHLH severity P200. A strange case of hyperferritin: from management to treatment P201. Idiopathic systemic hyperinflammation, a formidable diagnostic challenge P202. A case of macrophage activation syndrome associated with juvenile systemic lupus erythematosus in a 7-year old girl J. Varghese 1 , E. Landy 2 , V. Dang 1 , E. Peauroi 3 , L. Eisenlohr 3 , S. Canna 1 1 The Children’s Hospital of Philadelphia, Philadelphia, 2 University of Pittsburgh, Pittsburgh, 3 University of Pennsylvania, Philadelphia, United States Correspondence: S. Canna Introduction: Systemic Juvenile Idiopathic Arthritis (SJIA) and Macrophage Activation Syndrome (MAS) are associated with both low peripheral Natural Killer (NK) cell numbers and NK dysfunction. They are also associated with chronically elevated and unopposed peripheral blood levels of the inflammasome-activated cytokine IL-18. NK cells constitutively express the IL-18 receptor and canonically their response to IL-18 is to augment the effects of other cytokines (like IL-12) on Interferon gamma (IFNg) production and cellular cytotoxicity. Objectives: To determine the effects of chronic IL-18 excesson NK cell phenotype, distribution and function. Methods: We assessed NK cells by flow cytometry and RNAseq in mice with transgenic expression of mature, excretable IL-18 ( Il18tg mice). Results: Similarly to observations in SJIA/MAS, Il18tg mice demonstrated decreased numbers of NK cells in peripheral blood, spleen, and liver. Such mice also demonstrated a concomitant increase in activated CD8 T-cells in these same organs. Transcriptionally, NK cells from Il18tg mouse spleens showed increases in a few innate and NF-kB-related pathways, with effects on specific cytokines like Csf2 (encoding GM-CSF). These findings were paralleled by in vitro stimulation of WT NK cells with various cytokine combinations, including IL-18. However, the main transcriptional program was dominated by increases in cell cycle and gene expression programs, suggesting rapid cellular turnover. Additionally, Il18tg NK cells show a likely compensatory downregulation of Il18r1 at the mRNA and protein levels. This effect was diminished in Il18tg mice with selective deletion of Il18r1 on T cells ( Il18tg;Il18r1 delT ), suggesting T-cell responses to IL-18 affect NK cell homeostasis. Type 1 Innate Lymphoid cells showed similar transcriptional changes but no appreciable diminution of numbers. In vitro, NK cells from Il18tg mice died quickly after isolation, clouding assessment of in vitro killing or cytokine production. In vivo, Il18tg;Il18r1 delT were not protected from immunopathology induced by repeated stimulation through Toll-like Receptor 9 (TLR9-MAS) but rather showed more severe disease by some parameters, suggesting non-T cell response to IL-18 contribute to the more severe TLR9-MAS observed in Il18tg mice. The DNA poxvirus ectromelia causes an abortive infection in WT mice, but severe viral immunopathology in perforin-deficient or NK-depleted mice. Il18tg mice infected with ectromelia failed to expand NK cells or upregulate activation markers like CD49b or NKG2D, but nevertheless clear virus similarly to WT mice and did not develop immunopathology at early timepoints. Conclusion: Il18tg mice may be a valid model to examine the effects of chronic IL-18 on NK cell homeostasis and the inter-relationship of NK and CD8 T-cells. Preliminary results suggest CD8 T-cell expansion may overcome NK cell deficiency/dysfunction in the early fight against viral infection, but may nevertheless predispose to MAS-like immunopathology. Patient Consent: Not applicable (there are no patient data) Disclosure of Interest : J. Varghese: None declared, E. Landy: None declared, V. Dang: None declared, E. Peauroi: None declared, L. EisenlohrR. Erkens 1,2 , M. Jansen 2 , J. Calis 1 , J. van Loosdregt 1 , S. Vastert 1,2 1 Center for Translational Immunology, UMC Utrecht, 2 Pediatric Rheumatology, Wilhelmina Children’s Hospital, Utrecht, Netherlands Correspondence: R. Erkens Introduction: Recently several cases of Systemic Juvenile Idiopathic Arthritis (SJIA) with hepatic inflammation have been described and linked to Macrophage Activation Syndrome (MAS) or delayed drug reactions (DDR) to biologicals. Objectives: To identify characteristics that aided in the differential diagnostic process between two forms liver involvement in SJIA. Methods: Here we present two recent cases of liver involvement in SJIA and describe their clinical presentation, disease course, and response to installed treatment. Results: Patient 1 A girl of 2 years and 10 months, at the start of 2020 diagnosed with SJIA complicated by severe MAS. DNA HLH-panel, functional and cytolytic cell analysis showed no signs of underlying HLH associated abnormalities. Clinical improvement was induced by the combination of corticosteroids (GC), cyclosporine (CSA) and anakinra. Medication was tapered without problems. Early 2021 there was a flare of SJIA with a re-emergence of MAS, methylprednisolone (MP) pulses were given and anakinra was switched to canakinumab. Initially the patient seemed to improve clinically and fever disappeared, but the patient subsequently developed signs of hepatitis with conserved liver function and increasing transaminases, Galectin 9 and CLXL10 levels. Viral and autoantibody causes of hepatitis were excluded. Liver biopsy showed a mononuclear infiltrate with focal erytrophagocytosis. The combination of an activated INFy pathway and infiltration of activated monocytes and T cells on histology together with the history of MAS pointed to hepatic MAS. Canakinumab was continued, MP pulses initiated and CSA was added which resulted in clinical improvement. She is now in remission on canakinumab and CSA in tapering. Patient 2 A previously healthy girl of 1 year and 8 months was diagnosed with SJIA in 2021. First line treatment with Anakinra was started with a good initial response. 10 days after start of anakinra there was a reemergence of fever. The anakinra dose was increased with a short lasting improvement. The patient developed a pruritic rash, hepatomegaly, increasing transaminases and eosinophilia without appearing clinically ill. Liver function remained intact. Infectious and autoantibody causes of hepatitis were excluded. The HLH/MAS criteria were carefully monitored, DNA and functional HLH tests were initiated. A PET-CT scan was performed that showed lymphadenopathy with increased metabolic uptake, suspicious for lymphoma. The pathology report of the lymph extirpation showed no signs of lymphoma but a striking eosinophilia. This together with a distinctly elevated CCL17/TARC and no clinical picture of MAS prompted the suspicion of a DDR. Anakinra was stopped and GC were added. Hepatitis improved after this but the SJIA flared as the patient developed a spiking fever. Canakinumab was started and clinical outcome improved. Patient is currently in remission and GC are being tapered. Conclusion: These two cases show that patients with liver involvement in the setting of SJIA can have a similar presentation with different implications for treatment. Differentiation between DDR and hepatic MAS is important, but can be challenging as a clear definition of hepatic MAS is lacking. Interestingly, both patients are carriers of the DRB1*15:01 allele, that has been associated with DDR to biologicals. Key factors that aided in the differential diagnosis were the extent of eosinophilia in blood and biopsy, the pruritic rash, and relatively low HLH parameters for a DDR. The history of recurrent MAS, IFNγ pathway activation and monocytic infiltration in the liver for the diagnosis of hepatic MAS. A broadly supported and well defined definition of Hepatic MAS is needed to boost collaborative research into this rare complication of SJIA. Patient Consent: Yes, I received consent Disclosure of Interest : R. Erkens: None declared, M. Jansen: None declared, J. Calis: None declared, J. van Loosdregt: None declared, S. Vastert Consultant with: Sobi and Novartis
E. Krekhova 1 , E. Alexeeva 1 , I. KriulinI. Kriulin Introduction: Macrophage activation syndrome (MAS) is a life-threatening complication of systemic juvenile idiopathic arthritis (sJIA), self-diagnosis of which can significantly improve the outcomes of this complication. MAC, like sJIA, can have a continuously recurrent character of disease. Knowledge of predictors of relapse in patients with MAS in anamnesis will allow to suspect MAS in time and start therapy. Objectives: To develop a model for predicting MAS recurrence in patients with systemic juvenile idiopathic arthritis. Methods: Predictors of clinical and laboratory features of 100 patients with sJIA-associated MAS were collected in our study. In the analyzed cohort of 100 patients 114 cases of MAS were registered, 100 primary and 14 repeated. The method of multiple logistic regression analysis was used to predict the occurrence of MAS relapses in patients with sJIA. All clinical and laboratory manifestations of MAS were collected as prospective predictors. As a model of logistic regression, the dependence of the logarithm of the chance of occurrence of the predicted event on a linear combination of predictor variables was used. The interpretation of the logistic regression parameters was performed on the basis of the odds ratio (OR) with a 95% confidence interval. The sensitivity and specificity of the predictors were evaluated using the analysis of ROC curves. Results: For the development of MAS relapse, a statistically significant value was found among the following predictors: lymphadenopathy (OR =0.038; CI 95% 0.003-0.572), decrease in the number of red blood cells (OR =18.915; CI 95% 3.472-103.056), platelets (OR =0.988; CI 95% 0.980-0.995), chloride levels (OR =4,401; 95% CI 1,401-1,823) and increase lactate dehydrogenase activity (OR =0.996; 95% CI 0.992-1,000). The coefficient of determination (R2) for this model was 0.636. The specificity of the model is 98.0%. The total percentage of correct predictions was 95.6%. According to the results of constructing the ROC curve, the AUC index was 0.954±0.027 (95% CI 0.902 – 1,000; p <0.001). Conclusion: According to the results of our study, the parameters for predicting the recurrence of MAS in patients with sJIA can be: lymphadenopathy, decrease in number of red blood cells, platelets, chloride level and increase LDH activity. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
M. V. Mastrolia 1 , S. Boscia 2 , L. Galli 3 , L. Lodi 2 , L. Pisano 2 , I. Maccora 1 , S. Ricci 2 , I. Pagnini 1 , E. Marrani 1 , C. Azzari 2 , G. Simonini 1 1 Rheumatology Unit, Meyer Children’s University Hospital, 2 Immunology and Molecular Microbiology Unit, Meyer Children’s University Hospital , 3 Pediatric Infectious Diseases Unit, Meyer Children’s University Hospital, Department of Health Sciences, University of Florence, Florence, Italy Correspondence: M. V. Mastrolia Introduction: The measurement of the CD8 + CD38 high /HLA-DR + population in children with acute onset of shock and multisystem organ failure represents an important diagnostically useful parameter, to readily distinguish Hemophagocytic lymphohistiocytosis (HLH) from sepsis or healthy controls. A cut-off value > 7% of CD38 high /HLA-DR + cells among CD8 + T cells differentiates HLH from sepsis with a positive predictive value of 96%, and a negative predictive value of 100%. Therefore, this novel biomarker may be helpful in the differential diagnosis of critical patients, for which a timely treatment appears crucial. Objectives: To confirm the role of CD38 high /HLA-DR + CD8 + T cells as precocious HLH diagnostic biomarker Methods: We measured the percentage of CD38 high /HLA-DR + among CD8 + T cells by flow cytometry assay in 4 Italian children suffering from HLH secondary to visceral Leishmaniasis Results: Four patients, all coming from Tuscany, 3 males and one female, mean age of 20.25 months (IQR 24.25), were included. No one reported a recent stay in endemic areas, recent insectbites, contact with wild animals. All of them met the HLH 2004 diagnostic criteria 2 at hospital admission. Signs of hemophagocytosis were detected on the bone marrow (biopsy performed in 3/4) in all cases. The Leishmaniosis diagnosis was performed by the polymerase chain reaction positivity of the protozoan genome in all subjects: on bone marrow blood in 1 subject; on peripheral blood in the other 3, in 1 out of them subsequently confirmed even on the bone marrow. Serological tests resulted positive IgG and IgM in all patients. Meanwhile infectious tests were ongoing, due to life-threating clinical conditions, immunosuppressive therapy was started to control of the fast-evolving HLH. Steroids were administered in all patients, and immunoglobulin (2g/kg over 12 hours) and anakinra (5 mg/kg/daily) were added in 3.After positive results for Leishmania, liposomal amphotericin B was started according to standard protocol in 3 children with 7 infusions (3 mg/kg/dose) on days 1-5, and after at 14 and 21 days. In one patient,10 doses were required due to massive immunosuppression. All patients promptly recovered and immunomodulatory therapy was weaned and completely stopped in 4 weeks (range 2-6). CD38 high /HLA-DR + cells were measured at the disease onset before starting treatment, reporting a mean percentage value of 36.95% (IQR 32%). CD38 high /HLA-DR + levels were also assessed over the disease course in 3 out 4 children. A decreasing trend has been documented with a complete normalization at 4 weeks (range 3-5) from the HLH onset. However, the reduction up to the normalization resulted slower if compared to the clinical and biochemical HLH parameters normalization, including ferritin. Conclusion: Our results confirm the pivotal function of CD38 high /HLA-DR + cells as precocious HLH biomarker in the clinical setting of children presenting fever and multisystem organ failure, thus allowing an early initiation of the appropriate immunomodulatory treatment. The curve of the CD38 high /HLA-DR + cells over the HLH course seems to suggest that this specific CD8+ T cell subset may be a valuable parameter for monitoring the response to treatment. Further prospective studies will be needed to better define the expansion of CD38 high /HLA-DR + T cells in this groupS. Salugina, E. Fedorov, Z. KolkhidIt is well known that viral infections may be associated with an increased risk of flare in children with rheumatic diseases (RD), including probability of developing macrophage activation syndrome (MAS) in predisposed patients (pts). The pandemic of novel coronavirus disease 2019 (COVID-19) represents a source of concern for the management of pts with RD and the history of MAS. Objectives: To analyze in a retrospective study the course of COVID-19 in pts with RD and the history of MAS. Methods: The study included all pts with RD and the history of MAS who had COVID-19. The diagnosis of COVID-19 was confirmed either by positive polymerase chain reaction (PCR) or positive of serologic tests for SARS-CoV-2 (immunoglobulin (Ig) G, IgM or both). Results: During the period from March 2020 to April 2022 COVID-19 was verified in 12 pts with RD and the history of MAS (3 males/9 females). 8 pts had systemic juvenile idiopathic arthritis (sJIA), 2 – systemic lupus erythemathosus, 1 – juvenile dermatomyositis, 1 – JIA, polyarthritis. The median age of RD onset was 6.75 y.o. [IQR 5.5; 9.0]. 8 pts had 1 episode of MAS in history, 3 – 2 episodes, 1 – 3 episodes. Total 17 episodes of MAS was verified (11 – at onset, 4 – associating with flare of RD, 1 – after surgery, 1 – accompanied with viral infection H1N1). The median of disease duration at the time of COVID-19 was 6.0 y [5.75; 9.25]. Prior to COVID-19 8 pts received glucocorticoids per os (6 pts - 0-10 mg/day, 2 pts > 10 mg/day), 10 pts received DMARDs (methotrexate - 6, cyclosporine A - 2, mycophenolate mofetil - 1, hydroxychloroquine - 2), 10 pts - biologics (B) (tocilizumab – 6 pts, canakinumab – 2 pts, abatacept – 1 patient, infliximab – 1 patient). All pts received stabile therapy and had an inactive status of RD before COVID-19. 3 pts were asymptomatic COVID-19 (only PCR+, planned before hospitalization or after contact). 7 pts had a mild course of COVID-19: 7 pts had rhinorrhea, fatigue, 4 - anosmia, 3 - abdominal pain, 2 - diarrhea, 1 - headache. They received symptomatic treatment. 2 pts required hospital admission: 16 year-old boy with sJIA (duration of disease – 9 years) and interstitial lung disease (ILD) (initial manifestations verified 18 months before COVID-19) and 12 year-old girl with sJIA (duration of disease – 5 years). Earlier both pts had on 2 episodes of MAS, they received canakinumab in current mode (the boy as a 4th B after secondary inefficiency of tocilizumab, rituximab and anakinra, the girl as a 2nd B after infusion reaction on tocilizumab). Symptoms of COVID-19 in these pts included fever, headache, fatigue, arthralgia. The boy had COVID-associated pneumonia (ground-glass opacities of 45% of lung on CT scan images) with cough, chest pain and dyspnea. The girl had flare of sJIA and Epstein-Barr virus superinfection with persistence of elevated TA (ALT- 577-335 U/l, AST-268-154 U/l) during 2 months after COVID-19. Both pts received COVID-19–related treatments: glucocorticoids IV, azithromycin, canakinumab with reduced interval; acyclovir and intravenous immunoglobulin were also prescribed to the girl. No pts had critical course of disease. Conclusion: In our study the majority of pts had a mild course of COVID-19 infection despite a history of MAS. This may be due to the development of COVID-19 against the background of an inactive status of RD and stable therapy. The small number of pts in our study, however, does not allow us to reliably judge the impact of the history of MAS on the outcomes of COVID-19 and the further course of RD. As understanding of the COVID-19 pandemic appears to change, prospective follow-ups will provide additional data on the long-term impact of COVID-19 on the course of RD in these pG. Prencipe 1 , A. De Matteis 2 , M. Colucci 3 , M. N. Rossi 1 , I. Caiello 1 , P. Merli 4 , N. Tumino 5 , V. Bertaina 4 , M. Pardeo 2 , C. Bracaglia 2 , F. Locatelli 6 , F. De Benedetti 2 1 Laboratory of Rheumatology, 2 Division of Rheumatology, 3 Renal Diseases Research Unit, 4 Department of Onco-Haematology and Cell and Gene Therapy, 5 Department of Immunology, Ospedale Pediatrico Bambino Gesù, 6 Department of Onco-Haematology and Cell and Gene Therapy, , Ospedale Pediatrico Bambino Gesù; Sapienza, University of Rome, Rome, Italy Correspondence: G. Prencipe Introduction: CD8 + T-cell activation has been demonstrated to distinguish patients with primary and infection associated hemophagocytic lymphohistiocytosis (pHLH and iaHLH) from patients with early sepsis. Objectives: In this study, we evaluated the activation profile of CD8 + T cells in patients with various forms of secondary HLH (sHLH), including macrophage activation syndrome (MAS) complicating systemic juvenile idiopathic arthritis (sJIA). Methods: Flow-cytometry analysis was performed on peripheral blood mononuclear cells (PBMCs) isolated from children with inactive sJIA (n=17), active sJIA (n=27), MAS in sJIA (n=14), iaHLH (n=7) and with other forms of sHLH (n=9). Analyses were also performed in a prospectively enrolled validation cohort including 9 patients with active sJIA and 16 patients with sHLH. Results: PBMCs immunophenotyping revealed the presence of a significantly higher percentage of CD8 + T cells co-expressing the CD4 marker (CD4 dim CD8 + T cells) in patients with MAS or with other forms of sHLH, compared to patients with inactive or active sJIA. ROC curve analysis showed that the percentage of CD4 dim CD8 + T cells reliably discriminated patients with all forms of sHLH (AUC=0.92, p<0.001) as well as MAS patients (AUC=0.90, p<0.001) from those with active sJIA. We found that, compared to CD8 + T cells not expressing CD4 (CD4 - CD8 + T cells), a markedly higher percentage of CD4 dim CD8 + T cells expressed high levels of CD38 and HLA-DR activation markers. In addition, a significantly higher frequency of CD4 dim CD8 + T cells expressed the activation/exhaustion markers CD25, PD1, and CD95. We also found that the frequency of IFNγ positive cells was significantly higher among CD4 dim CD8 + T than among CD4 - CD8 + T cells from peripheral blood of MAS patients. Accordingly, peripheral CD4 dim CD8 + T cells cultured ex vivo tended to spontaneously release higher amount of IFNγ compared to CD4 - CD8 + T cells. Supporting the pathogenic role of these cells in sHLH, the frequency of CD4 dim CD8 + T cells significantly correlated with most of the laboratory parameters of disease severity, including ferritin, and with circulating levels of IL-18 and CXCL9. We confirmed our results in the validation cohort, by showing that the percentage of CD4 dim CD8 + T cells was significantly higher in patients with MAS/sHLH than in patients with active sJIA (p<0.001) and was able to distinguish sHLH patients from patients with active sJIA (AUC=0.93, p<0.001). In the validation cohort, we also analyzed the TCR Vβ repertoire by using a flow cytometric approach. We did not find expansion of any particular TCR Vβ family in CD3 + T cells of patients with MAS/sHLH compared to those of patients with active sJIA, suggesting that in sHLH CD8 + T cell activation is antigen-independent. Finally, we found that the frequency of CD4 dim CD8 + T cells was significantly associated with the clinical severity score (r=0.56, p<0.0001), based on endpoints including death, length of hospitalization, stay in intensive care unit and dose of glucocorticoids. Conclusion: Altogether, our data showing that CD4 dim CD8 + T cells are increased in patients with MAS/sHLH and associated with disease severity strongly support their involvement in MAS/sHLH pathogenesis. They also suggest a potential prognostic relevance of the assessment of CD4 dim CD8 + T cells in HLH/MAS syndrome and support the rationale for novel therapeutic strategies targeting activated CD8+ T cells. Disclosure of Interest : None declared
M. T. Riccio 1 , A. Catzola 1 , M. Alessio 1 , R. naddei 2 1 Department of Translational Medicine, Section of Pediatrics, University of Naples Federico II, Naples, 2Macrophage Activation Syndrome (MAS) is a severe hyperinflammatory condition, belonging to the spectrum of Haemophagocytic Lymphohistiocytosis, whose secondary forms occur in the context of pre-existing disorders such as infectious, rheumatic and oncological diseases. It may clinically manifest as fever, cytopenia, hypertriglyceridemia, hyperferritinemia, reduced fibrinogen, coagulopathy due to self-activation of T-cells, macrophages and overproduction of pro-inflammatory cytokines. MAS can have a fatal course, therefore an early diagnosis is necessary for rapid therapeutic intervention, including immunosuppressive and anti-inflammatory drugs such as corticosteroids, cyclosporine and biological drugs. Objectives: the aim is to demonstrate how HLH can be secondary to lymphomas and how anakinra is crucial in treatment. Methods: Child, 12 years old, severely obese (BMI 41), hospitalized in another structure for fever, joint pain, lymphadenopathy, where was suspected MAS secondary to Systemic Juvenile Idiopathic Arthritis (sAIG) and started therapy with methylprednisolone EV and cyclosporine. He is therefore transferred to our department for treatment and follow-up. The presence of fever, laterocervical lymphadenomegaly, skin rash in association with thrombocytopenia, hyperferritinemia, hypertriglyceridemia, increased LDH and hypofibrinogenemia confirmed the suspicion of MAS, but the presence of lymphadenomegaly and pruritus that did not respond to antihistamine treatment put the diagnosis in doubt. So it was decided to perform echography of the lymph nodes, histological examination if necessary, and serial checks of ferritin values and other laboratory indices. Results: Ultrasonography of the lymph nodes and abdomen revealed hepatosplenomegaly and diffuse lymphadenomegaly (dmax 43x21 mm), and consequently the right supraclavicular lymph node was biopsied. The clinical and laboratory profile showed a rapidly worsening evolution despite the early start of treatment: ferritin values progressively increased (>80,000 ng/ml), plurilinear cytopenia, hypofibrinogenemia (103mg/dl) and increased LDH (7720U/L), transaminases and bilirubin. Subcutaneous therapy with Anakinra 4 mg/Kg was therefore started first, then, due to lack of clinical-laboratory improvement, intravenous at a dose of 8 mg/kg. The shift in the treatment method led to an amelioration of the clinical and laboratory parameters. The histological examination, arrived during the phase of clinical and laboratory improvement, established the diagnosis of anaplastic T-cell lymphoma. Conclusion: The case report highlights aspects related to the management and treatment of MAS and its aetiology. Although MAS is frequently a complication of sAIG, it can be associated with other diseases, including malignancies, which should always be considered in the differential diagnosis. Anaplastic T-cell lymphoma, although rare, should be suspected as it may begin with MAS. Another consideration arises from the lack of response to therapy with Anakinra subcutaneously: the brilliant response to the intravenous shift may be due to severe obesity, which has reduced the efficacy of the drug by reducing its bioavailability. Disclosure of Interest : None declared
R. Stander, C. Scott, T. De Wit Paeditaric rheumatology, university of cape town, cape town, South Africa Correspondence: R. Stander Introduction: Here we describe a hyperinflammatory syndrome presenting as recurrent episodes of inflammation with a homozygous PRF1 mutation suggestive of a familial hemophagocytic lymphohistiocytosis (HLH). Objectives: A 5 year old previously well female presented with a history of intractable fever, rash, abdominal pain and arthralgia. Clinically she had lymphadenopathy and liver enlargement but no other supporting clinical features of systemic JIA, MIS-C or acute rheumatic fever. Laboratory features were suggestive of inflammation with striking hyperferritinemia, persistently raised CRP and raised d-dimers. She had mild anaemia and a bone marrow that was normocellular and showed no features of MAS. Methods: Sepsis and disseminated TB were excluded, her COVID PCR and COVID-Ab were negative. Initial therapy included intravenous immunoglobulins (IVIG), 10mg/kg of IV methylprednisolone for 3 days and IV antibiotics. Her temperature spikes persisted and cyclosporin was commenced to which she responded and was discharged home with a working diagnosis of idiopathic hyperinflammation. Over the next 3 months she had patchy recurrent episodes of inflammation which responded to non-specific immune suppression (a second pulse of IVMP with maintenance prednisone and cyclosporin). She was initiated on an IL-6 inhibitor Tocilizumab for ‘presumed’ systemic JIA. She followed up in rheumatology clinic one month later with severe back pain; and MRI showed a wedge collapse of T5 vertebra and a subligamentous collection; Staph Aureas and TB treatment were commenced in view of the immunosuppression and radiological findings. Results: During this admission she deteriorated; experiencing another hyperinflammatory episode with refractory shock and cardiac arrests; which responded to Anakinra. She developed a dense vitritis of the left eye and purpura fulminans with auto-amputation of her finger digits and toes. She remains symptom free on Tocilizumab and cyclosporin. Conclusion: The genetic panel was unable to identify pathogenic variants in known HLH genes. But here we describe a patient with an immune dysregulation syndrome and uncontrolled inflammation resembling familial HLH, but which may form part of an extended syndrome with a subacute onseT. Vasilev, M. Ganeva, K. Temelkova, A. Telcharova-Mihaylovska, V. Kostova, A. Dasheva, D. Hristova, S. Stefanov Rheumatology and Cardiology, Medical University of Sofia, Sofia, Bulgaria Correspondence: T. Vasilev Introduction: Macrophage activation syndrome (MAS) is a potentially life-threatening complication of rheumatic diseases, including juvenile systemic lupus erythematosus (jSLE). The incidence of MAS in jSLE ranges from 5.5 to 9 %.(1) Objectives: The aim of this clinical case report is to describe the clinical and laboratory features of MAS which occurred at the time of diagnosis of jSLE. Methods: We report the case of a child who presented with MAS at the onset of highly active SLE and who developed steroid-induced diabetes, which required adjustment in therapy. Results: We present a 7-year old girl with unremarkable history, who was admitted due to photosensitive skin lesions over the body. In the last two months the child had lost weight. Fever and painful ulcerative lesions over the oral cavity and hard palate appeared, as well. On admission the child was ill appearing, with erythematous-annular and papular lesions over the face, body and limbs. Erosions of varying depth over the hard palate and crusts on the lower lips were observed. Generalized lymphadenopathy and enlarged liver of 4 cm below the rib margin were detected. Laboratory tests revealed cytopenia - anemia Hgb - 89.0 g/L with positive direct Coombs test and thrombocytopenia Plt - 72.0 x10^9/L; impaired renal function - GFR 79.9 ml/min/1.73; low complement levels - C3 0.25 g/L and C4 < 0.020 g/L; positive ANA 1:640, anti-ds DNA 191 U/ml and anti SS-A > 200 antibodies. The diagnosis of jSLE was made based on fulfilling 5 clinical (acute cutaneous lupus, oral ulcers, renal involvement, hemolytic anemia, thrombocytopenia) and 4 immunological Systemic Lupus International Collaborating Clinics (SLICC)-12 criteria.(2) Furthermore, fever and elevated liver enzymes were noticed - AST - 676 U/L and ALT - 91 U/L with elevated triglycerides 170.15 mg/dL, hypofibrinogenemia 162 mg/dL and hyperferritinemia 4113.1 ng/mL. The child fulfilled 2016 Classification criteria for MAS complicating sJIA applicable to jSLE.(3) No infectious triggers for MAS were detected - negative serology for EBV; CMV; HSV; SARS-CoV-2. Pulse therapy with methylprednisolone (15 mg /kg /day) for 3 consecutive days was initiated, followed by 2 mg /kg /day. Moreover, seizures were observed with blood pressure up to 200/120 mmHg. Brain MRI revealed normal image. Anticonvulsant and antihypertensive therapy was started. The child was transferred to ICU. During follow up steroid-induced diabetes was diagnosed. Insulin therapy was started and discontinued on steroid tapering. Cyclosporine A was added to the treatment. After a 40-day stay at the Hospital, the girl was discharged. Conclusion: The presented clinical case shows the difficulties faced by the clinicians when it comes to managing life-threatening complications of a serious disease such as jSLE. References: 1. Sato S, Uejima Y, Arakawa Y, et al. Rheumatol Adv Pract. 2019 May 14;3(1). 2. Petri M, Orbai AM, Alarcуn GS, et al. Arthritis Rheum. 2012;64:2677–86. 3. Ravelli A, Minoia F, Davı S, et al. Arthritis Rheumatol. 2016 Mar;68(3):566-76P203. Musculoskeletal ultrasound in juvenile dermatomyositis – a proposal for a stratified evaluation method P204. Internal consistency and interrater reliability in musculoskeletal ultrasound in children P205. Qualitative and semiquantitative characterization of videocapillaroscopy evaluation of pediatric patients with perniosis 206. Comparison of coronary artery diameters on CT coronary angiography with 2D-Echocardiography in children with Kawasaki disease: a single center experience from Chandigarh, North India P207. High-frequency ultrasound and clinical correlation of disease activity in juvenile localized scleroderma: preliminary data P208. Ultrasound and muscle elastography could be a promising tool to assess disease activity in juvenile dermatomyositis patients? P210. Phalangeal microgeodic syndrome. A case report M. Krumrey-Langkammerer 1 , S. Bechthold 2 , T. Fröhlich 3 , B. Hügle 1 , A. Jansson 2 , R. Jones 4 , M. J. Laaths 5 , T. Lutz 6 , C. Schütz 7 , J.-P. Haas 8 1 German Centre for Pediatric and Adolenscent Rheumatology, Garmisch-Partenkirchen, 2 Dr. von Hauner´sches Kinderspital, Ludwig-Maximillians University, Munich, 3 Practice for Pediatric Rheumatology, Forchheim, Germany, 4 Pediatric rheumatology, Landeskrankenhaus, Salzburg, Austria, 5 Pediatric rheumatology, Children´s hospital, Amberg, 6 Practice for Pediatric Rheumatology, Heidelberg, 7 Pediatric Immunology and Rheumatology, Gustav-Carus University , Dresden, 8 German Centre for Pediatric and Adolescents Rheumatology , Garmisch-Partenkirchen, Germany Correspondence: M. Krumrey-Langkammerer Introduction: Juvenile dermatomyositis (JDM) is a chronic autoimmune disease in childhood that presents with vasculitis and inflammation of the muscles causing significant muscular damage if untreated. So far, the gold-standard for assessing disease activity and damage is magnetic resonance imaging. However, muskuloskeletal ultrasound (MSUS) is inceasingly used to assess inflammation in JDM. Objectives: To develop a scoring system to standardize evaluation of the observed changes in muscle patterns seen in MSUS. Methods: A single-center cross-sectional study included a cohort of 15 JDM patients examined between November 2021 and February 2022. We used a standardized procedure for MSUS (Canon Aplio i800®, linear probe 14MHz) in six different muscles. All MSUS and the interpretation of data were performed by two pediatric rheumatologist experienced in muskuloskeletal ultrasound, using a proprietary evaluation scheme designed for the interpretation of inflammatory myositis (see table 1). Pearson’s correlation was used to correlate MSUS scores with different markers of activity and damage in JDM including CMAS, MMT-8, DM-DAS (dermatomyositis disease activity score) and MDI (myositis damage index). Results: 15 JDM patients (9f/6m) have been included. Patients were 10,95 years (4,69-18,52) old at the time of examination with a mean diseaseduration of 6,11 yrs. (0,82-13,45). Mean values were: CMAS: 42,4; MMT-8: 68,91; CHAQ: 1,1; Phy-Global: 2,27; Pat/Par-Global 2,4; CK 531 U/l. Five exhibited a monocyclic, 7 a chronic-active and 3 a polycyclic course of JDM. Myositis specific antibodies had been detected in 10 patients (8xNXP-2, 1xMDA5, 1xTIFF-1g). The proposed evaluation score was found to have the highest correlation with the DAS (p=0,64). Increasing inhomogenity, thickening of fascia and hypervascularisation of muscle and fascia were found to indicate inflammatory disease activity. Chronic perimysial inflammation and necrotic myofibrils were represented by spotted or focally altered echogenity. Conclusion: This proposal of a scoring-system represents a first attempt at interpretation of MSUS in JDM. It should help to define disease activity and assess damage in JDM patients. Further prospective studies are required in order to adjust this score. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
D. S. Lazarevic 1,2 , J. Vojinovic 1,2 , C. Malattia 3 , L. Rossi-Semerano 4 , B. Sozeri 5 , M. Tsinti 6 , S. Lanni 7 , C. Host 8 , D. Windschall 9 , A. Snipaitiene 10 1 Pediatric Rheumatology and Immunology, Clinic of Pediatrics, University Clinical Center , 2 Pediatrics, Faculty of Medicine, University of Nis, Nis, Serbia, Nis, Serbia, 3 IRCCS Instituto Giannina Gaslini, Genoa, Italy, 4 Department of Pediatric Rheumatology, CEREMAIA, Bicêtre Hospital, AP-HP, Le Kremlin Bicêtre, France, 5 Health Sciences University, Umraniye Education and Research Hospital, Istanbul, Turkey, 6 Aghia Sophia Children’s Hospital, Athens, Greece, 7 Fondazione IRCCS Cà Granda Ospedale Maggiore Policlinico, Milan, Italy, 8 Aarhus University, Aarhus, Denmark, 9 University of Halle-Wittenberg and St. Josef-Stift Sendenhorst, Sendenhorst, Germany, 10D. S. Lazarevic Introduction: Numerous publications provided rational for involvement of musculoskeletal ultrasound (MSUS) and several inflammatory biomarkers as the clinically meaningful prediction panel for juvenile idiopathic arthritis (JIA). This could help to achieve personalized treatment and define new sensitive outcome tool important for evaluation of disease activity and treatment response. Objectives: To examine internal consistency and interrater reliability of pediatric MSUS in JIA patients evaluated by experts in this field. Methods: DAISY study is a multicentric longitudinal study that will recruit 150 JIA patients (ILAR classification criteria) with active disease (JADAS 10 and 27) prior starting recommended treatment. At enrollment and during predefined follow up visits (3, 6, 12 months) patients are evaluated by JADAS 10 and 27, examined by gray-scale (GS) and power-doppler (PD) in 44 joints using OMERACT synovitis scoring by an expert in pediatric MSUS, while blood samples are collected for evaluation of inflammatory markers (cytokines, chemokines, S100A8, S100A9 and S100A12). To be eligible pediatric ultrasound experts must have at least intermediate EULAR certified MSUS course and at least 5 years of experience in MSUS in children. Study is conducted in 9 participating centers in 8 different countries after Ethics Committee Approvals and Informed Consents were obtained. Prior to the project start up all ultrasonographers accessed the study educational material on DAISY web-portal (US normal and pathological images with standardized scanning from 4 different age categories in children). Once reviewed the educational material, 9 ultrasonographers performed “test for ultrasonographers” which represents calibration web-based reliability exercise on 56 MSUS still images of different aged categories with different GS and PD grades of synovitis using OMERACT synovitis scoring (to optimize interrater reliability). To pass the test, ultrasonographers must have more than 80 percent’s of right answers. We assessed the internal consistency reliability with Cronbach’s alpha. The inter-observer interclass correlation coefficient (ICCs) was calculated to estimate agreement between nine raters. ICCs were calculated and tested with a significance level of 5%. Cronbach’s alpha> 0.9 and ICC ≥0.8 were considered excellent. Results: Cronbach alpha was very high for all joints and reflects good internal consistency (α>0.9). The interrater ICCs for all joints (elbow, wrist, MCP II, hip, knee, tibiotalar, talonavicular, subtalar, MTP) was high with confidence interval 95%. The observed values for inter-observer analysis showed an excellent (ICC ≥ 0.80) agreement, with the strongest one on the hip (ICC =1), and a little bit lower, but still excellent on tibiotalar (ICC=0.828) and subtalar joint (ICC=0.861). Conclusion: The internal consistency reliability and interobserver reliability were excellent, pointing out that all ultrasonographers were highly reliable in grading paediatric musculoskeletal ultrasound. Trial registration identifying number: Funding: This study is supported by the Foundation for Research in Rheumatology (FOREUM). Disclosure of Interest : None declared
A. K. Leos Leija, R. C. Calderón Zamora, A. V. Villarreal Treviño, F. García Rodríguez, N. Rubio Pérez aediatric Rheumatology , Hospital Universitario “Dr. José Eleuterio González”, Monterrey, Mexico V. Villarreal Treviño Introduction: Perniosis is a disease caused by exposure to damp cold, which particularly affects unprotected areas of the skin. Although the exact cause of the disease is unknown, it may be idiopathic or secondary to other systemic diseases such as systemic lupus erythematosus (SLE). Nailfold videocapillaroscopy (NVC) is an important tool for patients with rheumatic diseases, evaluating morphological changes of the nailfold capillaries, such as capillary density, avascularity and abnormal capillaries. During the fourth wave of COVID19 pandemic we observed an increase of cases of perniosis in previously healthy pediatric patients, so capillaroscopic evaluation was performed. Objectives: To describe qualitative and semiquantitative capillaroscopic findings in pediatric patients with perniosis. Methods: Eight fingers, except the thumb, of 10 patients from a pediatric rheumatology department of a tertiary center in Monterrey, Nuevo León, México from December 2021 to March 2022 were included. There were analyzed the most representative 1 mm images of each one with x200 videocapillaroscope (Optilia), analyzing a total of 80 images by the same observer. Demographic data such as sex and age were included and capillaroscopic qualitative data such as pattern, density, abnormal shapes, microbleeding and edema, and semiquantitative characteristics such as length and width were obtained. Autoantibodies for the evaluation of connective tissue diseases were obtained for each patient. Results: Ten patients were included of which 8 (80%) were female, the mean age was 13 years. Perniosis was present in 100% of patients. Antinuclear antibodies (ANA) were present in 3 patients (30%) of which 2 (20%) had DFS70 pattern and 1 (10%) had nuclear fine speckled pattern. The rest of autoantibodies were negative in all patients. Among the semiquantitative characteristics, we found no capillaroscopic changes. Right hand finger No. 2 to No. 5 =R2, R3, R4, R5. Left hand finger No.2 to No. 5 = L2, L3, L4, L5. Conclusion: This study shows the semiquantitative data of the NVC analysis, and therefore it is proposed as a possible tool for the diagnosis and monitoring of perniosis, not only in patients with rheumatic diseases, but also in those previously healthy individuals with no clinical signs or symptoms of an autoimmune disease. Despite being a study with a small sample of patients, there is currently a lack of literature in pediatric patients where these characteristics are evaluated in individuals with perniosis, so it is recommended to carry out studies with a greater sample to obtain more dataK. Pilania 1 , M. Singhal 2 , H. Chaudhary 1 , A. Sil 1 , A. K. Jindal 1 , P. Vignesh 1 , S. Singh 1 1 Pediatric Allergy Immunology Unit , 2 Department of Radiodiagnosis and Imaging , PGIMER, Chandigarh, India Correspondence: R. K. Pilania Introduction: 2D echocardiography (2DE) has hitherto been standard of care for evaluation of coronary artery abnormalities (CAAs) in children with Kawasaki disease (KD). CT coronary angiography (CTCA) is an emerging imaging modality in this field. Objectives: To compare coronary artery dimensions on CTCA with 2DE in children with KD. Methods: Records of children with KD were retrieved wherein both CTCA and 2DE had been carried out within 48 hours of 2DE. Comparison of proximal coronary artery dimensions on both modalities was made. 2DE was carried by clinical fellows experienced in coronary artery assessment, while CTCA were performed by an experienced cardiac radiologist blinded to findings of 2DE. Statistical analysis was done on SPSS software version 23 and paired t-test was used to compare mean diameters recorded on two imaging modalities. Results: There were 61 children with KD who fulfilled the inclusion criteria. Twenty-four children were imaged at presentation, while 37 were imaged during convalescence/ follow-up. There was a significant difference in left circumflex artery (LCx) measurements between CTCA and 2DE (t=2.23, p=0.04). On an average, LCx measurements on CTCA were 1.06 mm higher than on ECHO (95% CI [0.04, 2.09]). There was no significant difference between left main coronary artery (LMCA) dimensions measured through 2DE and CTCA (p=0.534). Similarly, no significant difference was noted between left anterior descending (LAD) and right coronary artery (RCA) dimensions measured through 2DE and CTCA (p=0.675 and p=0.198 respectively). Conclusion: Dimensions of coronary arteries derived from CTCA in proximal segments of LMCA, RCA and LAD were comparable with 2DE, while LCx dimensions were significantly higher on CTCA. This may be due to difficulties in assessment of LCx on 2DE. CTCA thus has potential to become standard of care for diagnostic evaluation children with KD. Trial registration identifying number: ND. G. P. Piotto, R. L. F. D. Andrade, G. Clemente, M. M. Fraga, C. A. Len, M. T. R. Terreri D. G. P. Piotto Introduction: Juvenile localized scleroderma (JLS) is a rare pediatric disease characterized by inflammation and skin thickening. High-frequency ultrasound (HF-US) shows great promise in helping clinical assessment of the disease activity and fibrosis in JLS. Objectives: The aim of this study was to evaluate HF-US findings in JLS patients and to correlate clinical and ultrasound activity. Methods: The data collected included presence of symptoms, disease features, and the Localized Scleroderma Cutaneous Assessment Tool (LoSCAT), the modified Localized Scleroderma Skin Severity Index (mLoSSI), the Localized Scleroderma Skin Damage Index (LoSDI), activity and damage Patient Global Assessment of Disease (PGAD). All US images were obtained using a 22 MHz HF-US (GE machine). The clinical and US data were blindly evaluated by 2 examiners specialized in Paediatric Rheumatology. The dermal thickness, echogenicity score and the presence of Power Doppler of the JLS lesional dermis were measured in each lesion with the highest score for erythema, skin thickness, and dermal atrophy. Measurements were compared to contralateral side of unaffected skin. Results: A total of 22 JLS were included, 68.2% female with a mean age of 13.6 years (SD ± 4.6 years). Mean age at symptoms onset was 6.6 years (SD ± 3.3 years), mean age at diagnosis 7.5 years (SD ± 3.2 years) and disease follow up time of 7.1 years (SD ± 4.7 years). The linear form was the most prevalent (68.2%), followed by the linear + morphea association (18.2%) and isolated morphea plaques (13.6%). Regarding the activity and damage PGAD of lesions, the means were 1.8 (SD ± 1.7) and 1.1 (SD ± 1.5) respectively. The mean Rodnan score was 1.5 (SD ± 2.1) and LoSCAT 14.6 (SD ± 9.7). We studied 77 images of HF-US and we could observe a reduction in the thickness of the dermal layer and subcutaneous tissue, with greater echogenicity of the dermis compared to the skin of the contralateral unaffected skin in those patients with longer duration of the disease. Inflammatory (erythematous) lesions presented increase in the dermal thickness and decrease in the echogenicity, while fibrosis was characterized by hyperechogenicity and increased (sclerotic lesions) or decreased (atrophic lesions) dermal thickness, compared with healthy skin. Doppler US did not reveal increased vascularization of the lesion in these patients. As the study has not yet been completed, a statistical analysis was not possible. Conclusion: Our findings suggest that 22 MHz HF-US allows quantitative evaluation of the JLS lesions in different stages. HF-US morphological evaluation provides a reliable and more comprehensive measurement of the JLS lesionR. L. F. de Andrade 1 , J. A. Mendonça 2 , D. G. P. Piotto 1 , P. P. Aires 1 , A. M. D. O. Rocha 1 , M. T. R. Terreri 1 1 Pediatric, Universidade Federal de São Paulo, São Paulo, 2 Rheumatology, UNICAMP, Campinas, Brazil Correspondence: D. G. P. Piotto Introduction: Juvenile Dermatomyositis (JDM) is the most common idiopathic inflammatory myopathy (IIM) in the pediatric age group. For diagnosis, we often need invasive tests that are difficult to be accepted by children. In addition, it is hard to define whether the disease is active or only sequelae lesions are present, which is important in determining the therapy. The introduction of new imaging exams has allowed a better assessment of muscle involvement in JDM, with ultrasound (US) being a promising method in the pediatric age group. US proved to be useful in helping to define the degree of disease activity and also in patient follow-up, demonstrating the severity and tissue damage associated with the myopathy. Regarding muscle elastography, that is a technique more recently incorporated into US, studies are scarce and have controversial results. In IIM, elastography seems to show an increase in muscle stiffness compatible with that found in MRI. More particularly in the pediatric age group, elastography was not satisfactory for detecting active myositis, although it showed a correlation between the elastographic abnormalities with the increase in muscle echogenicity found in MRI. Objectives: This study aimed to establish an association of disease activity through clinical muscle assessment (through validated instruments), laboratory and nailfold capillaroscopy (NFC) with quantitative and semiquantitative ultrasound and elastography through the Strain Elastography (SE) technique. Methods: Twenty-two JDM-patients and fourteen healthy controls, aged between 5 and 21 years, matched for age and sex were enrolled. Patients underwent clinical evaluation through the application of muscle assessment questionnaires (CMAS and MMT), global assessment of the disease through the application of a questionnaire (DAS) and visual analogue scale (VAS) scores by the physician and parents and patients and assessment of functional capacity through the CHAQ questionnaire. Patients were also submitted to NFC and measurement of muscle enzymes, as recommended in follow-up. All subjects in the study underwent US assessment using gray scale, application of Power Doppler and application of SE. Results: In the categorical and semi-quantitative gray scale evaluation of patients and controls, we observed a higher frequency of alterations in the patients’ exams, with the categorical gray scale being more altered in the quadriceps femoris (p=0.029) and the semi-quantitative gray scale presenting higher frequency of alterations in all evaluated muscle groups (p<0.001). When comparing the US assessment of patients with disease activity parameters (CK, MMT, CMAS and VAS) no robust correlations were found. From the gray scale, we proposed a cut-off point for elastography, from which we can consider the muscle with pathological stiffness. This cut-off point was 54.36, with 45.3% of sensitivity, 64.4% of specitivity, 18% of positive predictive value, 87.2% of negative predictive value and 61.6% of accuracy. Conclusion: USG was able, through semiquantitative grayscale evaluation, to differentiate patients with JDM from healthy patients, however there was no correlation between ultrasound, grayscale, PD and elastography findings and disease activity markers. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
O. Vougiouka 1 , I. Nikas 2 1 2nd dep. of Paediatrics Athens University School of Medicine, P A Kyriakou Hospital, 2 Pediatr Diagnostic Center, Bioiatriki Healthcare Group, ATHENS, Greece Correspondence: O. Vougiouka Introduction: Phalangeal Microgeodic Syndrome (PMS) is a rare syndrome affecting finger phalanges, mostly accounted in children, first described in 1970 by Maroteaux P. Objectives: The presentation of an 11year old girl with finger oedema, purple skin discoloration, mild stiffness and pruritus manifested in early March this year. Methods: The girl had already visited her pediatrician and an orthopedic surgeon who recommended a right hand MRI. Immunology profile was prescribed. Results: There were multiple areas of bone marrow oedema on the whole finger phalanges on presented MRI compatible to PMS. Anti-dsDNA, ENA, RNP were all negative. Inflammatory indexes, blood cultures, quantiferon test, complement, TSH and PTH were normal. The girl, a rhythmic gymnastics’ athlete, was double COVID-19 vaccinated in January this year. COVID-19 or known contact were not reported. A frostbite condition diagnosed and conservative measures suggested. Conclusion: Radiographic features of PMS, as first described, show mild osteosclerosis with cortical irregularity and multiple small radiolucent spots of osteolysis in the diaphysis. Recently, MRI is helpful for the diagnosis. In PMS, the bone marrow of the phalanges shows diffuse low signal intensity on T1-weighted images and high signal intensity on STIR images which indicates bone oedema. Frostbites attributed to cold exposure, chilblains because of COVID-19 and Raynaud’s phenomenon are conditions with microgeodic findings on imaging, which we should be aware of in order to avoid extented investigations and reassure parents and patients. Trial registration identifying number: References: Maroteaux P. A microgeodic disease of unknown etiology affecting the finger phalanges in infants: report of five cases. Ann Radiol (Paris) 1970;13:229–36. Nishino et al. Two rare cases of adult-onset phalangeal microgeodic syndrome with magnetic resonance imaging-proven bone edema transiently occurring in winter. Joint Bone Spine 80 (2013) 523–524. Ramani S L et al. Musculoskeletal involvement of COVID-19: review of imaging. Skeletal Radiology 2021 Sep;50(9):1763-1773. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
P211. Rheumatology transition of young people in Switzerland – the heroes study 12. Functional state of the pulmonary system in children with juvenile idiopathic arthritis according to dynamic monitoring P213. The current evidence for transitional care in young people with chronic pain: a systematic review 4. Cardiopulmonary screening in connective tissue disease: UK multi-centre audit 5. Educating and empowering young patients with JIA using a board game in group sessions P216. Clinical evolution of adults with childhood onset rheumatic diseases: analysis of a 10-year experience from a transitional unit in Spain L. Berben 1 , M.-L. Daly 1 , T. Daikeler 2 , A. Wörner 1 1 University Children’s Hospital Basel, 2 University Hospital Basel, Basel, Switzerland Correspondence: L. Berben Introduction: About half of all children with rheumatic diseases need continuous medical care during adolescence and adulthood. An effective and well-planned transition built on individual, structured management plans is crucial for successful transition into adult services. Although transition principles have been described and implemented, outcome data are still scarce. Objectives: The overall objective of the HEROES study is to develop, implement and evaluate a transitional care (TC) program for adolescents and young adults (AYA) with a rheumatic disease moving from pediatric to adult settings in Switzerland. Specific aims are: assess AYAs’ and parents’ experiences and unmet needs related to current TC practices assess stakeholder, healthcare professional, setting and system barriers and facilitators to implementing a TC program develop and implement a TC program that includes a nurse TC coordinator evaluate the effectiveness of the TC program in relation to disease-related outcomes evaluate AYA-, parent- and healthcare professional-reported outcomes and care experiences in relation to the TC program evaluate the implementation outcomes of the TC program in relation to adoption, implementation and sustainability evaluate economic outcomes of the TC program Methods: This study which includes all 10 Swiss pediatric rheumatology centers and their adult counterpart, will use a hybrid effectiveness-implementation type 2 design. For the different parts of the study, specific designs will vary. For the qualitative data (i.e. AYA’ and parents’ experiences and unmet needs, acceptance and appropriateness of the intervention and assessment of contextual factors), an explanatory sequential mixed method design will be used. The quantitative data, i.e. the intervention-effectiveness outcomes, in this study will be assessed using a multi-center quasi experimental pre-post design . To maximize the acceptability and sustainability, a participatory partnership approach will be used, i.e. people that the intervention aims to help and those who will implement it are involved throughout the process. Intervention outcomes will be measured at the AYA (e.g. quality-of-life, treatment adherence), parent (e.g. care satisfaction, giving responsibility for care to the AYA), healthcare professional (e.g. time for AYA, work satisfaction), setting (e.g. billing of consultations) and system (e.g. costs) levels. Results: The (poster) presentation will describe the study methodology. Conclusion: This innovation has a potential to improve TC for children with rheumatic diseases as well as serving as a model for TC in other chronS. Shevchenko 1,2 , T. Holovko 1,2 , L. Bohmat 1,2 , V. Nikonova 1T. Holovko Introduction: Pulmonary manifestations of rheumatic diseases in children are uncommon. At the same time, there is a high risk of involvement of the respiratory organs in the pathological process, which manifests itself mainly as interstitial lung disease and pulmonary arterial hypertension. These pathological conditions can occur both as a result of the underlying disease and as a result of drug toxicity of basic therapy. In the future, they may be the cause of worsening the prognosis and increasing disability and mortality. Objectives: For timely diagnosis of pulmonary complications, lung ventilation function was assessed in children with juvenile idiopathic arthritis. Methods: The study included 38 children aged 7 to 18 years, 12 boys and 26 girls with oligoarticular and polyarticular variants of juvenile idiopathic arthritis. Spirometry was performed to determine the indicators of external respiration twice, with an interval of 12 months. Results: It was found that in a quarter of patients (26.31 ± 2.80%) there was a decrease in the function of external respiration. In all cases, the violations were restrictive in nature, mild. The frequency of disorders did not have a significant relationship with gender and subtypes of the disease. All patients with respiratory disorders had the disease for more than 3 years, 80.0% of them had ANA-positivity, which persisted for a long time and testified to the immunological activity of the process. Patients received methotrexate (15-20 mg / week), the duration of therapy was at least 2.5 ± 0.30 years. Re-examination after 12 months revealed a decrease in reduced pulmonary ventilation function in 23.74 ± 2.62%. In this group, ANA-positivity occurred in 66.7% of patients, which indicated the preservation of the activity of the pathological process and the need for further basic therapy. Conclusion: In children with juvenile idiopathic arthritis under the age of 18 years, there are signs of restricted external respiration, which persists for a long time, indicating involvement in the pathological process of the lung. The detected changes persist and occur after one year of illness and are associated with the duration and activity of JIA. Disclosure of Interest : None declared
L. Huckerby 1 , J. E. McDonagh 1,2,3 , R. R. Lee 4,5 1 Royal Manchester Children’s Hospital, 2 National Institue of Health Research Biomedical Research Centre, Manchester University Hospital NHS Trust, 3 Centre for Epidemiology Versus Arthritis, Centre for Musculoskeletal Research, University of Manchester, 4 National Institute of Health Research Biomedical Research Centre, Manchester University Hospital NHS Trust, 5Manchester, United Kingdom Correspondence: L. Huckerby Introduction: Paediatric chronic musculoskeletal pain presents a significant individual and societal burden, with an estimated prevalence of 11-38% (1,2,3). A large proportion of adolescents with chronic pain are likely to have unresolved pain which continues into adulthood (4,5). Transitional care is a lengthy process, starting in early adolescence and includes preparation and support for the move from paediatric to adult centred services. To date, there is limited evidence identifying the extent to which transitional care for adolescents with chronic pain has been developed or investigated in research (6). Objectives: To review the current evidence for transitional care in young people with chronic pain. Methods: Studies were identified by searching 4 databases: EMBASE, Medline, CINAHL and PsycINFO. The PEO tool was used to develop a search strategy, and terms such as “Adolescent”, “Persistent long-term pain”, and “Transition* (or variations of such words) were implemented. Inclusion criteria were: sample population age between 10-24 years, a confirmed diagnosis of a condition characterised by chronic pain, any health care setting, any service provider, published peer reviewed, English language. Excluded were case reports, editorials, abstracts, meta-analyses, books or book chapters. Searches took place between September and December 2021. Results: 98 papers were identified by the search, 14 were selected after abstract screening. Two independent reviewers then screened papers for inclusion, extracted data, and assessed the quality of the studies followed by a senior reviewer. Of the 14 papers, full text review found that none of the papers looked specifically at the evidence with respect to transitional care for young people with chronic pain. Of those which did not meet the inclusion criteria, there were 4 papers which informed our discussion. Conclusion: We found a lack of research considering chronic pain in the context of transitional care. Chronic pain is a feature of many long-term health conditions. It remains unknown as to whether there are any pain-specific aspects of transitional care. How pain management is addressed in existing transitional care provision and the relationship of pain to outcomes of transitional care needs further research. If effective interventions can be provided during these crucial years, the trajectory of these young people as adults can potentially be improved (5). References 1. King S, Chambers CT, Huguet A, et al. The epidemiology of chronic pain in children and adolescents revisited: a systematic review. Pain; 2011;152:272938. 2. Szer IS. Musculoskeletal Pain Syndromes That Affect Adolescents. Arch Pediatr Adolesc Med; 1996;150:740–7. 3. Weiss JE, Stinson JN. Pediatric Pain Syndromes and Noninflammatory Musculoskeletal Pain. Pediatr Clin North Am. 2018 ;65:801–826. 4. Walker LS, Dengler-Crish CM, Rippel S, Bruehl S. Functional abdominal pain in childhood and adolescence increases risk for chronic pain in adulthood. Pain; 2010; 150: 568–72. 5. Kashikar-Zuck S, Cunningham N, Peugh J, et al. Long-term outcomes of adolescents with juvenile-onset fibromyalgia into adulthood, and impact of depressive symptoms on functioning over time. Pain; 2019; 160: 433–41. 6. Dolezalova P, et al. The European network for care of children with paediatric rheumatic diseases: care across borders. Rheumatology (Oxford); 2019; Jul 1;58(7):1188-1195Lythgoe 1 , K. Mageean 2 , P. Lawrence 1 , S. Mayell 1 , D. Luciano 1 , P. Duong 1 , J. Walsh 3 , E. Twynam-Perkins 3 , M. Ahmid 3 , H. Sansby 3 , C. Anderson 4 , F. Ritchie 4 , L. Crosby 2 , C. Longthorpe 2 , L. McCann 1 , C. Pain 1 1 Alder Hey Children’s NHS Foundation Trust, Liverpool, 2 Bristol Royal Hospital for Children, Bristol, 3 Royal Hospital for Children, Glasgow, 4 Royal Hospital for Children and Young People, Edinburgh, United Kingdom Correspondence: H. Lythgoe Introduction: Cardiopulmonary involvement in connective tissue disease (CTD) manifests in many ways, may be asymptomatic and can have a significant impact on morbidity and mortality. Investigation of cardiopulmonary symptoms and signs in these patients is therefore important and screening asymptomatic patients may facilitate early recognition and treatment. Objectives: To describe cardiopulmonary involvement in patients with four CTDs (juvenile systemic lupus erythematosus (JSLE), juvenile dermatomyositis (JDM), juvenile systemic sclerosis (JSSc) and juvenile mixed connective tissue disease (JMCTD), define audit standards and measure performance against them. Methods: Current patients seen within the last twelve months from four tertiary paediatric rheumatology centres were included. Available guidance was reviewed and both generic and disease-specific audit standards were defined. Results: 82 patients were included with a median follow-up of 2.8 years. Most patients had JSLE (33) and JDM (32) with smaller numbers of JSSc and JMCTD patients (6 and 11 respectively). Guidance on cardiopulmonary involvement was identified for JSLE (Single Hub and Access Point for Paediatric Rheumatology in Europe (SHARE) and British Society for Rheumatology (BSR) guidance), JDM (SHARE) and JSSc (SHARE). No guidelines were identified for JMCTD. Screening for cardiopulmonary involvement was inconsistent and standards were largely not met (table 1). Abnormal findings did not always lead to further/repeat investigation; HRCT was not performed for all patients with persistently abnormal lung function. Pulmonary involvement was identified in 24.4% and was most frequent in JSSc (83%). Two patients had ILD. Cardiac involvement was less frequent (9.8%) with pericardial effusion the most common manifestation and one patient with pulmonary arterial hypertension. Conclusion: Guidance for cardiopulmonary screening is limited but cardiopulmonary screening is inconsistently performed for both symptomatic and asymptomatic patients and standards are not met. Service evaluation and development with multi-disciplinary team engagement is required to increase screening and improve long-term outcomes forPieler, H. K. Ipsen, A. Estmann Christensen, P. Toftedal H.C. Andersen Childrens Hospital, Odense , Denmark orrespondence: A. Pieler Introduction: During the process of improving transition to adult rheumatology it became apparent that some of the adolescents had little insight and understanding of their disease or of disease management. Objectives: To evaluate group sessions in adolescents using a board game designed to facilitate discussions on JIA. To optimize the young patients knowledge about their diagnosis and treatment. Methods: The nurses interviewed a random sample of the adolescents during their ordinary visit to the outpatient clinic, in order to get an understanding of what would make them attend and take an active part in a group session. The most optimal format seemed to be one-time-only sessions of maximum duration of two hours in the evening as to not interfere with school. The young persons needed a unifying element in the sessions that made it feel safe for them to initiate talking and sharing with others while in an un-known setting. We created a board game about JIA as a way to begin the sessions and facilitate a conversation about JIA. Nurses from the pediatric rheumatology team organized and hosted group sessions with 4-8 adolescents. All the participants knew the nurses in advance. No parents or doctors were present in these group sessions. The adolescents were invited by mail to participate in a group session on a day of an ordinary visit. An anonymous evaluation format was used at the end of the session. Results: Since the first group session was held in 2019, there has been six totally. Between two and five adolescents attended each session. Getting the adolescents to attend has proven to be a challenge. Many are skeptical about being in a setting with strangers and without their parents. The younger the adolescents are the easier it is to get them to attend as they do not have the same pressure of not missing school and are still very influenced by their parents, who often want them to attend. Feedback shows that they all gain something from attending and find it has meaning for them. The board game works very well as a means to convey information about disease, complications and treatment and to facilitate discussion and thoughts about the future. Sometimes it was used throughout the session, and other times just as a preliminary means to get the conversation started. Conclusion: The most appropriate age for group session seemed to be at the age of 15-16 years. The patients older than this were more obliged regarding school. Feedback from those attending shows that the group sessions give meaning for the adolescents. They gain knowledge that they did not have before. For most of the attending adolescents, the session was their first meeting with other young persons with JIA. Meeting and talking with others in a similar situation was of high value for them. Though the groups have been smaller than intended, it may have had a positive effect on the level of engagement. Almost all of the adolescents have participated actively during the sessions. Disclosure of Interest : None declared
C. Udaondo 1 , L. Nuño Nuño 2 , C. Millan - Longo 1 , C. Muñoz - Gomez 1 , B. Diaz - Delgado 1 , S. Murias 1 , R. Alcobendas 1 , A. Balsa 2 , A. Remesal 1 1 Paediatric Rheumatology, 2 Rheumatology, Hospital La Paz, Madrid, Spain Correspondence: C. Udaondo Introduction: Most childhood-onset rheumatic diseases continue into adulthood and in some cases they have sequelae of their disease, with clinical features that differ from those of paediatric age. Transitional units as well as specific training for rheumatologists who will follow up these patients are needed to ensure the successful control of the disease. Objectives: To describe the characteristics, complications and clinical evolution of patients followed up in transitional consultation and identify characteristics associated with a better prognosis of the disease. Methods: We performed a retrospective, longitudinal observational study of patients followed up in a transitional clinic in a reference hospital in Madrid from 2012 to 2022. Patients were seen together by paediatric rheumatologists and an adult rheumatologist in the paediatric unit for 2-4 visits, and were subsequently transferred to an adult rheumatology clinic with the same adult rheumatologist, who did long-term follow-up. Medical charts were reviewed. Clinical and treatment data were collected and patients with loss to follow-up were contacted. A descriptive analysis was carried out and differences were analysed using contingency tables for qualitative variables and t-tests for independent samples for quantitative variables. Results: 87 patients were included in follow-up at the transitional unit in the study period (72.4% female), with an age at diagnosis of 9±5 years, and a mean time of disease evolution of 10±5 years. The most frequent diagnosis was oligoarticular JIA. At the time of transfer, patients had a mean age of 19±1 years (16-24 years), 85.1% receivied immunomodulatory treatment and 62.1% biologic therapy (etanercept 23%, adalimumab 18.4%, tocilizimab 8%, golimumab and anakinra and infliximab 2.3% each, abatacept and certolizumab 1.1% each). Two patients were being treated with 2 biologics simultaneously due to high disease activity. 15 (17.2%) were in prolonged clinical remission without treatment, showing a longer time of disease progression (13±4 years vs. 8±5 years; p=0.07). No differences were found between prolonged remission off medication and sex or age at onset. There was only a loss to follow-up in 5 cases (5.7%). As important complications, there was an unwanted pregnancy in patient with active SLE and a Felty’s syndrome that required intensive care unit admission and was related to poor adherence to treatment. Conclusion: The combined paediatricians-rheumatologists model seems to be well accepted by patients and relatives, with a low number of losses to follow-up. There was a high number of patients on biologic therapy. There is a need for additional resources to avoid major complications. Disclosure of Interest : None declared
P217. Treat-to-target strategies for the management of Familial Mediterranean Fever in children P218. Implementing a treat to target strategy for pediatric lupus: design of an intervention L. Ehlers 1 , E. Rolfes 1 , M. Lieber 1 , D. Müller 1 , E. Lainka 2 , F. Gohar 3 , G. Klaus 4 , H. Girschick 5 , J. Hörstermann 6 , J. Kümmerle-Deschner 7 , J. Brunner 8 , K. Palm-Beden 3 , K. Tenbrock 9 , L. von Wrangel 10 , M. Faßhauer 11 , N. Blank 12 , R. Trauzeddel 13 , S. L. von Stuckrad 1 , S. Higgins 14 , T. Welzel 7 , T. Lutz 12 , V. Hentgen 15 , D. Föll 16 , H. Wittkowski 16 , T. Kallinich 1 1 Charité University Hospital, Berlin, 2 University Hospital Essen, Essen, 3 St. Josef-Stift Sendenhorst, Northwest German Center for Rheumatology, Sendenhorst, 4 KfH Center of Paediatric Nephrology, Marburg, 5 Vivantes Klinikum Friedrichshain, Children’s Hospital, 6 Deutsches Rheuma-Forschungszentrum (DRFZ), An Institute of the Leibniz Association, Berlin, 7 University Hospital Tübingen, Tübingen, Germany, 8 Medical University Innsbruck, Innsbruck, Austria, 9 RWTH Aachen, Aachen, 10 Patient representative, Berlin, 11 Hospital St. Georg GmbH Leipzig, Academic Teaching Hospital of the University of Leipzig, Leipzig, Leipzig, 12 University Hospital of Heidelberg, Heidelberg, 13 Helios Klinikum Berlin-Buch, Berlin, 14 Paediatric medical practice Hürthpark, Hürth, Germany, 15 Versailles Hospital, Versailles, France, 16 University Hospital Münster, Münster, Germany Correspondence: L. Ehlers Introduction: Familial Mediterranean fever (FMF) is a monogenic autoinflammatory disease presenting with recurrent fever episodes accompanied by serositis and elevated inflammatory parameters. Amyloidosis as a sequela of persistent inflammation represents the main cause of mortality. 5-10% of patients do not achieve satisfactory disease control on colchicine monotherapy. Increasing therapeutic options with the recent approval of IL-1 antagonists underline the need for disease activity-guided treatment recommendations. Objectives: The objective of this PRO-KIND initiative of the German Society for Paediatric Rheumatology (GKJR) was to develop consensus-based treat-to-target (T2T) strategies for FMF to improve patient care and long-term outcome. Methods: An initial set of statements was developed in accordance with existing management recommendations. Following a systematic literature review, these preliminary statements were adapted in line with the current state of knowledge and evaluated by means of a Delphi survey. Applicability of the approach in daily practice was ensured via a survey conducted among paediatric rheumatologists in Germany. Data from the national AID-Net registry were analysed with respect to therapeutic response. Results: This T2T initiative yielded a total of 26 statements supported by evidence and validated by expert agreement that cover the following categories: i) diagnosis, ii) differential diagnosis, iii) treatment targets, iv) colchicine treatment, v) monitoring, vi) colchicine intolerance / inadequate colchicine response, and vii) colchicine reduction / termination. Multidimensional treatment targets incorporating objective and subjective reported outcome measures were developed. They comprise attack frequency, severity of the single attack, number of school days missed due to FMF, level of inflammatory markers in the attack-free intervals, occurrence of chronic sequelae, as well as the subjective patient and physician reported outcome measure of satisfaction with the current disease status. A composite score of these items categorises disease severity as follows: remission or minimal disease activity, mild/moderate/severe disease activity. According to this evaluation, management recommendations are provided in the form of a flow chart to guide individual treatment decisions following the respective paths: continue colchicine, persisting attacks / inflammation, colchicine intolerance, persisting arthritis, colchicine reduction and adjustment/reduction of biologics. Data from the national AID-Net showed that colchicine therapy was successfully terminated in 63% of patients (27/44) with heterozygous MEFV mutations. Conclusion: The proposed consensus treatment plan for the management of FMF incorporates multidimensional targets allowing transparent treatment decisions, which will promote personalised disease management and increase adherence to therapy. Patient Consent: Not applicable (there are no patient data) Disclosure of Interest : L. Ehlers: None declared, E. Rolfes: None declared, M. Lieber: None declared, D. Müller: None declared, E. Lainka Consultant with: Mirum, Albireo, F. Gohar: None declared, G. Klaus: None declared, H. Girschick: None declared, J. Hörstermann: None declared, J. Kümmerle-Deschner Grant / Research Support with: Novartis, SOBI, Consultant with: Novartis, SOBI, J. Brunner: None declared, K. Palm-Beden: None declared, K. Tenbrock Grant / Research Support with: Pfizer, Consultant with: Pfizer, Novartis, L. von Wrangel: None declared, M. Faßhauer: None declared, N. Blank Grant / Research Support with: Novartis, SOBI, Consultant with: Novartis, SOBI, R. Trauzeddel Consultant with: Novartis, S. L. von Stuckrad: None declared, S. Higgins: None declared, T. Welzel: None declared, T. Lutz: None declared, V. Hentgen Grant / Research Support with: Novartis, SOBI, D. Föll: None declared, H. Wittkowski Grant / Research Support with: Takeda, Consultant with: Novartis, Takeda, T. Kallinich Consultant with: Roche
E. A. Smitherman 1 , J. G. Harris 2 , A. O. Hersh 3 , A. Chapson 4 , S. Oscar 4 , K. Wiegand 5 , E. M. Morgan 6 , J. M. Burnham 7 1 University of Alabama at Birmingham, Birmingham, 2 University of Missouri Kansas City, Kansas City, 3 University of Utah, Salt Lake City, 4 parent co-author, n/a, n/a, 5 Cincinnati Children’s Hospital, Cincinnati, 6 University of Washington, Seattle, 7 University of Pennsylvania, Philadelphia, United States Correspondence: E. A. Smitherman Introduction: Achievement of a low disease activity state, including remission,systemic lupus erythematosus (SLE). No prior studies have operationalized disease activity measures for clinical use. Objectives: Our objective was to design an intervention to standardize measurement of lupus disease activity and implement a Treat to Target strategy for lupus in the pediatric rheumatology clinic. Methods: Our team of 4 pediatric rheumatologists from 4 centers and 2 parents of children with lupus participated in bimonthly coaching sessions with a quality improvement specialist and advisor from May to November 2021. A scoping literature review was conducted to determine disease activity definitions with the best evidence for clinical use. We utilized the Model for Improvement and implementation mapping to design toolkit materials. Finally, we completed a baseline assessment using the National Implementation Research Network’s Hexagon Tool, Acceptability of Intervention Measure, Intervention Appropriateness Measure, and Feasibility of Intervention Measure. Each domain was assessed using a Likert scale 1-5 with 5 as the most positive response, and the mean response was calculated for each item. Results: We selected the Lupus Low Disease Activity State (LLDAS) and Definition of Remission in SLE (DORIS) definitions to operationalize for clinical use. We identified 4 key drivers for improving the proportion of clinic visits with disease activity and glucocorticoid targets assessed: 1) timely and complete data collection and documentation, 2) usable tools for data interpretation, 3) collaborative, efficient, and effective care teams, and 4) informed, activated, and engaged patients and families. We developed process maps and failure modes effect analysis around expected barriers to collecting disease activity measures in clinic. We defined process and outcome measures for a Lupus Treat to Target intervention and designed a phased implementation approach (Table 1). We co-produced a handout with information on lupus disease activity for patients and parents with our parent partners, as well as clinic staff educational material. On the Hexagon Tool, the highest ranking domain was Fit (4.3), followed by Capacity (4.2), Supports (4.0), Usability (4.0), Need (3.5), and Evidence (3.3). We found the intervention to be acceptable (4.9), appropriate (4.8), and feasible (4.4) among our team members. Conclusion: We designed and evaluated a Lupus Treat to Target Toolkit. Our next step is to pilot test implementation. Our long-term goal is to form a multi-center Pediatric Lupus Quality Collaborative with potential for significant impact on health outcomes in pediatric lupus. Disclosure of Interest : None declared
P219. Multitarget therapy in lupus podocytopathy: case and literature review P220. Oral midazolam before intra-articular corticosteroid injections in juvenile idiopathic arthritis P221. JAK inhibitors for the treatment of rheumatologic diseases in children: a retrospective case series P222. Impact of medications’ side effects on jia patients’ health related quality of life and well-being P223. Use and outcome of intra-articular steroid injections in juvenile idiopathic arthritis P224. Neridronate treatment in a pediatric cohort P225. Safety, and disease activity after live attenuated measles-mumps and rubella booster vaccination in children with juvenile idiopathic arthritis treated with immunosuppressive therapy: a retrospective long term follow up study P226. Female susceptibility to elevated ALT-levels and high methotrexate intolerance severity scores in juvenile idiopathic arthritis – a cross-sectional study P227. Worldwide evaluation of Clinical Practice Strategies (CLIPS) in Juvenile Inflammatory Rheumatisms (JIR) through the JIR-CLIPS network 28. Persistent hypogammaglobulinemia after rituximab therapy in children P229. Rapid response to pulse methylprednisolone in an adolescent girl with IGG4 disease related orbital mass P230. Preferences of pediatric rheumatologists for tapering biologic dmards in children with Juvenile Idiopathic Arthritis (JIA) – results of a clinical vignette study 31. Comparison of the effectiveness of the structured 3d exercise and the conventional exercise program for scoliosis in children with rheumatic disease P232. Treat-to-target approach in JIA, SLE and JDM: parent-reported outcomes and treatment satisfaction, first data of the ProKind-rheuma project P233. Safety of vaccination under canakinumab in patients with autoinflammatory periodic syndromes - interim analysis of the reliance registry 234. Uveitis as predictive factors of relapse after TNF-inhibitor withdrawal in a cohort of JIA 5. What matters most to pediatric rheumatologists in deciding whether to withdraw biologics in a child with juvenile idiopathic arthritis: a best-worst scaling study 6. Strategic use of levofolinic acid for methotrexate-induced side effects in juvenile idiopathic arthritis: a prospective observational study P237. Use of intravenous iloprost for the treatment of digital ulcers in juvenile systemic sclerosis: a pediatric clinical case P238. Strategy for stopping tnf inhibitor in juvenile idiopathic arthritis does not affect effectiveness after re-treatment P239. What after the first biologic agent in JIA: preliminary data P240. Sirolimus in a case of generalized lymphatic anomaly P241. Overview of anakinra therapy in multisystemic inflammatory syndrome in children associated with COVID-19 (MIS-C) from tertiary pediatric rheumatology clinics perspective P242. A single center experience in biological therapy for juvenile idiopathic arthritis in Bulgaria , S. Rodriguez Aguayo, E. Faugier Fuentes, A. K. Primero Nieto, H. W. Bermudez Canales, A. Guzman Revilla Rheumatology, HOSPITAL INFANTIL DE MÉXICO, FÉDERICO GÓMEZ, Ciudad de México, Mexico Correspondence: M. A. V. Barba Aguilar Introduction: Lupus Nephritis is a major cause of morbidity and mortality in adult and pediatric patients with systemic lupus erythematosus. Approximately 20% to 75% of children with will develop nephritis. While, the pathogenesis of LN indicates the formation of immunocomplexes by complement activation and Fcy receptors and relates to the classification of Lupus Nephritis according to the 2003 International Society of Nephrology/Renal Pathology Society criteria. Additional histologic subtypes have now been described. Lupus Podocytopathy has been described in 1-1.6% of renal biopsies compatible with lupus nephritis as an additional histologic subtype of this disease, characterized by effacement of the podocyte footprint observed on electron microscopy and characterized by histopathologic lack of subendothelial and subepithelial immunocomplex deposits, occasional presence of mesangial deposits on immunofluorescence; clinically it is characterized by severe nephrotic range proteinuria. Currently, comparative studies have been carried out with the use of multitarget therapy with mycophenolate and tacrolimus as therapy for this entity, observing remission and reduction of relapse. Objectives: To present a case of Lupus Podocytopathy treated with multitarget therapy with mycophenolate and tacrolimus in a patient with lupus nephritis. Methods: Clinical case description and literature review. Results: A 14 year old boy who started with acute renal failure and Nephrotic Syndrome, was treated with hemodialysis due to dialysis urgency. Systemic Lupus Erythematosus was diagnosed, so methylprednisolone and steroid were administered orally and multitarget therapy with mycophenolate 2 gr/day and cyclophosphamide 750 mg/m2/dose, during hospitalization he presented cytomegalovirus infection and due to persistent activity gamma globulin and plasmapheresis was administered for 5 sessions. He presented Thrombotic Microangiopathy and 5 sessions of plasmapheresis were indicated again. A renal biopsy was performed which reported class III lupus nephropathy, activity index 4/24 and chronicity 2/23, immunofluorescence with mesangio IgG+, IgM+, scarce deposits. Proteinuria persisted in nephrotic range after 6 boluses of cyclophosphamide, so rituximab 375 mg/m2 (4 weeks) was started with refractoriness. Electron microscopy was performed with a report of effacement of filtration diaphragms, consistent with lupus podocytopathy. Multitarget therapy was started with tacrolimus 0.07 mg/kg and mycophenolate with remission of proteinuria, negative anti DNA, normal C3 and C4, inactive sediment. Conclusion: Podocytopathy should be suspected in those patients with proteinuria persisting in the nephrotic range and electron microscopy should be performed. In addition, it should be considered that in spite of not being included in the classification there are cases in the literature in adults and children with this entity. Multitarget therapy in the case of this patient achieved complete remission of the disease and SLEDAI of 0 points, which could suggest a therapy option in those refractory cases, even those treated with rituximabBurrone 1 , M. Guazzi 1 , R. Naddei 1 , M. Spelta 1 , C. Malattia 1 , N. M. Disma 2 , A. Ravelli 3 , A. Consolaro 1 1 UOC Clinica Pediatrica e Reumatologia, 2 UOC Anestesia e Terapia del dolore Acuto e Procedurale, 3 Direzione Scientifica, Istituto Giannina Gaslini, Genova, Italy Correspondence: M. BurroneMidazolam, a short acting benzodiazepine, has been found to be an effective premedication in children by reducing anxiety and mitigating uncooperative behavior with a large margin of safety. Midazolam is primarily administered intravenously; however, recently a new oral solution designated for procedural sedation in children has been introduced. Several studies in this field have shown that a child’s level of anxiety before a painful medical procedure is directly correlated with the amount of pain experienced. Objectives: We aimed to determine the efficacy of oral midazolam as premedication to improve tolerance of IACI in children with JIA Methods: In this one month pilot study, all consecutive patients who were prescribed one or more IACIs by the caring physician at the study Unit were proposed to participate in the study. Premedication with midazolam was offered to an unselected group of children based on the availability of the procedural anesthesia team of the Gaslini Institute. All children received local anesthesia. Patients in Group 1 received 0.25 mg/kg (maximum 15 mg) of oral midazolam solution and local anesthesia with lidocaine/prilocaine 5% cream, 30 to 45 minutes before IACI; patients in Group 2 received only local anesthesia. Patients’ pain experienced before and at the end of the procedure were assessed using a 0-10 21-circle visual analog scale (VAS). Subjective rating of the ease of performing the procedure by the operator was recorded on a 4 points Likert scale (“Very easy”, “Easy”, “Quite difficult”, “Very difficult”). The level of anterograde amnesia was recorded on a 3 points Likert scale (“No amnesia”, “Partial amnesia”, “Complete amnesia”). During procedure and until two hour after, vital signs and oximetry were monitored and recorded in children receiving midazolam. Results: Seven patients were enrolled in Group 1 and seven patients in Group 2. Patients receiving only local anesthesia were older (mean age at procedure 16 years for Group 2, 12 years for Group 1, p = 0.04); 28% of patients received multiple IACIs in both groups. Pain level prior the procedure was similar in the two groups (p = 0.7). Patients received midazolam reported less pain (mean score 2.1) compared those receiving local anesthesia alone (mean score 3.4) at the end of the procedure, although the difference was not significant (p = 0.25). In 28% of cases physician described the procedure as difficult in Group 2, compared to 14% in Group 1. All patients receiving midazolam reported some degree of anterograde amnesia (28% reported complete amnesia, 72% partial amnesia) when recalling the procedure. No adverse events were recorded in both groups. Conclusion: In this small cohort of children, although not significantly, oral midazolam reduced pain during IACIs and increased the ease of the procedure. Oral midazolam is a safe and effective premedication before IACI in patients who require or prefer sedation and improves the child’s perception of the procedure, by inducing anterograde amnesia. This pilot study opens to further investigation, aiming to contribute to improvement in patient care and outcomes. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
D’Agostin 1 , A. Pin 2 , A. Tesser 2 , S. Pastore 2 , A. Taddio 2 , A. Tommasini 1,2 1 University of Trieste, 2 Institute of Maternal and Child Health – IRCCS Burlo Garofolo, Trieste, Italy Correspondence: M. D’Agostin Introduction: Based on their mechanism of action and clinical experiences in small series, JAK inhibitors (JAKinibs) have been proposed to treat paediatric diseases characterized by an interferon-mediated inflammation, when conventional anti-inflammatory therapies have failed (1,2). Objectives: We aim to describe our clinical experience with JAKinibs in the treatment of rheumatologic diseases in children. Methods: We enrolled all the paediatric patients who underwent a treatment with JAKinibs for a rheumatologic condition at the Institute for Maternal and Child Health IRCCS Burlo Garofolo (Trieste), between December 2016 and April 2022. The benefit was evaluated considering the reduction of clinical symptoms, the need of corticosteroids treatment and the number of concomitant medications. Changes in IFN score and inflammation biomarkers were assessed. Finally, adverse effects were reported and evaluated. Results: Overall, we treated thirteen patients (10 females; mean age 13.6 ± 5.9 years) for a mean duration of 2.1 ± 1.5 years. Nine patients were treated with baricitinib (mean dose 0.17 ± 0.11 mg/kg/day), the remaining four patients with tofacitinib (mean dose 0.30 ± 0.05 mg/kg/day). The treatment indications were heterogeneous. Half patients had monogenic disorders associated with increased interferon signalling: two COPA syndrome, one monogenic lupus due to DNAse2 deficiency, one STAT1 gain-of-function disease with systemic lupus erythematosus (SLE)-like phenotype, one histiocytosis-lymphadenopathy plus syndrome, one CANDLE (chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature) syndrome. The others had multifactorial disorders with high interferon score and poor response to first-line treatment: two polyarticular juvenile idiopathic arthritis (JIA), one SLE, one Weber-Christian panniculitis, one juvenile systemic sclerosis, one localized scleroderma and one scleroderma-like chronic cutaneous graft versus host disease. The mean age at disease onset and at the start of treatment were respectively 5.6 ± 4.3 years and 11.4 ± 5.3 years. Almost all the patients, except one, had a refractory disease that had not responded to conventional antirheumatic drugs and biologics. Eleven patients had tried several medications before JAKinibs (mean 3.6, range 0–9 medications). Moreover, ten patients out of 13 (76.9%) were receiving corticosteroids before the start of JAKinibs treatment. Daily prednisone dose decreased significantly after the start of JAKinibs from 0.52 mg/kg/day (range 0.2 – 1 mg/kg/day) to 0.07 mg/kg/day (range 0 - 0.2 mg/kg/day, p-value <0.001). Interesting, for six patients out of 10, the treatment with corticosteroids was not necessary once they started JAKinibs therapy. Moreover, the number of concomitant medications used after the start of JAKinibs tended to be lower than before (mean 2.07, range 1-3) (p=0.56). We obtained a good control of the disease in ten patients (76.9%), with clinical improvements. In three cases we attended a progression of disease. A reduction of interferon signalling was recorded in eight out of ten subjects at last follow-up, irrespectively from clinical improvements. Three patients developed drug-related adverse events, which were considered serious just in one case: two of them had a worsening of lymphopenia and one a transient increase of gammaglutamyl transferase and dyslipidaemia. Conclusion: Our experience confirms how JAKinibs may be a valuable treatment for severe interferon-mediated inflammatory disorders in children and it is confirmed by an improvement of symptoms, a reduction of daily prednisone dose and of the number of concomitant medications. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
M. Dellepiane 1 , E. Pescio 1 , M. Spelta 1 , R. Naddei 1 , M. Burrone 2 , C. Trincianti 2 , F. Ridella 2 , R. Cuttica 3 , A. Estmann 3 , S. Kamphuis 3 , A. Ravelli 2,4 , N. Ruperto 3 , A. Consolaro 1,2 1 Clinica Pediatrica e Reumatologia, IRCCS G. Gaslini, 2degli Studi di Genova, 3 UOSID Centro Trial Printo, 4 Direzione Scientifica, IRCCS G. Gaslini, Genoa, Italy Correspondence: M. Dellepiane Introduction: In recent years, there have been remarkable advances in the management of JIA, which comprise the advent of medications that are capable of inducing extended periods of complete disease quiescence. However, it is well known that prolonged treatment with immune-modulatory medications may be associated with the presence of side effects (SEs). It was not yet explored the impact of SEs on patients’ perception of the disease status. Objectives: Aim of the study is to explore the impact of the presence and number of medications’ SEs on the health related quality of life of JIA children. Methods: We included subjects in the EPidemiology, treatment and Outcome of Childhood Arthritis (EPOCA) study, receiving medications to treat JIA. The EPOCA dataset is made of more than 9.000 patients with JIA from 118 pediatric rheumatology centres in 49 countries. The presence and classification of SEs was collected by the juvenile arthritis multidimensional assessment report (JAMAR). When available, patient-reported data were considered; differently, parent proxy-reported data were retained. Disease features and parent/patient reported outcomes (PROs) were compared between subjects reporting and those not reporting medications SEs. The frequency of each side effect was compared as well: school related problems; compliance to prescribed therapy; satisfaction with current disease state. Finally, we compared the level of well-being measured with a 0-10 VAS and the level of health related quality of life measured with the Pediatric Rheumatology Quality of Life (PRQL) scale according to the number of SEs reported by the patient or parent. Results: We included in the analysis 6,911 JIA patients receiving treatment for JIA; 2,041 (29.5%) reported at least one side effect of medications. “Nausea” (12%), “Mood swings (excitement, depression, anxiety)” (7%), “Pain or burning feeling in the stomach” (7%), and “Headache” (7%) were the SEs more frequently reported. Children with SEs had older age at onset and higher JADAS, and were receiving more frequently synthetic DMARDs and corticosteroids (p < 0.001). Table shows the comparison of main PROs based on the presence of SEs. Patients reporting school related problems had more frequently SEs (43% vs 24%) and subjects satisfied with disease outcome and reporting compliance to therapy had less frequently SEs of medication (27% vs 39% and 30% vs 48%, respectively). The Well-Being VAS and the PRQL score progressively worsened when subjects reported no SE, 1 SE, 2 SEs, and >2 SEs (p < 0.001 for both comparisons). Conclusion: The presence of medications SEs had a significant impact on patients’ health related quality of life, and in particular on school-related problems, compliance to therapy, and satisfaction with disease state. Parents and patients give weight to the presence and the number of SEs when scoring the well-being VAS. Patient Consent: Yes, I received consent Disclosure of Interest : M. Dellepiane: None declared, E. Pescio: None declared, M. Spelta: None declared, R. Naddei: None declared, M. Burrone: None declared, C. Trincianti: None declared, F. Ridella: None declared, R. Cuttica: None declared, A. Estmann: None declared, S. Kamphuis: None declared, A. Ravelli Speaker Bureau with: AbbVie, Angelini, BMS, Pfizer, Hoffman LaRoche, Novartis, Pfizer and Reckitt Benckiser, N. Ruperto Grant / Research Support with: Bristol Myers and Squibb, Eli-Lilly, F Hoffmann-La Roche, Novartis, Pfizer, Sobi, Consultant with: 2 Bridge, Amgen, AstraZeneca, Aurinia, Bayer, Brystol Myers and Squibb, Celgene, inMed, Cambridge Healthcare Research, Domain Therapeutic, EMD Serono, Glaxo Smith Kline, Idorsia, Janssen, Eli Lilly, Novartis, Pfizer, Sobi, UCB, A. Consolaro Grant / Research Support with: Pfizer
A. Dghaies, K. Maatallah, H. Ferjani, L. Kharrat, S. Miladi, W. Triki, D. Ben Nessib, D. Kaffel, W. Hamdi Rheumatology, Mohamed Kassab Institute, Manouba, Tunisia Correspondence: W. Hamdi Introduction: The intra-articular steroid injections are a commonly used tool in the management of juvenile idiopathic arthritis (JIA). Objectives: This study aimed to assess the efficacy of intra-articular steroid injections in JIA patients. Methods: It was a retrospective study including children with JIA (according to the International League of Associations for Rheumatology (ILAR)) followed in the rheumatology department of Kassab institute Tunisia. General demographic and clinical information were collected; family history of chronic inflammatory diseases, child’s current age, age at disease onset, duration of disease progression, JIA subset, visual analog scale (VAS), inflammatory biomarkers (sedimentation rate (SR), c reactive protein (CRP), and concomitant treatments. Intra-articular steroid injections and their efficiency were noted. Results: Among 43 patients with JIA, 11 children (8 boys) needed intra-articular steroid injections and one of them needed two injections. The mean age was 12.2 ±4.2 years old [4-19]. The mean duration of the disease was 2.5 years, with extremes varying from one to 4 years. The mean age at disease onset was 9.7 ±4.3 years [2-15]. Family history of a chronic inflammatory disease was noted in 3 cases and psoriasis in 2 cases. The frequency of each JIA subset was as follows: polyarticular with rheumatoid factor (n=1), psoriatic arthritis (n= 1), enthesitis-related arthritis (n=6), and oligoarthritis (n= 3). One patient had a positive antinuclear antibody. As for the treatment course, 9 patients received non-steroidal anti-inflammatory drugs (naproxen in 3 patients, diclofenac in 5 patients, and naproxen then diclofenac in 3 patients). Seven patients received methotrexate with a mean dose of 10mg per week and 3 patients received biological treatment (etanercept in 2 cases, adalimumab in 1 case). The mean patient VAS was 4.7±2.4, the mean parent VAS was 2± 1.42, and the mean doctor VAS was 5±1.4. The mean SR was 26.5 ±29 mm/h, and the mean CRP was 12.1 ±13.2. Among the eleven intra-articular steroid injections performed, 2 injections were in the sacroiliac joints, 6 in hip joints, and 4 in knee joints. The corticosteroid used was triamcinolone hexacetonide in 3 cases for the hip joint and betamethasone in the other cases. There was a negative significant association between the use of the injection and the duration of the disease (p=0,04). Patients were evaluated during the next visit after a mean duration of 2.6± 1.2 weeks. Six patients reported improvement of the pain (mean patient VAS dropped to 3.2 ±1.3) with a mean improvement rate in joint pain of 53.3% [40-80]. There was no improvement and no worsening of the pain for the rest of the patients. Conclusion: The result of our study showed that Intra-articular steroid injections helped to reduce pain in JIA patients. Those injections may be useful, especially as a bridge therapy, in non-yet controlled disease. Disclosure of Interest : None declared
M. Ricci 1 , A. Marino 2 , M. Gattinara 2 , C. Chighinzola 1 , I. Pontikaki 2 , R. Caporali 1 , R. Cimaz 1 , T. Giani 1 1 University of Milan, 2 Gaetano Pini Hospital, Milan, Italy Correspondence: T. Giani Introduction: Bisphosphonates (BPs) have been increasingly used in pediatrics for the treatment of different disorders characterized by bone fragility, even if long-term efficacy and safety studies are still lacking. Currently, the use of Neridronate in Italy is licensed for adult patients affected by Paget’s disease and complex regional pain syndrome type 1 (CRP1), and under 18 years of age, for the treatment of Osteogenesis Imperfecta (OI). Objectives: We analyzed the safety profile of Neridronate in a pediatric cohort, and secondary we evaluated its efficacy in reducing bone lesions observed at Magnetic Resonance Imaging (MRI). Methods: In this retrospective observational study, we included 25 patients (17 females, 8 males), with a median age of 11 years and 6 months (range 1 year and 2 months- 17 years and 4 months) who received Neridronate for one of the following conditions: CRP1 (13), chronic recurrent multifocal osteomyelitis, CRMO (6), OI (2), osteochondritis (1), sacroiliac bone edema (1), avascular necrosis (AVN) of the femoral head (1), reduced bone mineral density associated with Neurofibromatosis type I (1). Neridronate was intravenously administered at a standard dose of 2mg/kg. The treatment regimen included a single infusion every 3 months or a cycle of 4 infusions over 14 days, eventually followed by a 3-month booster dose. Results: Fifteen out of 25 children (60%) had some side effect; flu-like symptoms were the most frequent one, observed in 14/15 cases. These patients complained of arthralgia and low-grade fever, that developed within the first 24-48 hours from the infusion and lasted 1-2 days. One patient referred itching during the first minutes of the first infusion in the absence of cutaneous signs, one presented bilateral conjunctivitis that resolved in one day, one referred significant nausea, which spontaneously resolved in a few hours, and one presented transient neutropenia (lowest value 700/mmc), normalized within 2 weeks. The majority of the patients who presented some symptoms (14/15, 93.3%) developed them at the first infusion. In 21/25 patients a post-Neridronate treatment MRI was available. A clear improvement or a complete recovery of the radiological findings detected at the pre-Neridronate MRI was observed in 9/13 patients with CRP1 , 3/6 with CRMO, 2/2 with OI, and 1/1 with bone edema at the sacroiliac joint. Conclusion: In this pediatric cohort side effects linked to intravenous use of Neridronate, although not unusual, resulted to be mild, rapidly self-limiting, and commonly associated with the first infusion. Despite our limited and heterogeneous data, Neridronate seems to be effective. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
M. Hamad Saied, J. W. van Straalen, M. Jansen, G. de Joode-Smink, N. M. Wulffraat Department of Paediatric Immunology and Rheumatology, Wilhelmina Children’s Hospital, University Medical Center Utrecht, Utrecht, The Netherlands, Utrecht, Netherlands Correspondence: M. Hamad Saied Introduction: Vaccines, especially live-attenuated vaccines, in immunocompromised patients pose a great challenge, due to the hypothetical risk of infection with the live-attenuated pathogen, lower immunogenicity due to treatment and the fear that vaccination itself may lead to disease flare[1]. In the Netherlands, JIA patients receive a measles, mumps and rubella (MMR) booster vaccine around age nine years as part of the Dutch National Immunization Program. This practice has been supported by a randomized control trials and EULAR recommendations [2]. Objectives: To study long-term disease activity, vaccine-related adverse events in a large cohort of JIA patients who received the MMR booster vaccine while being treated with immune modulatory drug therapy . Methods: Clinical and drug therapy data were collected for two visits prior and five visits after the 9 th birthday from electronic medical records of JIA patients treated with corticosteroids, synthetic disease-modifying antirheumatic drugs and biologicals since 2007 at the Wilhelmina Children’s Hospital. Frequency of patient-reported adverse events following MMR booster vaccination was determined. The adjusted effect of MMR booster vaccination on MMR antibody levels, the active joint count, physician global assessment of disease activity, patient-reported visual analogue scale (VAS) for disease activity and clinical Juvenile Arthritis Disease Activity Score (cJADAS) was analyzed using linear mixed effects models with a random intercept per patient and a random slope for MMR booster vaccination. Missing values were handled by multiple imputation. Results: Data were collected for 202 patients and 1254 visits. Following MMR booster vaccination, 5 patients (2.5%) reported fever and/or flu like symptoms, 1 patient (0.5%) reported joint pain and 6 patients (3.0%) reported injection site reactions. Disease activity after MMR booster vaccination was not higher than before vaccination (Table 1). Analysis of MMR antibody levels is still in progress. Conclusion: MMR booster vaccination was safe and did not result in worse disease activity in a large cohort of patients with JIA under immune modulatory drug therapy during long-term follow-up. Currently we are performing MMR serology to test the long term immunogenicity of the MMR booster References 1. Heijstek MW, Kamphuis S, Armbrust W, Swart J, Gorter S, de Vries LD, et al. Effects of the Live Attenuated Measles-Mumps-Rubella Booster Vaccination on Disease Activity in Patients With Juvenile Idiopathic Arthritis. JAMA. 2013 Jun 19;309(23):2449. 2. Heijstek M, Ott de Bruin L, Borrow R, van der Klis F, Koné-Paut I, Fasth A, et al. Vaccination in paediatric patients with auto-immune rheumatic diseases: A systemic literature review for the European League against Rheumatism evidence-based recommendations. Autoimmunity Reviews. 2011 Dec;11(2):112-22. Disclosure of Interest : None declared
C. Wibrand 1 , N. Kyvsgaard 2 , A. E. Christensen 3 , T. Herlin 1,* 1 Department of Pediatrics, Aarhus University Hospital, Aarhus N, 2 Department of Pediatrics, Goedstrup Hospital, Herning, 3 Department of Pediatrics, Odense University Hospital, Odense, Denmark Correspondence: T. Herlin Introduction: Methotrexate (MTX) plays a key role in the treatment of juvenile idiopathic arthritis (JIA), but MTX-intolerance (including nausea) is a significant clinical challenge, affecting up to 50% of the treated children. It is well established that MTX-treatment might affect the liver in JIA, illustrated by elevated levels of alanine aminotransferase (ALT), and that nausea is a common but unspecific symptom of liver affection. Objectives: Our study aimed to investigate the association between ALT-levels during MTX-treatment and MTX-intolerance in children with JIA. Methods: Children aged 9 years or above, diagnosed with JIA and treated with MTX for at least 6 weeks, were eligible for enrollment. Enrollment period was December 2013 - July 2016. MTX-intolerance was assessed on the date of enrollment using the Methotrexate Intolerance Severity Score (MISS), completed by the children’s parents. The MISS ranges from 0-36 points, and a child was categorized as MTX-intolerant if the MISS total was 6 or above with at least 1 point for a behavioral, anticipatory and/or associative symptom. ALT-levels were determined at the date of enrollment. ALT-levels were categorized as elevated if above the upper normal limit. Results: A total of 121 children were enrolled (82 girls;39 boys). The MISS-questionnaire was not completed for one child, and for two children no ALT-value in proximity to the enrollment date was available, leaving 118 patients for analysis (Table 1). MTX-intolerance was registered in 61%. There was no statistically significant difference between the MTX-intolerant group and the MTX-tolerant group on any of the demographic parameters. Additionally, ALT-levels did not differ between the MTX-intolerant group (mean = 29.8 U/L [95%CI: 21.4-38.1]) and the MTX-tolerant group (mean = 29.4 U/L [95%CI: 19.7-39.2]; t-test p= 0.96). MTX-intolerance was prevalent in around 60% of both boys and girls, and the MTX-intolerant girls and the MTX-intolerant boys did not differ statistically significantly on any of the demographic parameters. Nine (18%) of the MTX-intolerant girls had elevated ALT-levels, compared to none of the MTX-intolerant boys. The MTX-intolerant girls also had higher numerical ALT-values (mean = 33.9 U/L [95%CI: 19.9-47.8]) than the MTX-intolerant boys (mean = 19.4 U/L [95%CI: 15.6-23.1]; t-test p=0.05). Furthermore, the MTX-intolerant girls had a higher MISS (median = 14.0 [IQR: 9.3-17]) than the MTX-intolerant boys (median = 10.0 [IQR: 7.3-12]; Mann-Whitney-U-test; p = 0.009). Conclusion: No overall association between MTX-intolerance and ALT-levels was found. However, our results suggest a female susceptibility to MTX treatment recorded as high MISS and elevated transaminase levels. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
M. Hofer 1,2 , D. Gorczyca 3 , R. Cimaz 4 , S. Georgin-Lavialle 5 , V. Hentgen 6 , F. Hofer 7 , T. Kallinich 3 , K. Theodoropoulou 1,2 , H. Wittkowski 8 , S. Kamphuis 9 1 Pediatric Rheumatology Unit of Western Switzerland, Lausanne University Hospital (CHUV), Lausanne, 2 Geneva University Hospital, Geneva, Switzerland, 3 Pediatric Pneumology and Immunology, Charité University Medicine, Berlin, Germany, 4 †, †, Italy, 5 Department of Internal Medicine, French reference center for autoinflammatory diseases (CEREMAIA), Tenon Hospital, Paris, 6 Department of General Pediatrics, French reference center for autoinflammatory diseases (CEREMAIA), Versailles Hospital, Versailles, France, 7 JIRcohorte, Foundation RES, Etoy, Switzerland, 8 Pediatric Rheumatology and Immunology, University Hospital Muenster, Muenster, Germany, 9 Pediatric Rheumatology, Erasmus MC, Sophia’s Children Hospital Rotterdam, Rotterdam, Netherlands Correspondence: M. Hofer Introduction: Juvenile Inflammatory Rheumatism (JIR) is a family of rare and chronic diseases that carry the risk of significant morbidity and early mortality. Many affected patients will need lifelong medication. Although evidence or consensus-based recommendations for diagnosis and treatment exist, they are difficult to implement in a real-life setting due to the wide array of (country-specific) medical systems and financial capabilities. Additionally, physicians in many countries need support in decision-making processes for these rare diseases in childhood Objectives: The objectives of this proposal are: 1) to build a network of pediatric and adult rheumatology to foster exchange of clinical experiences, training, and clinical research between European and non-European countries; 2) to assess, based on this network, the diagnostic procedures, treatment algorithms, and outcome measures applied among patients with five medical conditions representative of the large spectrum of JIR-diseases (lupus nephritis, vasculitis, Still’s disease, biological treatments in monogenic periodic fever syndromes, PFAPA); 3) to analyze and compare these clinical practice strategies (CliPS) within and between the different countries. Methods: The JIR-CliPS project was launched in October 2021. Working groups for each of the five medical conditions cited above were formed and online questionnaires for the surveys developed. These include questions on participants’ demographics and local/national constraints as well as specific questions on the diagnosis, therapy, and general care for these five medical conditions. We are now starting a pilot data collection in four countries (Brazil, Ghana, New-Zealand, and Switzerland) to test the questionnaires and the methodology. Results: This exchange of expert experiences will help to better harmonize the CliPS used for the five studied JIR medical conditions and should help to improve the outcome of children with Juvenile Inflammatory Rheumatism worldwide. Conclusion: -Höppener 1 , S. Veldkamp 2 , M. De Groot 3 , S. Haitjema 3 , J. Drylewicz 2 , J. J. Boelens 1,4 , C. Lindemans 5 , J. Montfrans 1 , A. Van Royen-Kerkhof 1 , M. Jansen 1 1 Pediatric Rheumatology and Immunology, Wilhelmina Children’s Hospital, University Medical Center Utrecht, 2 Center for Translational Immunology, 3 Central Diagnostic Laboratory, University Medical Center Utrecht, Utrecht, 4 Department of Pediatrics, Memorial Sloan Kettering Cancer Center, New York, 5 Blood and Bone Marrow Transplantation, Princess Máxima Center for Pediatric Oncology, Utrecht, Netherlands Correspondence: S. Höppener Introduction: Hypogammaglobulinemia (HG) occurs regularly as a result of rituximab therapy (RTX). Although post-RTX Hypogammaglobulinemia and its associated risk factors have been well defined in adults, research on this complication in children is still scarce. Objectives: This study aims to determine the prevalence, risk factors and clinical consequences of HG in children after RTX therapy. Methods: A retrospective cohort study was conducted at the University Medical Center Utrecht including all patients aged ≤ 18 years treated with RTX between 2000 and 2020. Data including patient characteristics, treatment characteristics, recurrent infections, need for IVIG supplementation, and Immunoglobuline (Ig) levels were collected from the Utrecht Patient Oriented Database and evaluated. Patients were classified as having HG when (1) IgG levels were less then 1.96 standard deviations below the mean of age matched healthy controls, or (2) during IVIG therapy for any indication. Results: 145/171 eligible patients met the inclusion criteria. RTX indications included post-HSCT complications (n=67), autoimmune disease (n=43), haematological malignancies (n=13), kidney disease (n=11), post-solid organ transplantation complication (n=6) and immunodeficiencies with immune dysregulation (n=5). HG post-RTX was observed in 84/116 patients of whom IgG follow-up data was available (72%). Median follow-up after last RTX dose was 2.0 years (IQR 0.7-4.6 years), with 23 patients not having recovered from HG at this time (39%). Persistent HG (>6 months after last RTX dose) was observed in 61/95 patients (64%) and occurred more frequently in children treated due to post-HSCT complications compared to autoimmune disease (OR=8.34, p<0.001). Low baseline IgG, IgA and IgM levels were associated with persistent HG (p=0.002, p=0.005, p=0.024, respectively), while multiple logistic regression identified low baseline IgG levels (p=0.024) and post-HSCT complication as RTX indication (p<0.001) as significant risk factors for persistent HG. 89/145 patients received IVIG supplementation during follow-up. Recurrent infections were observed in 18 patients, which could be directly attributed to RTX-related HG in 7 patients. Twenty-three patients died during follow-up, with 2 deaths due to, or complicated by, proven bacterial infections during persistent HG. Secondary Ig class switch impairment was observed in 2 patients, one of whom died of bacterial meningitis after discontinuing IVIG supplementation against medical advice. ConclusionTrial registration identifying number: In this study, 72% of children developed HG following RTX therapy. This rate is higher than reported rates in adults, and in line with recent paediatric studies. Furthermore, a majority developed persistent HG, for which children post-HSCT and/or with low pre-RTX IgG levels were especially at risk. Children with HG regularly need IVIG supplementation, can suffer from (lethal) recurrent infections and, rarely but importantly, can develop secondary Ig class switch impairment after RTX. Evaluation of pre-RTX Ig levels and close monitoring of Ig levels after treatment are therefore highly recommended. Disclosure of Interest : None declared
B. Kasap-Demir Pediatric Nephrology and Rheumatology, İzmir Katip Çelebi University, İzmir, Turkey Kasap-Demir Introduction: Immunoglobulin G4-related disease (IgG4-RD) is a rare systemic, immune mediated, fibroinflammatory disease caused by monoclonal proliferation of IgG4(+) plasma cells. Orbital involvement is frequent in children. We have reported a case with an orbital mass related to IgG4-RD. Objectives: Case: A 12-year-old girl presented with a swelling in the right eye for the last five months, which was intensified in the last two weeks. In the orbital magnetic resonance, there was a heterogeneous and intensely enhancing mass lesion located in the right orbital superior extraconal area, extending to the medial and lateral extraconal areas, measuring 45x43x20 mm in the widest part. The lesion surrounds the superior rectus muscle and is in contact with the lateral and medial rectus muscles. She was referred to neurosurgery for biopsy, which revealed IgG4 related disease. In her physical examination, she had a huge mass in her right eye. All other systemic findings were normal. Laboratory test revealed normal complete blood count test and urinalysis. Biochemical markers were within normal limits. C3 and C4 were normal; ANA, p-ANCA and c-ANCA were negative. IgG4 level was normal. Abdominal, thyroid and parotid ultrasonography were negative. We treated her with three pulse metylprednisonone (PMP) and the mass retarded dramatically in a few days, but she had ptosis in the . Concomittantly, we have prescribed mycophenolate mofetil and used corticoteroids with tapering doses. However, residual orbital tumor was defined in the control orbital MRI and debulking surgery was performed. As the latest current status, she was on MMF and the lowest dose of corticosteroids. Methods: - Results: - Conclusion: Conclusion: PMP is may be an effective treatment of choice for huge orbital masses associated with IgG4-RD as the first step. Surgery may be another option for cases resistant to medical treatment. Trial registration identifying number: - Patient Consent: Yes, I received consent Disclosure of Interest : None declared
J. A. van Til 1 , M. M. Kip 1,2 , M. Twilt 3 , E. Schatorjé 4,5 , K. Groothuis-Oudshoorn 1 , S. Warta 1 , G. Currie 3 , D. A. Marshall 3 , J. F. Swart 2,6 , R. S. Yeung 7 , S. M. Benseler 3 , S. J. Vastert 2,6 , N. Wulffraat 2,6 , M. J. IJzerman 1 on behalf of on behalf of UCAN CAN-DU and UCAN CURE Consortium 1 University of Twente, Enschede, 2 University Medical Center Utrecht, Utrecht, Netherlands, 3 University of Calgary, Calgary, Canada, 4 Radboud University Medical Center, 5 St. Maartenskliniek, Nijmegen, 6 Utrecht University, Utrecht, Netherlands, 7 University of Toronto, Toronto, Canada Correspondence: M. M. Kip Introduction: Conclusive evidence on clinical and biologic determinants for successfully withdrawing (i.e. discontinuing or tapering) biologic DMARDs (bDMARDs) in patients with JIA in remission is lacking. In clinical practice, each decision to withdraw bDMARDs is based on the clinical judgement of individual pediatric rheumatologists. Objectives: To determine the influence of specific disease and clinical characteristics on the timing of the decision of pediatric rheumatologists to withdraw bDMARDs in children with JIA. Methods: A clinical vignette design was used to study predictors of clinician decision making regarding the timing of bDMARD withdrawal. We included the following nine disease and clinical characteristics: time to treatment response with the current bDMARD, rheumatoid factor (RF), flare history, bDMARD treatment failure, joint damage in the current treatment period, uveitis in the current treatment period, spine involvement, temporomandibular joint (TMJ) involvement, and patient/parent preference. An experimental design was used to systematically vary levels of the characteristics over 16 clinical vignettes. These clinical vignettes were distributed in an online survey among pediatric rheumatologists in the Netherlands and Canada. Respondents were first asked about their shortest treatment time after reaching clinical remission (6-18 months in 3 month intervals) in a child without complications. For each vignette, they were asked whether they would taper/stop bDMARDs at that time, or for how long they would continue. Statistical analysis included descriptive statistics, logistic and interval regression analysis. Results: Twenty-nine pediatric rheumatologists responded (response rate 35%) to the survey. Pediatric rheumatologists in Canada continue bDMARDs in an uncomplicated child in remission longer compared to pediatric rheumatologists in the Netherlands (median of 13.5 vs. 9.0 months, p=0.009). Mean treatment time before withdrawing bDMARDs for the clinical vignettes is 11.6 months. Logistic regression analysis shows that the strongest predictors of continuing bDMARDs are: patient/parent preference for continued treatment (OR 4.8; p<0.001), uveitis in the current treatment period (OR 3.7; p<0.001), bDMARD treatment failure (OR 2.2; p=0.017), and RF positive JIA (OR 1.8; p=0.033). Pediatric rheumatologists who indicate a longer treatment time in a child without complications in the clinical vignettes are significantly less likely to continue bDMARDs (OR 0.19; p=0.005). Interval regression analysis showed that, on average, pediatric rheumatologists wait 11.6 months before withdrawing bDMARDs. In a child with RF positive JIA, bDMARDs are withdrawn 4.9 months later than in a child with RF negative JIA. In a child with uveitis in the current treatment period, bDMARDs are withdrawn 3.7 months later than in a child with no history of uveitis. Conclusion: Of the characteristics included in the clinical vignettes, only TMJ involvement had no influence. Pediatric rheumatologists with shorter treatment times in a child without complications are more likely to continue treatment in a child with complications. We aim to integrate these results in a decision support tool for pediatric rheumatologists, and thereby reduce unwanted treatment variation among children with JIA on bDMARDP. Kısa 1 , E. Tarakcı 2 , G. Leblebici 3 , O. Kasapcopur 4 1 Health Science Faculty, Department of Physiotheraphy and Rehabilitation, Biruni University, İstanbul, 23 Health Science Faculty, Department of Physiotheraphy and Rehabilitation, Medeniyet University, 4 Cerrahpasa Faculty of Medicine, Department of Pediatric Health and Diseases, Department of Pediatric Rheumatology, Istanbul University Cerrahpasa, Istanbul, Turkey Correspondence: E. P. Kısa Introduction: Arthritis is one of the most common chronic diseases in childhood. It is one of the important causes of short and long-term sequelae. Problems such as weakness or pain may occur, especially in the muscles around the trunk. These conditions may lead to abnormal biomechanics in children with rheumatism. Delayed neuromuscular development, muscle weakness, foot problems, ligament laxity and growth disorders are other factors that contribute to changes in the musculoskeletal system (MS). Problems in the MS in children with rheumatism may cause displacement of the center of gravity, deterioration of biomechanics, and muscle imbalance. All these situations can lead to scoliosis, which we often encounter in children with rheumatism. Scoliosis is an important health problem, cosmetic, social and psychological problems related to the deformity that is mostly seen in adolescents. When we look at the literature, there are many recent studies describing various 3 dimension (3D) exercise methods (SEAS, Schroth, Dobomed, BSPTS, Side-shift, Lyon, etc.) effective on scoliosis. However, no study was found in the literature that applied these exercise methods in children with rheumatism. Objectives: In this study, it was aimed to compare the effectiveness of the conventional exercise program against the personalized structured 3D exercises in children with scoliosis with rheumatic disease. Methods: Children randomly divided into 2 groups. Structured 3D scoliosis exercises were taught to the first group (n=25), and conventional physiotherapy exercises (posture exercises and core stabilization exercises) were taught to the second group (n=25). Demographic characteristics were questioned with the “Sociodemographic data form”. Pain were evaluated using the visual analog scale (VAS), and trunk rotation angles (ATR) were evaluated using a scoliometer. Cobb angle was measured using the SubrotoAngleAid application on x-ray, and risser degrees were measured by the physician. Body awaraness was evaluated with Walter Reed Scale (WRS). Quality of life was questioned with Scoliosis Research Society Scale-23 (SRS-23). Statistical analysis of the data was performed using the SPSS 24.0 (Statistical Package for Social Sciences) program. P≤0.05 was considered statistically significant in all analyzes. Results: Of the children participating in the study, 47 were diagnosed with JIA, 2 with scleroderma, and 1 with dermatomyositis. The mean age of the first group was 11.76±2.26, and the second group was 11.40±2.73. There was no significant difference between the groups between age, height, BMI and VAS. Risser was measured as 2.56±1.38 in Group 1 and 2.38±1.20 in Group 2, and there was no difference between the groups. While Cobb angle and ATR improved in both groups, Group 1 showed a more significant improvement compared to Group 2. A positive correlation was found between disease duration, Cobb angle and ATR. Cobb angle and ATR increased as the disease duration increased (r: 0.55, 0.45, p≤0.001, respectively). A significant correlation was found between the decrease in Cobb angle and ATR and WRS (r:0.57, r:0.44, p≤0.001). Conclusion: Cobb angle, rotation angle, and body image components have an important place in determining the severity, progression or treatment method of scoliosis. The results of this study, which we planned to investigate the effectiveness of structured 3D scoliosis exercises, showed that this method is effective in reducing Cobb angle and rotation angle in scoliosis in children with rheumatism, increasing body image and quality of life. Being the first study in this field makes our study valuable. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
J. Klotsche 1 , K. Vollbach 2 , S. Eulert 1 , K. Tenbrock 2 , D. Foell 3 , J.-P. Haas 4 , F. Weller-Heinemann 5 , P. Oommen 6 , D. Windschall 7,8 , K. Moenkemoeller 9 , T. Kallinich 10 , M. Hufnagel 11 , I. Foeldvari 12 , T. Hospach 13 , M. Klaas 14 , M. Rühlmann 15 , R. Trauzeddel 16 , C. Schütz 17 , J. Kuemmerle-Deschner 18 , A. Klein 19,20 , K. Minden 1 , G. Horneff 19,20 1 Epidemiology, German Rheumatism Research Center Berlin, a Leibniz Institute, Berlin, 2 Klinik für Kinder- und Jugendmedizin, Universitätsklinikum Aachen, Aachen, 3 Klinik für Pädiatrische Rheumatologie und Immunologie, Universitätsklinik Münster, Münster, 4 Zentrum für Schmerztherapie junger Menschen, Deutsches Zentrum für Kinder- und Jugendrheumatologie, Garmisch-Partenkirchen, 5 Prof-Hess- Kinderklinik, Klinikum Bremen-Mitte, Bremen, 6 Klinik für Kinder-Onkologie, -Hämatologie und Klin. Immunologie, Bereich Pädiatrische Rheumatologie, Heinrich-Heine-Universität, Düsseldorf, 7 Clinic of Paediatric and Adolescent Rheumatology, St.-Josef-Stift Sendenhorst, Sendenhorst, 8 Medical Faculty, Martin- Luther-University Halle-Wittenberg, Halle, 9 Klinik für Kinder- und Jugendmedizin, Kinderkrankenhaus der Stadt Köln, Köln, 10 Department of Pediatric Respiratory Medicine, Immunology and Critical Care Medicine, Charité - Universitätsmedizin Berlin, Berlin, 11 Zentrum für Kinder- und Jugendmed, Universitätsklinik Freiburg, Freiburg, 12 Kompetenz-Zentrum für Uveiits und Sklerodermie im Kindes- und Jugendalter, Hamburger Zentrum für Kinder- und Jugendrheumatologie, Hamburg, 13 Zentrum für pädiatrische Rheumatologie, Olgahospital Stuttgart, Stuttgart, 14 Klinik für Kinderund Jugendmedizin, Vivantes Klinkum Friedrichshain, Berlin, 15 Kinderarztpraxis, Göttingen, 16 Fachambulanz Kinderrheumatologie, Helios Klinikum Berlin-Buch, Berlin, 17 Klinik und Poliklinik für Kinder- und Jugendmedizin, Universitätsklinikum Carl Gustav Carus, Dresden, 18 Klinik für Universitätsklinik für Kinder- und Jugendmedizin, autoinflammation reference center Tübingen und Abteilung für Pädiatrische Rheumatologie, Universitätsklinikum Tübingen, Tübingen, 19 Department of Pediatrics, Asklepios Kinderklinik St. Augustin, St. Augustin, 20 Department of Pediatric and Adolescents Medicine, University Hospital of Cologne, Cologne, Germany Correspondence: J. Klotsche Introduction: There is growing evidence that a treat-to-target (t2t) approach can improve the prognosis of juvenile chronic inflammatory rheumatic diseases (IRD). Therefore, this therapeutic strategy is recommended and increasingly implemented for the most common IRD [1,2]. The acceptance and outcomes of a t2t-approach in clinical practice are currently being investigated in the ProKind-Rheuma project of the GKJR. Objectives: To assess outcomes and parent-reported treatment satisfaction in a prospective cohort of children and adolescents with recent-onset IRD. Methods: ProKind-Rheuma is a multicenter prospective non-interventional observational study. Patients with recently diagnosed juvenile idiopathic arthritis (JIA), systemic lupus erythematosus (SLE), and juvenile dermatomyositis (JDM) have been enrolled in 18 centres in Germany since January 2020 and prospectively followed during the first year of treatment. Physician- and parent-reported data are collected up to five times in a standardized way; data from patients with at least one follow-up visit through May 2, 2022, were included. Results: A total of 420 patients included so far, 173 from 18 rheumatology sites with follow-up (FU) were considered for this analysis. Patients with JIA (n=147), SLE (n=16) and JDM (n=10) were enrolled 0.7±1.1 months after diagnosis and followed for 7.5±3.9 months until their last documented FU. Patients showed substantial health improvement over time (cJADAS-10, SLEDAI, Myositis-DAS). Parent-reported outcomes improved significantly (see table). Almost half of patients were treated with glucocorticoids and 66% with DMARDs. Most families (83%) were satisfied with the drug treatment (80% of JIA, 93% of SLE, and 100% of JDM patients), with 47% being very satisfied and 15% being extremely satisfied. 82% reported taking medications regularly or as recommended, with higher adherence in patients with JDM (100%) and SLE (93%) than in those with JIA (79%). 57 families (36%) reported medication-related adverse events at last follow-up, of which nausea (43%), stomach pain (41%), and sleep disturbances (21%) were the most common. The most frequently reported adverse events in SLE and JDM were different from those in JIA. In patients with SLE and JDM, weight gain and headache or increased body hair dominated in addition to gastrointestinal complaints. Conclusion: Patients with IRD improve rapidly in their health status during the first months under targeted therapy. Parents are largely satisfied with drug treatment, although adverse events are not uncommon. Satisfaction with therapy and treatment adherence are higher in patients with SLE and JDM. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
J. B. Kuemmerle-Deschner 1 , J. Henes 2 , B. Kortus-Goetze 3 , T. Kallinich 4 , P. T. Oommen 5 , J. Rech 6 , T. Krickau 7 , F. Weller-Heinemann 8 , G. Horneff 9 , A. Janda 10 , I. Foeldvari 11 , F. M. Meier 12,13 , C. Schuetz 14 , F. Dressler 15 , M. Borte 16 , M. Hufnagel 17 , M. Fiene 18 , J. Weber-Arden 19 , N. Blank 20 1 Department of Pediatrics, Division of Pediatric Rheumatology, 2 [email protected], University Hospital Tuebingen, Tuebingen, 3 Division of Nephrology, University of Marburg, Marburg, 456 Department of Internal Medicine 3 - Rheumatology and Immunology, Friedrich-Alexander University (FAU) Erlangen-Nuernberg and Universitaetsklinikum Erlangen, 7 Pediatrics, Friedrich-Alexander Universität Erlangen-Nürnberg (FAU), ErlangeDepartment of Pediatrics, University Hospital Ulm, Ulm, 11 Centre for Pediatric and Adolescence Rheumatology Hamburg, Hamburg, 12 Division of Rheumatology, University Hospital Frankfurt, Goethe University, Frankfurt am Main, 134 Pediatrics, Medizinische Fakultaet Carl Gustav Carus, Technische Universitaet Dresden, Dresden, 15 Division of Pediatric Pneumology, Allergology and Neonatology, Hannover Medical School, Hannover, 16 ImmunoDeficiencyCenter Leipzig (IDCL), Hospital St. Georg gGmbH Leipzig, Leipzig, 178 Rheumatology Center Greifswald, Greifswald, 19 NOVARTIS, Nuernberg, 20 Rheumatology, University Hospital Heidelberg, Heidelberg, Germany Correspondence: G. Horneff Introduction: Treatment of autoinflammatory periodic syndromes with the interleukin-1β inhibitor canakinumab (CAN) has been shown to be safe and effective in clinical trials and in practice. Patients are recommended to be vaccinated against common infections (including influenza, covid-19, pneumococcus) while on therapy, as is the general population. It is known from the literature that severe local and systemic inflammatory reactions can occur frequently in patients with immunosuppressive therapy, especially after pneumococcal vaccination [1]. Objectives: In this study, in addition to general safety parameters, the safety of the recommended vaccinations in patients with CAPS, FMF, HIDS/MKD and TRAPS under CAN therapy was investigated in clinical practice. Methods: RELIANCE is a prospective, non-interventionalautoinflammatory periodic syndrome who routinely receive CAN. Efficacy and safety parameters were recorded at baseline and assessed at 6-month intervals. Results: The interim analysis includes data from N=199 patients with autoinflammatory diseases enrolled in the RELIANCE registry between October 2017 and December 2021. The mean age of the overall cohort is 24.4 years (2-79 years; N=104 female patients [53%]) and the median duration of CAN treatment before study entry was 2 years (0-12 years). During the study, N=87 patients received a total of 130 vaccinations. Vaccination reactions were reported for N=16 patients, and N=8 patients were classified as suspected adverse drug reactions (Table 1). In no case was the vaccination reaction classified as severe. Covid-19 vaccination was given to N=42 patients (N=6 Comirnaty, N=1 Spikevax, N=36 not reported; 1 patient received 2 different vaccines). Of these, a vaccination reaction was reported for N=6 patients, which were not considered drug-related or classified as severe. Conclusion: The interim data from the RELIANCE study confirm the safety of long-term treatment with canakinumab in the entire study population. Vaccination with CAN therapy also did not reveal any new safety signals beyond known vaccine side effects. Disclosure of Interest :T. Kallinich Speaker Bureau with: Roche, P. T. OommenF. Weller-Heinemann: None declared, G. Horneff Grant / Research Support with: AbbVie, Chugai, Merck Sharp & Dohme, Novartis, Pfizer, Roche, Speaker Bureau with: AbbVie, Bayer, Chugai, Merck Sharp & Dohme, Novartis, Pfizer, Roche, A. Janda: None declared, I. Foeldvari Consultant with: Novartis, F. M. Meier Speaker Bureau with: Novartis, C. Schuetz: None declaredM. FieneI. Maccora, V. Accardo, E. Marrani, M. V. Mastrolia, I. Pagnini, G. Simonini 1 Rheumatology Unit, Meyer Children’s University Hospital, NeuroFARBA Department, University of FLorence, Florence, Italy Correspondence: I. Maccora Introduction: TNF-inhibitors (TNFi) have radically modified the prognosis of Juvenile Idiopathic arthritis (JIA). However, when, and how discontinuing the treatment is still a challenge. Objectives: The purpose of this study is to describe a cohort of patients with JIA treated with TNFi (Adalimumab (ADA) and Etanercept (ETA)) in whom these drugs were discontinued due to persistent remission on medicationsHospital, Florence. The study involved patients with a diagnosis of polyarticular and oligoarticular JIA according to ILAR criteria treated with a first course of ADA or ETA, that were then discontinued for persistent remission. We collected demographic, clinical and laboratory data at onset and at biologic starting. Relapse was entered when a JIA children flared after stopping medication due to arthritis, due to uveitis or both. We recorded the time between the onset of symptoms and diagnosis, diagnosis and start of biologics, start of biologics and the achievement of inactivity, presence of flare, duration of treatment, of disease inactivity, presence of relapse after withdrawal, time intercourse between withdrawal and relapse, and discontinuation modality (distinguishing increase of interval of administration, reduction of dose and abrupt discontinuation). Statistical Analysis were performed using SPSS v27 Results: 62 patients were enrolled (54 female), the 51.6% had an oligoarticular JIA and 48.4% polyarticular, 41.9% had uveitis, and 85.5% had ANA positivity. The median age at onset was 3 years old (R 1-11y), with a median value of ESR of 39 mm/h (R 2-100), of CRP 1.34 mg/dl (0.23-9.1) and of JADAS10 16.5 (R 6.4-34). The first course of the TNFi inhibitors was started after a median time of disease of 15 months (R 2-93m) and at a median age of 6 years (R 1-13 y). The 61.3% of patients (38) were treated with ADA, while the 38.7% (24) with ETA, of whom the 79% (49) were treated with a concomitant therapy of Methotrexate and the 14.5% (9) with corticosteroids. All the patients achieved the disease remission after median time of 4 months (R 1-32). The last item that achieved remission was arthritis in 79% (49) of patients, uveitis in 14.5% (9) and both in 6.5% (4). The therapy was discontinued after a median time of treatment of 30 months (R 7-62) and a median time of inactivity of 22 months (R 9-60). TNFi were stopped in the 56.5% increasing the interval of administration, in the 38.7% reducing the dose, and in the 19.4% abrupt discontinuation. The 80.6% (50) of patients relapsed after a median time of 5.5 months (R 0.5-96), because of arthritis in 64% (32) of patients, uveitis in 22% (11), both in 14% (7). We observed that patients who relapsed had significantly more frequently a positive anamnesis for uveitis (χ 2 3.9, p 0.04) and were younger when the TNFi were started (5.84 vs 7.67, p 0.021), while no difference were evaluated in the modality of suspension. The chance to relapse after TNF withdrawal resulted significant higher in JIA children with a history of uveitis (Log Rank χ 2 = 5.54 p = 0.019). Conclusion: The presence of uveitis seems to be significantly more frequent in subjects who relapsed, children with an history of uveitis had a shorter period of remission out of medicaR. Currie 1 , K. Groothuis-Oudshoorn 2 , M. Twilt 1 , M. M. Kip 3 , M. J. IJzerman 3 , S. M. Benseler 1 , J. F. Swart 4 , S. J. Vastert 4 , N. M. Wulffraat 4 , R. S. Yeung 5 , D. A. Marshall 1 on behalf of on behalf of UCAN CAN-DU and UCAN CURE Consortium 1 University of Calgary, Calgary, 2 University of Twente, Enschede, Canada, 3 University of Twente, Enschede, 4 University Medical Centre Utrecht, Utrecht, Netherlands, 5 University of Toronto, Toronto, Canada Correspondence: M. M. Kip Introduction: The care of children with JIA has significantly been transformed in the biologics era, however, these medications carry important (although rare) risks and are costly. There is little clinical guidance for physicians to identify which patients in clinical remission can safely have their biologic withdrawn (by stopping or tapering). Therefore we examined what characteristics of the child or their context are important to pediatric rheumatologists when making the decision to discuss the possibility of withdrawal of biologicals in JIA. Objectives: To identify what characteristics of the child, or their context,s in JIA. Methods: We conducted a survey including a best-worst scaling (BWS) exercise in pediatric rheumatologists in Canada and the Netherlands to assess the relative importance of 14 previously identified characteristics of children or their health care context. A balanced incomplete block design was used to generate choice sets, and respondents were asked to evaluate 14 sets of 5 characteristics of a child with JIA, and identify which among each set was most and least important in their decision to offer withdrawal. Results were analysed using conditional logit estimation and expressed as odd-ratios. Results: Fifty-one pediatric rheumatologists participated, with an overall response rate of 65%. The most important three characteristics were how challenging it had been to achieve remission, a history of established joint damage and the time spent in disease remission. The least important three characteristics in the opinion of pediatric rheumatologists for this issue were history of temporomandibular joint involvement, accessibility of biologics and the patient’s age. Conclusion: These findings give quantitative insight into the factors that are important to the decision making of pediatric rheumatologists about biologic withdrawal, an area where there is currently little clinical guidance. In addition to high quality clinical evidence, further research is needed to understand the perspective of patients and families to help inform shared decision-making about withdrawal of biologics in JIA patients with clinically inactive diseaMartini, A. Meneghel, M. Fastiggi, F. Dell’Apa, F. Tirelli, F. Vittadello, F. Zulian Department of Pediatrics, University Of Padova, Padova, Italy Correspondence: G. Martini Introduction: Low-dose methotrexate (MTX) is a pivotal therapy in Juvenile Idiopathic Arthritis (JIA) and in other autoimmune diseases. Around 50% of treated patients report side effects, mainly gastrointestinal symptoms, such as nausea and vomiting. These symptoms can occur after drug administration or even be anticipatory thus suggesting a psychological origin in these cases. Different methods have been suggested to reduce side effects such as use of anti-emetics, folic and levofolinic acid (LVF) but strategies are highly variable from centre to centre. Objectives: In our centre all patients receiving MTX take a single dose of LVF 48 hours after MTX administration but, despite this, some of them still report gastrointestinal discomfort. Our aim was to evaluate the efficacy of a supplemental dose of LVF, administered 48 hours before MTX, in reducing side effects without interference with drug efficacy Methods: A prospective observational study was performed including consecutive JIA patients reporting significant gastrointestinal side effects or discomfort post-MTX and excluding patients with anticipatory symptoms. At study entry (T0) patients were suggested to take a supplemental dose of LVF 48 hours before MTX administration and were followed every 3-4 months (T1 to T5). At each visit the following data were collected: efficacy of LVF on gastrointestinal symptoms, disease activity (JADAS, ESR, CRP values) and treatment (MTX reduction/increase/stop and biological agents start/reduction/increase) Results: 21 patients were recruited: 15 oligoarticular (12 ANA+), 5 polyarticular (1 RF+) and 1 enthesitis-related arthritis (ERA). At study entry (T0) mean MTX treatment duration was 40 months (median 22, range 2-185 months) and 7 patients also received a biological agent (5 adalimumab, 2 tocilizumab). In all patients, MTX was administered subcutaneously (mean dose 9.54 mg/m2) and was associated with LVF 48 hours later (mean 6.5 mg, range 2.5-7.5 mg). Side effects were nausea in 14 patients, vomiting in 5, nausea and diarrhoea (1) and vomiting and diarrhoea (1); 7 patients used anti-emetics (ondansetron). All patients were followed for at least 1 year (range 12- 29 months). Complete remission of the side effects was reported in 13/21 at T1, in 18/21 patients at T2, in 20/21 at T3, in 12/14 at T4, in 7/7 at T5). Friedman test for repeated measures from study start to T4 demonstrated statistically significant differences for JADAS and CRP values (p=0.006 and 0.008 respectively). Over the observation period, two patients started adalimumab (1 for disease relapse after SARS-CoV2 infection, 1 for persistent disease activity), in all remaining patients MTX dose was tapered and in 8 of them it was withdrawn for remission. Conclusion: Our study suggests that LVF administered 48 hours before MTX is effective in reducing MTX-induced gastrointestinal side effects. Furthermore, although MTX mechanism of action is to target folate pathway, increasing doses of LVF were not associated with a decreased efficacy of the drug. Taken together our results suggest that this strategic use of LVF may improve patients’ compliance and quality of life. Disclosure of Interest : None declared
Nardini 1 , C. Borzacchiello 1 , E. Coppola 1 , L. Martemucci 2 , F. Orlando 2 , M. Tardi 2 1 Department of Translational Medical Sciences, Pediatrics, Federico II University, 2 Department of Pediatrics, AORN Santobono Pausilipon, Naples, Italy Correspondence: G. Nardini Introduction: Juvenile Systemic Sclerosis (JSSc) is a rare connective tissue disease which affects skin, blood vessels, heart, lungs, kidneys, gastrointestinal tract and musculoskeletal system. Skin involvement has different phases: edematous, fibrotic, and eventually atrophic. In all phases there are: reduction of peripheral perfusion and risk of Digital Ulcers (DU). The management of this complication in adult population consists of different pharmacological options such as Calcium antagonists, Phosphodiesterase 5 inhibitors, Prostanoids and Endothelin 1 receptor antagonists, until even surgery. Objectives: The objective of this case report is to present a case of pediatric JSSc with DU successfully treated with intravenous Iloprost. Methods: We present the case of M., 4 years old, female, in apparent good health, who came to our attention for bilateral acute necrosis of foot fifth finger. At clinical examination on admission, DU with advanced necrotic evolution of both fifth fingers of feet, livedo reticularis, hardening and xerosis of the skin (especially on the face and extremities of inferior limbs), rhinolalia, thin hair, several necrotic areas (earcup, scalp) and right knee arthritis were observed. Blood tests showed a positive C Reactive Protein (22,43 mg/L, normal value <5), severe hypernatremia and hyperchloremia (Na + 165 mMol/L, Cl- 133 mMol/L) and hypoalbuminemia. At abdominal ultrasound hepatomegaly and mild left kidney hypotrophy were shown. Chest CT scan was performed and showed atelectasis areas in superior and medium right lobe. Inferior limbs angio CT scan showed normal vascularization. An onset of JSSc was hypothesized, so M., after performing a bone marrow examination, started pulse intravenous methylprednisolone therapy (30 mg/kg/die), followed by daily intravenous dose of 2 mg/kg and subcutaneous methotrexate (15 mg/mq/wk). Results: For DU M. performed a cycle of five infusions of intravenous Iloprost (2 ng/kg/min), subsequently shifted to oral Nifedipine. Clinical response was excellent with progressive improvement of most ulcerative lesions until almost complete healing and no new lesions. Nevertheless, necrosis of fifth finger of the left foot persisted, so surgical curettage was performed with good clinical and esthetic result. Conclusion: Iloprost is an analogous of Prostacyclin (PGI2), which inhibits platelet aggregation and adhesion, dilates arterioles and venules, actives fibrinolysis and reduces the release of oxygen free radicals. There is evidence of effectiveness of intravenous Iloprost in treatment of DU in adult patients with Systemic Sclerosis. So far, poor data about its use in pediatric population are available. We present a pediatric case of a patient with DU in JSSc successfully treated with intravenous Iloprost. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
S. S. Pelk, M. J. Doeleman, J. F. Swart, S. de RoockS. S. Pelk Introduction: Discontinuation and re-treatment with tumor necrosis factor inhibitors (TNFi) is common during the treatment course of children diagnosed with JIA. These medications are either gradually tapered over a longer period by increasing treatment intervals (tapering) or immediately discontinued [1]. Although there is some evidence that re-treatment with TNFi after withdrawal remains effective [2], there is no scientific consensus on the association of withdrawal strategy (taper or immediate discontinuation) with response to re-treatment with TNFi. Objectives: To determine if tapering or immediate discontinuation of TNFi affects drug efficacy after re-treatment. Methods: This is a monocentric retrospective cohort study of patients diagnosed with JIA, re-treated with adalimumab, etanercept, or golimumab during or after withdrawal. Data was collected from the Wilhelmina Children’s Hospital. Patient characteristics were compared between patients where TNFi treatment was tapered or immediately discontinued. Adequate response to re-treatment was defined as an active joint count (AJC) equal to 0 after approximately 6 months. Nonresponse was defined as a switch to another biological agent or the inability to achieve an AJC equal to zero at the 6 month visit. Association of withdrawal strategy prior to re-treatment and response to re-treatment with TNFi was analyzed using multiple logistic regression, adjusted for sex, age, AJC at re-treatment, and length of TNFi-free period prior to re-treatment. Results: 55 patients were included in this study, 28 patients tapered before re-treatment and 27 patients discontinued immediately. Patient characteristics of the two groups are presented in Table 1. Multiple logistic regression demonstrated no significant association of withdrawal strategy with response to re-treatment (taper vs. immediate discontinuation OR 1.62, 95% CI 0.35-7.57). The length of TNFi-free period prior to re-treatment (in months) was significantly associated with the probability of response to re-treatment (OR 0.91 95% CI 0.84-1.00). Conclusion: Our data suggest that tapering or immediate discontinuation was not significantly associated with the probability of response to re-treatment with TNFi. The length of TNFi-free period prior to re-treatment was negatively associated with the probability of response. Nevertheless, our results are limited by a small sample size. Future efforts will focus on increasing our sample size and investigation of other patient and treatment characteristics that could influence the efficacy of re-treatment, including the development of anti-drug antibodies and TNFi dosage. References: 1. Halyabar O, Mehta ·J, Ringold S, et al. Treatment withdrawal following remission in juvenile idiopathic arthritis: A systematic review of the literature. doi: 10.1007/s40272-019-00362-6. 2. Klotsche, J., Klein, A., Niewerth, M. et al. Re-treatment with etanercept is as effective as the initial firstline treatment in patients with juvenile idiopathic arthritis. Arthritis Res Ther 23, 118 (2021). doi: 10.1186/s13075-021-02492-0M. R. Pellico 1 , E. A. Conti 2 , F. Minoia 3 , G. Filocamo 3 , M. V. Gattinara 4 , C. B. Chighizola 4 , R. Caporali 4 , A. Marino 4 on behalf of on behalf of the Pediatric Rheumatology Associated Group of the Milan Area (PRAGMA) 1 Reumatolgy Department, 2 Pediatrics Department, University of Milan, 3 Pediatric Rheumatology, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, 4 Unit of Pediatric Rheumatology, ASST Pini-CTO, Milan, Italy Correspondence: M. R. Pellico Introduction: Biologic agents (bDMARDs) have dramatically changed the disease course of juvenile idiopathic arthritis (JIA). Nowadays, TNF-α inhibitors (TNFi) are frequently used as first-line treatment, while there are no clear recommendations on the choice of the second bDMARDs. Indeed, little is known about the prescription pattern and efficacy of second bDMARDs in JIA patients. Objectives: To describe the prescription pattern of the second bDMARDs in a cohort of non-systemic JIA patients and to report data on the efficacy and safety of TNFi vs. non-TNFi biologic agents as second-line bDMARDDemographic and clinical data were collected from medical charts. A descriptive analysis was then undertaken. Results: The study cohort included 30 patients (3 males); the mean age at disease onset and at diagnosis were 5 ± 4.9 (1-15) years and 5.3 ± 4,96 (1-15) years, respectively. Of the 30 patients, 6 (20%) had a diagnosis of polyarticular JIA, 19 (63%) of oligoarticular JIA, 3 (10%) of enthesitis-related arthritis and 2 (6%) of psoriatic arthritis. Fourteen patients (47%) had a history of uveitis (all with oligoarticular JIA). 87% of the patients received methotrexate (MTX) before the introduction of bDMARDs and 90% received TNFi as the first bDMARDs, etanercept and adalimumab being the most prescribed (13 and 12 patients, respectively). The mean age at the first bDMARDs was 7.3 ± 5.1 (1-17.3) years. Most of the patients (87%) received the first bDMARDs in combination with MTX. The causes of the first bDMARDs discontinuation were the following: articular flare (16 patients), uveitis flare (7 patients), both articular and uveitis flare (1 patient), and adverse events (6 patients). Three out of 13 patients treated with etanercept as the first bDMARDs withdrawn it because of uveitis occurrence. Adalimumab was the most prescribed second bDMARDs (13; 43.3%), followed by infliximab (7; 23.3%), abatacept (5; 16.7 %), etanercept (3; 10%) and tocilizumab (2; 6.7%). The most frequent switch was from etanercept to adalimumab (11 times). The mean age at the second bDMARDs start was 10.8 ± 5 (3-19) years. 63.3% of the patients received a second bDMARDs in combination with MTX. A non-TNFi was prescribed as second bDMARDs in 7 (23.3%) patients: 3 oligoarticular JIA and 4 polyarticular JIA. Comparison between TNFi vs. non-TNFi groups is shown in Table 1. Three patients of the non-TNFi group withdrawn the second bDMARDs due to articular disease. Among the TNFi group, 6 patients discontinued the second bDMARDs due to articular disease, 4 for uveitis, 1 for both articular and ocular disease, and 2 because of adverse events. Conclusion: Most of the patients in our cohort received a TNFi as second bDMARDs, with adalimumab being the most prescribed. No clear differences were seen between the non-TNFi and TNFi as for achievement of clinical inactive disease, rate of discontinuation, and duration of the second bDMARDs. Further studies are needed to explore the choice of the second bDMARDs in JIA. Disclosure of Interest : None declared
A. Prabhudesai 1 , A. Khan 1 , H. Panwala 2 , N. P. Maldar 1 , R. Khubchandani 1 1 Pediatric Rheumatology, 2 Pediatric Radiology, NH SRCC Children’s Hospital, Mumbai, India Correspondence: A. Prabhudesai Introduction: Generalized Lymphatic anomaly (GLA) is a rare, congenital, non-neoplastic condition characterized by abnormal proliferation of lymphatics. It is multisystemic with lungs and bones being most commonly involved. Thoracic involvement in children is associated with higher morbidity and mortality. With no evidence-based treatment protocol, emerging data suggests mammalian target of rapamycin (mTOR) inhibitor, Sirolimus, as a viable treatment option. mTOR,a serine/threonine kinase in the PI3K/AKT pathway governs cellular growth and angiogenesis. By inhibiting mTOR, Sirolimus reduces disease burden by causing partial or complete regression in bony disease, pericardial and pleural effusions. Objectives: Due to recurrent/ persistent symptomatology, it mimics rheumatological illnesses with polyserositis. Also, as sirolimus and other immunomodulators are common drugs in the rheumatologist/immunologist’s repertoire, rheumatologists are likely to be referred such patients for management. We describe a child with GLA with predominant thoracic disease referred as a diagnostic dilemma. She was diagnosed due to classic radiological features and showed improvement after 1 year of treatment with Sirolimus. Methods: An 11-year-old girl of average built with recurrent pleuropericardial effusions in the last 4 years, presented with severe respiratory distress, O2 saturation of 88% in room air and an intercostal tube in situ draining a massive right sided pleural effusion. The fluid was milky, exudative, with high triglycerides. Laboratory results showed hemoglobin 11g/L, total leucocyte count 910 9 /L, absolute lymphocyte count 2.210 9 /L and platelet count 2210 9 /L. The serum creatinine was 0.3 mg/dl with normal electrolytes and serum albumin of 3.7g/l. Spirometry showed moderate restriction. Whole body MRI showed diffuse confluent sheet like lesions in the anterior and middle mediastinum, extending superiorly up to the neck with bilateral peribronchovascular and interlobular septal thickening. Multiple hyperintense lesions were seen in the spleen and vertebral bodies, pointing towards a multisystem involvement. The characteristic radiology coupled with the clinical features led to a diagnosis of GLA. After parental counselling and informed consent, treatment with Sirolimus was initiated at 0.8mg/m 2 and doses adjusted to maintain trough levels between 10-15ng/ml. Lipid levels, blood counts and liver function were tested at baseline and monitored monthly. After 1 year of treatment with Sirolimus, the patient reported no exertional dyspnoea, no hospitalizations and improved lung function reflecting in the spirometry by a significant increase in the FEV1 and FVC. MRI showed partial reduction in the diffuse mediastinal infiltration and peribronchovascular and interlobular septal thickening. There was significant regression in the splenic and vertebral lesions. There was no major side effect of sirolimus except for hyperlipidemia with triglycerides 201mg/dl and cholesterol 249mg/dl, which was managed with statins. Sirolimus thus helped provide symptomatic relief and stabilized the disease in our patient without any major adverse effects. Results: Pre and post treatment radiological images shall be demonstrated in the poster Conclusion: GLA is a rare multisystemic condition which may present as a mimic to a rheumatologist. Alternately the rheumatologist may be called upon to opine on its treatment with sirolimus. Although Sirolimus is emerging as an effective and well tolerated option helping to stabilize and reduce the disease burden, hyperlipidemia may be encountered. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
S. caglayan 1 , H. E. Sonmez 2 , G. Otar Yener 3 , E. Baglan 4 , K. Ozturk 5 , K. Ulu 1 , V. Guliyeva 6 , S. Ozdel 4 , H. Bukulmez 7 , N. Aktay Ayaz 6 , B. Sozeri 1 1 Pediatric Rheumatology, University of Health sciences, umraniye education and research hospital, Istanbul, 2 Kocaeli University, Kocaeli, 3 Şanlıurfa Research and Training Hospital, Sanlıurfa, 4 Sami Ulus Research and Training Hospital, Ankara, 5 Istanbul Medeniyet University, Göztepe Research and Training Hospital, 6 Istanbul University, Faculty of Medicine, Istanbul, Turkey, 7 Case Western Reserve University, Cleveland, United States Correspondence: B. Sozeri Introduction: Multisystemic inflammatory syndrome in children (MIS-C) is a hyperinflammatory condition that has recently entered our lives after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and presents with Kawasaki disease (KD) and/or shock-like findings. MIS-C is a rare complication of COVID-19 with an incidence of 0.4% . A total of 6851 MIS-C cases have been reported and 59 deaths have been observed since May 2020. The underlying pathogenesis has not yet been fully elucidated and an abnormal immune response is blamed as the main factor in the pathogenesis of MIS-C Objectives: The study aimed to report the efficacy and safety of anakinra treatment in patients with refractory multisystemic inflammatory syndrome in children (MIS-C). Methods: This is a cross-sectional study consisting of pediatric patients diagnosed with MIS-C who were treated with anakinra. Results: Eighty-two (48 male/34 female) patients with MIS-C were evaluated. The median age of patients was 115 (6-214) months. The median duration of hospitalization was 15 (6-42) days. Sixty patients (73.1%) were admitted to pediatric intensive care unit. Patients were treated with a median dose of 2.7 mg/kg/day anakinra concomitant with IVIG and steroids. Intravenous anakinra was applied to 12 patients while 70 patients received it subcutaneously. Twenty-eight patients required high dose (4-10 mg/kg/day) anakinra. Anakinra was ceased within a median of 7 days. No injection site reactions were observed while elevated transaminase levels were detected in 13 patients. Seventy-three patients (89.1%) were discharged without any sequela or morbidity. Seven patients (8.5%) died. Abnormal echocardiographic findings continued in two patients (2.4%) (coronary artery dilatation in one, low ejection fraction in one) at discharge and became normal on the 2 nd month. Conclusion: MIS-C patients should be evaluated and treated dynamically due to its severe and potentially fatal course. In refractory MIS-C cases, anakinra provides significant improvement in both clinical and laboratory findings. The use of anakinra in refractory MIS-C cases is safe even at high doses. Disclosure of Interest : None declared
K. Temelkova, M. Ganeva, A. Teltcharova, V. Kostova, T. VasilevSofia, Bulgaria Correspondence: M. Ganeva Introduction: Biological therapy is indicated in the treatment of Juvenile Idiopathic arthritis (JIA) after failure of disease-modifying anti-rheumatic drugs (DMARDS) by the ACR/EULAR recommendations. Despite the availability of wide range of EMA approved biological agents (BA) for JIA, only three of them are allowed to be used in Bulgaria -Adalimumab, Etanercept and Tocilizumab. Objectives: The objective of the study is to describe the characteristics of Bulgarian patients with JIA at the initiation of biological therapy. Methods: This is a retrospective study of JIA patients treated with biological agents in University Children’s Hospital in Sofia, for a period of 12 years(2009 – 2021). Data was collected from the medical records of all JIA patients on biological therapy including: demographics, disease characteristics and concomitant therapy. Results: Тhree hundred and eighty-four patients were included in the study, of whom 237 (61%) were females .The average age of diagnosis of the patients was 6,5 years and the average age of initiation of biological therapy was 9,9 years. In one hundred and sixty-nine (44%) children polyarthritis was identified, making it the most common subtype of JIA. Oligoarthritis was found in 150 (39%) patients, predominantly in girls with Male:Female ratio 1:2. Thirty-eight (10%) children had systemic JIA (sJIA) and 27 (7%) had enthesitis related arthritis (ERA), with M:F ratios being 1,3:1 and 3,3:1 respectively. The average number of affected joints was 3.1, with maximum count of 13 joints and the most commonly involved were knee joints, followed by ankles and wrists. Rheumatoid factor (RF) was positive in 14 (3.6 %) patients and HLA B27 positivity was found in 41 (10.6 %) children. Antinuclear antibodies were detected in 101 (26 %) children with four times higher frequency in girls. Eye involvement was observed in 37 (9, 6 %) JIA patients with female predominance (21 girls to 16 boys) and 67 % of them were treated with Adalimumab. All of the children were treated with DMARDS - Methotrexate or Sulfasalazine, and all of them were also on concomitant corticosteroid (CS) therapy . Intraarticular CS injections were performed in 41 % of the patients during the disease course. The use of BA made possible tapering and discontinuation of the systemic CS therapy in 44 % of the children. TNF- inhibition was performed in 280 (73 %) of the patients and IL- 6 inhibition in 104 (27 %) patients. Most commonly used BA was Adalimumab (38 %) followed by Etanercept in 35 % and Tocilizumab in 27 % of JIA patients. As some of the JIA patients could not achieve remission despite using the prescribed biologicals, BA switching was required in 59 (15 %)children. The most common reason for BA switching was inadequate response (69 %).In 16 ( 28 %) patients eye involvement was observed during the disease course and was the reason for switching between different BA. Allergic reactions in 2 patients led to change in medication.BA were switched 2 times in 11 patients. Conclusion: Our data demonstrate real-world treatment practice in patients with JIA in tertiary care center. Availability of biological therapy was increased during the last years in Bulgaria, which led to better outcomes for our youngP243. The characteristics of 71 patients with chronic non-bacterial osteomyelitis (CNO): a single-centre cohort study P244. Lung involvement in children with Chronic Non-bacterial Osteomyelitis (CNO ) P245. Incidence of Chronic Recurrent Multifocal Osteomyelitis (CRMO) in the UK and republic of Ireland: intial results from 13 months of surveillance study P246. An unusual onset of chronic recurrent multifocal osteomyelitis in a 10- year-old boy P247. Effect of adjusting dxa scan results for body size on the diagnosis and management of low bone mineral density and osteoporosis in children P248. Chronic Non-bacterial Osteomyelitis (CNO) and bone mineral density 49. Osteoid osteoma presenting with chronic limb pain- our experience with noninvasive therapy E. D. Batu 1 , S. Sener 1 , A. E. Yıldız 2 , U. Kaya Akca 1 , M. Kasap Cuceoglu 1 , Z. Balık 1 , Y. Bayındır 1 , E. Aliyev 1 , O. Basaran 1 , Y. Bilginer 1 , Ü. Aydıngöz 2 , S. Ozen 1 1 Department of Pediatrics, Division of Rheumatology, 2 Division of Musculoskeletal Radiology, Department of Radiology, Hacettepe University Faculty of Medicine, Ankara, Turkey Correspondence: E. D. Batu Introduction: Chronic non-bacterial osteomyelitis (CNO) is an autoinflammatory bone disease. Objectives: We aimed to analyse the characteristics of CNO patients in our centre. Methods: Patients with CNO followed at Hacettepe University Paediatric Rheumatology Department in Ankara, Turkey, were included in this retrospective study. Results: 71 CNO patients (M/F:1.2/1) were included (Table). The median (min-max) ages at disease onset and diagnosis were 9 (0.2-17) and 10 (2-18) years, respectively. The median time from onset to diagnosis was 12 months. The median follow-up period was 26 months. Arthritis and enthesitis were present in 16 (22.5%) and 3 (4.2%) patients, respectively. HLA-B27 was positive in 9 (29%) out of 31 tested patients. Seven patients fulfilled the ILAR criteria for enthesitis-related arthritis. Skin lesions were present in 11 (15.5%) patients. 63 (88.7%) patients had whole-body MRI. There was long bone involvement in 65 (91.5%), sacroiliitis in 44(62%), and vertebral involvement in 23 (32.4%) patients. Mandible, clavicle, or sternum was involved in 2 (2.8%), 10 (14.1%), or 10 (14.1%) patients, respectively. Bone biopsy was performed in 23 (32.4%) patients, revealing mostly a fibroinflammatory reaction. The most frequently administered drug was NSAID (n=58; 81.7%), followed by methotrexate (n=47; 66.2%), etanercept (n=42; 59.2%), and pamidronate (n=20; 28.2%). The response rate to treatment was highest among patients who received etanercept (33/41; 80.4%). The response rate was 50% for pamidronate. Five patients were lost-to-follow-up; there was not enough time after the treatment to evaluate the outcome in seven patients. Complete and partial clinical remission was achieved in 52 (73.2%) and 6 (8.5%) out of 59 patients, respectively. One patient died from tuberculous meningitis. Conclusion: The frequent sacroiliac involvement and low frequency of skin lesions were the striking features of our cohort. The most effective treatment was anti-TNF drugs and the outcome was good in the majority of patients. References: Nuruzzaman F, Zhao Y, Ferguson PJ. Chronic nonbacterial osteomyelitis: insights into pathogenesis, assessment and treatment. Rheum Dis Clin North Am 2021;47:691-705 Aydıngöz U, Yildiz AE. MRI in the diagnosis and treatment response assessment of chronic nonbacterial osteomyelitis in children and adolescents. Curr Rheumatol Rep 2022;24:27-39. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
C. Celani 1 , V. Messia 1 , M. Pardeo 1 , P. Toma’ 2 , F. De Benedetti 1 , P. Moràn Alvarez 1 , A. Insalaco 1 1 Division of Rheumatology IRCSS Ospedale Pediatrico Bambino Gesù, 2 Department of Diagnostic Imaging, Bambino Gesù Children’s Hospital, IRCCS, Rome Italy , Rome , Italy Correspondence: C. Celani Introduction: Chronic non-bacterial osteomyelitis (CNO) is an idiopathic autoinflammatory disease characterized by osteolytic as well hyperostotic/osteosclerotic lesions. It is characterized by multifocal and often symmetrical pattern of osseous involvement. Other organ systems may be involved as well. Up to 20% of CNO patients develop skin manifestations (psoriasis, cystic acne, pustulosis), up to 10% of patients bowel disease and occasionally (up to 3% of patients) hepatospenomegaly and lymph node enlargement. Rarely pulmonary involvement may occur. Nowadays three cases of pulmonary involvement were reported: two children and one adult with CNO and pulmonary lesions described as parenchymal consolidations Objectives: To describe four cases of CNO with pulmonary involvement Methods: We describes four cases of children with CNO and pulmonary involvement discovered by WBMRIResults: We report four cases of children affected by CNO associated with pulmonary lesions. Median age at the time of diagnosis was 12.5 years (range: 10-17 years). The median time to develop pulmonary lesions from the CNO diagnosis was 21 months (0-72 months). The suspicion of CNO was risen by a clinical examination and the diagnosis was confirmed by Whole body MRI. All patients underwent a bone biopsy to exclude infections and bone malignancies .All of them presented with a multifocal pattern and 2 with vertebral involvement. At the onset of the disease, all patients presented with bone pain. Only one patient presented fever and increased inflammatory markers CRP (4 mg/dl) and ESR (43 mm/h). In 3 out of 4 patients, pulmonary lesions were discovered accidentally by WBMRI, just one patient presented with respiratory symptoms .The patients showed multiple pulmonary lesions with radiological characteristics of bronchiolitis obliterans organizing pneumonia (BOOP). Conclusion: We presented four cases of CNO with a BOOP pattern. This radiological finding was previously described in others rheumatic diseases in adults and in one case was reported in CNO adult patient. Our case reports suggest a possible link between these two inflammatory conditions that should be confirmed in a larger study population. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
Suo 1 , D. Chia 1 , A. Toms 2 , A. P. Sanghrajka 3 , A. V. Ramanan 4 , O. Killeen 5 , B. Jacobs 6 , C. Ilea 6 , K. Mahmood 7 , S. Compeyrot-Lacassagne 8 , K. Armon 1,9 1 Department of Paediatrics, University of Cambridge, 2 Department of Radiology, Cambridge University Hospitals, Cambridge, 3 Department of Truama and Orthopaedics, Norfolk and Norwich University Hospitals, Norwich, 4 Department of Paediatric Rheumatology, Bristol Royal Hospital for Children, Bristol, United Kingdom, 5 National Centre for Paediatric Rheumatology, Children’s Health Ireland, Dublin, Ireland, 6 Royal National Orthopaedic Hospital, London, 7 Department of Rheumatology, Alder Hey Children’s Hospital, Liverpool, 8 Great Ormond Street Hospital, London, 9 Department of Paediatric Rheumatology, Cambridge University Hospitals, Cambridge, United Kingdom Correspondence: D. Chia Introduction: Chronic Recurrent Multifocal Osteomyelitis (CRMO), also known as chronic nonbacterial osteomyelitis (CNO), is a rare autoinflammatory condition affecting the bones. It occurs primarily in children and teenagers and is characterised by bone pain and swelling in the absence of infection or tumour. The incidence of CRMO remains uncertain, with estimates ranging from 0.4-1 per 100,000 person years (1,2). Objectives: The primary aim of the study was to identify the incidence of CRMO in patients under the age of 16 in the United Kingdom (UK) and Republic of Ireland (ROI). Additional aims include describing the demographics, clinical features, treatment, and healthcare needs of patients with CRMO. Methods: A prospective surveillance study was undertaken via the British Paediatric Surveillance Unit. A monthly e-reporting card was sent to all registered paediatric consultants in the UK and ROI. A parallel surveillance study was sent via the British Society for Children’s Orthopaedics to identify patients managed solely by orthopaedics. A standardised questionnaire was sent to the reporting clinicians to collect further information. Results: During initial 13 months of surveillance, 168 cases were reported. The 23 questionnaires were not returned (13.7% of reported cases). After de-duplication, a preliminary analysis was performed to exclude cases outside the reporting time period and age-group. 82 confirmed and 8 probable cases were included in our interim results. The estimated incidence of CRMO is 0.605 cases /100,000 children per year. Median age at time of diagnosis was 10 years (range 3-16). 53 (58.9%) of cases were female. Median delay from symptom onset to diagnosis was 5 months and 16 patients (17.78%) had a delay of greater than 12 months. Most (48.9%) of the cases were diagnosed by paediatric rheumatology specialists. Other cases were diagnosed by orthopaedics (16.7%), general paediatricians (15.6%) or by a multidisciplinary team. 34 cases (37.8%) reported requiring admission related to CRMO. The most common presenting feature was bone pain (96.67%). 34 patients (37.8%) presented with clavicular pain, and thirty-one (34.4%) had unifocal bone pain. Patients also presented with bone swelling (52.2%), joint swelling (20.0%), fever (12.2%) and general malaise (13.3%). A median of 3 investigations were reported for each case, of which 61 cases (67.7%) had whole body MRI performed and 33 cases (36.67%) had bone biopsy. The most common treatment was NSAIDs (90.0%) and bisphosphonates (33.3%). Conclusion: Our results estimate the incidence of CRMO as 0.605 cases per 100,000 person years. The study will continue to capture new CRMO cases for a further 12 months. Reported cases will be followed up for 24 months. This will provide greater insight into the medium-term outcomes for patients diagnosed with CRMO and provide a snapshot into treatment strategies used by clinicians in the UK and ROI. These results will provide a valuable baseline for further research and improvement in care for patients with CRMO. 1. Jansson, A.F., V. Grote, and E.S. Group, Nonbacterial osteitis in children: data of a German Incidence Surveillance Study. Acta Paediatr, 2011. 100(8): p. 1150-7. 2. Walsh, P., et al., Chronic recurrent multifocal osteomyelitis in children: nine years’ experience at a statewide tertiary paediatric rheumatology referral centre. Rheumatology (Oxford), 2015. 54(9): p. 1688-91. Patient Consent: Not applicable (there are no patient data) Disclosure of Interest : C. Suo: None declared, D. Chia: None declared, A. Toms: None declared, A. Sanghrajka: None declared, A. Ramanan Consultant with: Abbvie, Eli Lilly, SOBI, Roche, UCB, Novartis, O. Killeen: None declared, B. Jacobs: None declared, C. Ilea: None declared, K. Mahmood: None declared, S. Compeyrot-Lacassagne: None declared, K. Armon: None declared
M. F. Gicchino 1 , G. Lodato 2 , A. N. Olivieri 1 1 Department of Woman, Child and General and Specialistic Surgery, University of the study of Campania Luigi Vanvitelli, 2 University of Naples Federico II, Naples, Italy Correspondence: M. F. Gicchino Introduction: Chronic recurrent multifocal osteomyelitis (CRMO), also known as chronic nonbacterial osteomyelitis, is a rare, noninfectious inflammatory disorder that causes multifocal lytic bone lesions with swelling and pain characterized by periodic exacerbations and remissions of unclear/unknown pathogenesis. Objectives: To describe an unusual CRMO onset in a 10 years old boy. Methods: A 10- year-old boy suffered from recurrent episodes of ocular pain associated with marked hyperaemia and periorbital oedema. Suspecting periorbital cellulitis these episodes were treated with antibiotics and steroids. Before came to our Department patient was evaluated by an ENT specialist who prescribed a computed tomography (CT) and a magnetic resonance imaging (MRI) of the orbits. The CT revealed an osteolytic lesion on the orbital process of the zygomatic bone. The MRI showed a lesion on the orbital process of the zygomatic bone, this lesion appeared hypointense in T1 sequence and hyperintense in T2 and STIR sequences, suggesting an inflammatory process. To perform a correct diagnosis a bone biopsy of this lesion was planned. Meanwhile the child developed back pain and since his brother was in follow up to our Department because of juvenile idiopathic arthritis the patient was evaluated by us for the first time. Clinical examination revealed pain on the pressure of dorsal column and sacroiliac joints. Laboratory examinations revealed increase of inflammatory parameters: erytrocyte sedimentation rate (ERS) 30mm/h (normal value <15mm/h), C-reactive protein (CRP) 2.5 mg/dL (normal value < 1mg/dL). Liver and kidney function, iron levels, C3/C4 levels, LDH, blood coagulation profileFaecal calprotectin was in the normal range. Both heart and abdomen ultrasound were in the norm. X-ray of the spine was in the norm. Spine MRI revealed abnormal marrow signal (low signal on T1, high signal on T2 and STIR) in T6, T11 vertebral bodies and in S2. A bone biopsy of T6 was preformed. Results: Bone biopsy revealed: infiltration by lymphocytes and neutrophils. No neoplastic cells were identified. All cultures were negative. Langerhans cells histiocytosis (LCH) was excluded since immunohistochemical evaluation of bone marrow for CD1a and S100 expression was negative. Clinical history, physical signs, instrumental investigations and histopathological pattern were highly suggestive of CRMO. In consideration of vertebral column involvement was started therapy with bisphosphonates. Conclusion: In a patient with recurrent bone pain CRMO should be considered. The diagnosis is of exclusion and it is based on the clinical and radiological data. Biopsy is needed to exclude infectious osteomyelitis, malignancy, LCH. Skeletal manifestations are unifocal or multifocal, and the involvement of clavicle, sternum or mandible suggest a CRMO diagnosis. Extra- articular manifestation are gastrointestinal and skin involvement (acne, pustolsis, psoriasis). The peculiarity of our case concerns the initial site of disease, the periorbital region. This primary localization caused difficulties in the differential diagnosis with other condition, in particular infections. The appearance of a second lesion on the vertebral column and the investigations performed, allowed the diagnosis of CRMO. The treatment of CRMO has been mostly empiric, NSAIDs are the first choice for CRMO treatment. When disease activity is high or there are complications therapy with Methotrexato, bisphosphonates or biologic drugs such as TNF antagonists (etanercept) should be considered. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
F. Hamblin 1 , K. Armon 2 , N. Crabtree 3 , A. Thankamony 4 , S. Walton-Betancourth 4 , E. Hendriks 1 1 Department of Paediatrics, University of Cambridge, 2 Department of Paediatric Rheumatology, Cambridge University Hospitals NHS Foundation Trust, Cambridge, 3 Department of Endocrinology and Diabetes, Birmingham Children’s Hospital, Birmingham, 4 The Weston Centre, Department of Paediatric & Adolescent Diabetes and Endocrinology, Cambridge University Hospitals NHS Foundation Trust, Cambridge, United Kingdom Correspondence: F. Hamblin Introduction: Bone mineral density (BMD) from Paediatric DXA scans are often reported as Z-scores based on an areal calculation of BMC and normative data for age. It does not take into account effect of stature on the areal bone density. Bone mineral adjusted density (BMAD) of lumbar spine adjusts for volume of the vertebra and therefore body size. We studied the effect of making this adjustment in a clinical population. Objectives: We aimed to re-evaluate DXA scan results of paediatric patients in Addenbrooke’s Hospital, Cambridge, UK by applying BMAD adjustments and up-to-date guidelines, and to assess whether this affected the diagnosis and management of reduced bone density and osteoporosis in these children. Additionally, we aimed to investigate whether clinical decisions made based on the standard BMD DXA scan results were appropriate given these adjustments. Methods: All children under 16 years who had a DXA scan in 2016-2018 at Addenbrooke’s Hospital were included. Data were retrospectively collected and included fracture history and treatment decisions concerning bone health. Lumbar spine DXA results were adjusted for vertebral size to derive age adjusted BMAD Z-scores and were compared with lumbar spine BMD Z-scores. Patients classified as having normal and low bone density by each method were also compared. Results: 153 patients with mean age 11.72 years (SD = 3.01) were included in the study. The Z-scores of lumbar spine BMD were significantly lower than lumbar spine BMAD Z-scores (-0.85 (SD = 1.23) vs -0.47 (SD = 1.16), p<0.001). In total 11 patients were classified as having low bone density by both measures. Fewer patients were categorised as having low bone density using BMAD compared to BMD (7.2% vs 17.0%) and only 42.3% found to have low BMD had this confirmed with BMAD (P<0.001). 16 patients had treatment changes based on low BMD. 8 of them did not have low BMAD and therefore change may not have been implemented had adjustment been used. Only 10 (33%) of patients categorised with low bone density had any form of vertebral imaging for crush fractures. Conclusion: The use of lumbar spine BMD alone in reporting DXA scans in paediatric patients is more likely to result in an inaccurate diagnosis of low bone density. It is essential to use adjustment methods, such as lumbar spine BMAD, in paediatric patients to derive bone density and evaluate along with fracture history (including potentially silent vertebral fractures) when making a diagnosis of osteoporosis and therefore any treatment decisions. Disclosure of Interest : None declared
M. Kasap Cuceoglu 1 , M. Canturk 2 , S. Sener 1 , Z. Balik 1 , Y. Bayindir 1 , E. Aliyev 1 , O. Basaran 1 , A. E. Yildiz 3 , E. D. Batu 1 , Y. Bilginer 1 , U. Aydingoz 3 , A. Ozon 2 , S. Ozen 1 1 Pediatric Rheumatology, 2 Pediatric Endocriology, 3 Radiology, Hacettepe University, Ankara, Turkey Correspondence: M. Kasap Cuceoglu Introduction: Chronic nonbacterial osteomyelitis (CNO) is an inflammatory bone disorder that affects children. 1 In the absence of accepted diagnostic criteria or clinical/radiological biomarkers, CNO remains a diagnosis of exclusion. 2 Objectives: We aim to show the relationship between CNO and bone mineral density and investigate whether osteoporosis with/without vitamin D deficiency can cause challenges in the treatment of CNO. Methods: In the study, 70 patients (<18yo) with CNO were reviewed using electronic files at Hacettepe University retrospectively between January 2015 and April 2022. Demographic and clinical characteristics of patients, 25 OH vitamin D level status (N:>20 ng/ml), radiological findings as well as medical treatments were evaluated. A score between -1.1 and -2.4 is classified as osteopenia (low bone mass). A score of -2.5 and below is defined as osteoporosis. In the pediatric population, osteoporosis is a clinical diagnosis and is reserved for those patients with a BMD Z- score less than or equal to -2.0 in combination with a clinically significant fracture. Results: A total of 70 patients (females 53%) were included in this study. The median age at diagnosis of CNO was 7 (1-17) years. Overall, vitamin D level was available before diagnosis in 46/70 patients, 28 of which were normal, while 18 patients had severe deficiency (25 OH vitamin D <10 ng/ml). Bone mineral density was studied in 24 patients, normal in 14, osteoporotic in 4, and osteopenic in 6 patients. 19 patients received pamidronate treatment, 13 of whom received methotrexate and 9 received anti-TNF before pamidronate. Among the patients receiving pamidronate, 7 had 25 OH vitamin D <10 ng/ml, 3 had osteopenia, 3 had osteoporosis in DEXA. Thirty-five patients received anti-TNF therapy (29 of them used ETA, 5 ADA, 2 INF). Although it was not statistically significant, there was no patient with osteoporosis in the group receiving synthetic DMARD/NSAID (pamidronate/anti TNF naive group, n=20). The median lumbar BMD z score was -0.90 (min-3.8, max 3.2) in the pamidronate/anti TNF group and 0.4 (min-1.8, max1.02) in the DMARD/NSAID group. Overall history of fracture was present on admission in ten patients (3 osteoporosis, 3 osteopenia, 3 normal bone mineral density, 1 NA). All patients with fracture received pamidronate as first option and they benefited from this treatment. (p=0.013). In the pamidronate/ anti TNF group, 25 OH vitamin D level was lower than synthetic DMARD/NSAID (pamidronate naïve) group, and the difference was nearly significant. (p=0.073) Conclusion: Patients with normal bone mineral density responded well to DMARD/NSAID, better than that of remaining patients, and they did not need alternative treatment. Vitamin D deficiency and/or osteopenia may lead to a more refractory course, thus it should be monitored effectively in patients with CNO. References 1. Zhao Y, Ferguson PJ. Chronic Nonbacterial Osteomyelitis and Chronic Recurrent Multifocal Osteomyelitis in Children. Pediatr Clin North Am . 2018;65(4):783-800. doi:10.1016/j.pcl.2018.04.003 2. Oliver M, Jayatilleke A, Wu E, et al. Establishing core domain sets for Chronic Nonbacterial Osteomyelitis (CNO) and Synovitis, Acne, Pustulosis, Hyperostosis, Osteitis (SAPHO): A report from the OMERACT 2020 special interest group. Semin Arthritis Rheum . 2021;51(4):957-961. doi:10.1016/j.semarthrit.2021.05.015Rubelj 1 , I. Radoš 1 , L. Novosel 2 , R. Vukojević 2 , M. Vidovic 1 1 Department of pediatric, Devison of rheumatology and clinical immunology, 2 Department of radiology, University Hospital Center Sestre milosrnice, Zagreb, Croatia Correspondence: K. Rubelj Introduction: Limb pain is common childhood complaint. Chronic and recurrent pain raises different diagnostic options osteoid osteoma (OO) being one of them. OO is the most common benign bone tumor of young adults and it’s relatively rare in children. It mostly affects long bones with chronic nighttime pain as predominant symptom. Current method of treatment is complete surgical excision of the nidus. This is the first time that the new method - percutaneous thermal radiofrequency ablation (RFA) with temperature of 90°C under control of conventional computerized tomography scan (CT-scan) or cone-beam CT-scan is used in pediatric population in Croatia. Objectives: In this paper we present pediatric patients in whom OO is successfully removed by percutaneous RFA. Methods: Nine patients hospitalized with chronic limb pain from 05/2020 to 10/2021 in the Department of rheumatology and clinical immunology, Clinic of Pediatrics Sestre milosrdnice University Hospital Center were diagnosed with OO and treated with percutaneous RFA in corroboration with Clinical department for diagnostic and interventional radiology. All procedures were performed under general anesthesia. The average age of the patient was 11.8 year with overhang of female gender (6/9) over male (3/9). Classical symptom in all patients was with sleep disturbance. Average time from first symptom to the diagnosis was 7.8 months. Patients underwent different diagnostic procedures including x-ray, ultrasound, multisided computed tomography (MSCT) or scintigraphy of the bones. Almost everyone (8/9) had the diagnosis confirmed with magnetic resonance imaging (MRI) with pathognomonic sclerotic lesion with nidus and perifocal bone and soft tissue subperiosteal edema. Results: Procedure was successful in all patients, with only occasional oral analgesia during 24-hour observation and no delayed complications. In all cases the diagnosis was confirmed by pathophysiological evaluation. Two patients had recurrence of pain in 2 month period following the ablation due to recurrence of the lesion. Complete pain relief was however achieved after a second ablation in both cases. Thus, our primary and secondary clinical success rates were 78 and 100%, respectively. Conclusion: One of the differential diagnostic options in children with chronic nighttime limb pain is OO. Complete surgical excision is preferred treatment option, providing pain cessation. However, surgery has some disadvantages, including the difficulties in locating the lesion intraoperatively, need for prolonged hospitalization, and the possibility of postoperative complications such as infection, fracture and unsatisfactory cosmetic result.. Percutaneous RFA as new method is safe minimally invasive, and extremely effective method for the management of OO in children and should be considered as primary option. Patient Consent: No, I have not receive consent Disclosure of Interest : None declared
P250. Fibrodysplasia ossificans progressiva : first case report in Libya P251. Livedo racemosa a cutaneous finding that could be an early presentation for Sneddon’s syndrome P252. Glutathione S-transferase gene polymorphisms and methotrexate effectiveness in patients with juvenile idiopathic arthritis 53. Lack of association of SAA1 RS12218 and IL1B RS1143634 gene polymorphisms with FMF and CAPS, but the presence of a correlation of IL1B RS1143634 gene polymorphism with the level of serum IL-18 in patients with FMF P254. Genetic analysis of whole exome sequencing in a cohort of children with refractory JIA reveals rare genetic risk factors for JIA at loci of known inflammatory diseases 1 Paediatric Rheumatology , Faculty of Medicine,Tripoli university , Tripoli Children’s Hospital , 2 Paediatric Rheumatology , faculty of Medicine, Tripoli university, Tripoli children’s hospital, Tripoli , Libya Correspondence: Y. Elfawires 2 Introduction: Fibrodysplasia ossificans progressiva (FOP) is a very rare genetic connective tissue disorder characterized by abnormal development of bone. Patients with FOP have characteristically malformed big toes that are present at birth (congenital). Other skeletal malformations may occur. Most cases of FOP is caused by the mutation of a gene (ACVR1) in the bone morphogenetic protein (BMP) pathway. To our knowledge so far there is no reported cases with FOP in Libya, and this case has been worked up initially as infectious or malignant causes behind the bone swelling. Here we describe the clinical features and management. Objectives: to highlight the importance of early diagnosis and recognition of FOP, and avoid unnecessary blood test, injections, biopsies and dental work minimizing the disease progression and early disability as much as possible Methods: the diagnosis was established mainly on typical clinical picture ( appearance of malformed big toes and the new bone formation at site of biopsy and trauma), the case was discussed with an international FOP awareness campaign and due to lack of facilities and the high cost of the gene test clinical diagnosis was found to be sufficient. Results: a two years and four months old Moroccan girl born of non-consanguineous healthy parents, presented to P/Rheumatology department Oct/2021 with history of neck lumps at level of lower cervical spine, associated with neck stiffness and limitation of movement. The neck lumps where first noted three to four months before presentation discovered accidentally and not preceded by a recognized trauma nor a special event. The lumps where about 2x2 cm, firm to hard in consistency, not tender, no redness, and no hotness, associated with cervical lymphadenopathy evident by examination and neck U/S, and associated with limitation of neck movement especially neck extension and decreased range of movement in both shoulders. The case was initially addressed as lyphadenopathy. Her initial lab results showed, WBC 14.28 x 103 (49.9% neutro: 39.9% lympho), Hgb 12.9g/dl, PLT 360 x103, blood film showed no significant abnormality. ESR 5 mm/hr, CRP negative, LDH 366 normal, Alkp 142 normal, serum Ferritin 61.7 ng/ml, Ca+2 9.8, CK 92 IU/L, Renal function and Liver function tests came within normal range. TB also was excluded, immunoglobulin level came within normal. Regarding the imaging U/S neck = normal study apart from non specific deep cervical lymphadenopathy. U/S chest wall/ abdomen/ pelvis = ill defined fusiform thickening of subcutaneous fat layers at Lt Lateral aspect of the chest wall, measuring 30x 14 mm suggesting post inflammatory residual edema. CT scan of chest/ abdomen/ pelvis = no significant abnormality. CT neck with IV contrast = bilateral multiple deep cervical enlarged lymph nodes. Her case was discussed with orthopedists, endocrine and infectious specialties and unfortunately a true cut Biopsy from the lesion was taken showing evidence of infantile myofibromatosis. She received IV and IM Abx during workup of the case. The illness progressed involving the elbow joints and both wrists, with pain disturbing her sleep but not her activity, also the site of injections and bone biopsy started to harden. Depending on the above mentioned scenario and the typically malformed big toes noticed on examination the diagnosis of FOP was made and the case was discussed regarding the need of gene testing putting in consideration the very limited resources. She was started on an induction course of steroids 1 mg/kg and NSAIDs ( Ibuprofene ) showing much improvement in her symptoms. Conclusion: FOP should be considered in any child with malformed big toes presenting at birth, to protect the child from any un needed interventional tests and procedures minimizing the early complications of diseaseY. AElfawires 2Tripoli , Libya Correspondence: Y. AElfawires Introduction: Sneddon’s syndrome is a rare disorder that most commonly affects young females, characterized by livedo racemosa and central nervous system disease. Livedo racemosa is differentiated from livedo reticularis by its shape, pattern in addition to its persistence on warming. Livedo racemosa in Sneddon syndrome is widespread and almost always involves the trunk and/or buttocks. Objectives: to highlight the importance of considering sneddon’s syndrome in young females with levido racemosa due to life threatening Ischemic events such as stroke and transient ischemic attack. Headache and hypertension are also known associated problem Methods: two case reports are described Results: case one , eight year-old Libyan female for non-consanguineous parents and no significant past nor family histories. Presented to Pediatric rheumatology clinic as referral from Dermatology clinic with livedo racemosa appeared one year ago on lower limbs, upper limbs and trunk later. Among the presenting symptoms she mouth ulceration and mild arthralgia rest of the systemic review was neg. Her peripheral pulses and BP was normal, ECG & ECHO also normal, U/S abdomen normal, MRI brain normal (changes are expected to be seen in 2 nd decade of life). Blood tests: WBC 9.1, HgB 11.9 g/dl, plt 436. ESR 60 mm/hr, CRP 23.4 mildly raised, LFT normal, U/E/C normal, LDH 187,ENA ( Ro/SS-A 52, Ro/SS-A 60 and La/SS reacted +ve), D-dimer normal, PT APTT INR normal, ANA +ve, antids- DNA neg,anticardiolipin IgG& IgM neg, anti phospholipid IgG&IgM neg, lupus anticoagulant weak +ve, C3&C4 normal range, coomb’s test normal. slit lamp eye examination normal. Skin biopsy and MRA brain has been ordered waiting for results. Currently she is on asprin tab 75mg. she received short course steroid 1mg/kg and hydroxychloroqin 4mg/kg earlier. Case two, 14 year- old Libyan female for non-consanguineous parents also neither significant family history nor past medical history. Referred to our clinic from dermatology clinic as a case of livedo racemosa involving both LL (since 2011 till 2017 when diagnosed), with progression and involvement of both UL and trunk. Initially she had only arthralgia affecting both ankles but started to have headache and despite normal BP and normal peripheral pulses she feels tingling, hotness and dull aching pain in LL. She had a Doppler scan for LL showed it post tibial vein heamangioma/venous angioma of both calf with no evidence of thrombosis. MRI & MRA brain both normal, ECHO & ECG normal, x-ray of both ankles and wrists showed soft tissue swelling only, MRI Rt ankle showed acute non destructive arthritis. Slit lamp eye examination normal. Blood tests: WBC 10, Hgb 10.7 g/dl, PLT 459, blood film normocytic hypochromic anemia with normal iron study. ESR 52mm/hr, CRP 25mg (weak +ve), LFT normal, U/E/C normal, CPK & LDH normal, PT APTT INR normal, D-dimer 607 ng/ml raised, fibrinogen 4.5 g/l just above normal range. Serology: ANA, anti ds DNA, ENA screen, C3&C4, anticardiolipin, lupus anticoagulants and anti beta2 glycoproteine1 Abs all came negative. Skin biopsy was requested many times but family was reluctant to do it, with poor compliance to follow up visits. She received short course steroid and Naproxen for arthritis and Aspirin 75 mg daily. Re-evaluation workup including new MRA brain and new serology was requested in last follow up considering she now is in her 2 nd decade of life. Conclusion: Ischemic events such as stroke and transient ischemic attack are a hallmark of Sneddon’s syndrome and usually appear later in adulthood. Early screening for patients with livedo racemosa with brain MRA & MRI, control of BP and starting anticoagulants can minimize cerebrovascular events and their impact on patients life. Also importance of skin biopsy for diagnosis in such cases. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
S. Huljev Frkovic 1 , M. Frkovic 1 , K. Crkvenac Gornik 2 , D. Rogic 2 , M. Jelusic 1 1 Department of pediatrics, UHC Zagreb / School of medicine Zagreb, 2 Department of laboratory diagnostics, UHC Zagreb, Zagreb, Croatia Correspondence: M. Frkovic 1 Introduction: Early control of the inflammatory process in juvenile idiopathic arthritis (JIA) correlates with more favourable treatment outcomes. In case of methotrexate, combination of heterogeneous treatment response and 3–6-month period of continuous administration required for clinical assessment of the drug effectiveness, result in closing the “window of opportunity” in more than 30 % of JIA patients. Deleting polymorphisms of glutathione S-transferase T1 (GSTT1) and M1 (GSTM1) genes influence the effectiveness of different drug combinations in a variety of rheumatic diseases. Objectives: To investigate the impact of GSTM1 and GSTT1 gene deletion polymorphisms on MTX effectiveness in JIA patients. Methods: The study included 109 JIA patients: 46 patients in stable clinical remission for a minimum 6-month period during MTX therapy and 63 patients who did not achieve stable clinical remission during MTX therapy. In all patients participating in the study, GSTM1 and GSTT1 deletion polymorphisms were determined. Results: Deletion polymorphism of the GSTM1 gene was detected in 40/63 (63.5%) of patients who did not achieve remission during MTX therapy and 27/46 (58.7%) of patients who achieved remission during MTX therapy. Deletion polymorphism of the GSTT1 gene was detected in 14/63 (22.2%) of patients who did not achieve remission during MTX therapy in 8/46 (17.4%) of patients who achieved remission during MTX therapy. The combination of deletion polymorphisms for the GSTM1 and GSTT1 genes was detected in 8/63 (12.7%) of patients who did not achieve remission during MTX therapy and in 6/46 (13.0%) of patients who achieved remission during MTX therapy. No statistically significant differences were observed in the distribution of deletion polymorphisms or their combinations among the study groups. In a subgroup of JIA patients who did not achieve remission during MTX therapy, statistically significant difference in frequency of GSTM1 deletion polymorphism was detected between subgroup with one or more changes of biologic disease modifying drugs (bDMARD) compared to the subgroup with one bDMARD in terms of achieving remission: 64.3% to 28.6%; P=0.026. Conclusion: According to this study, the determination of GSTM1 and GSTT1 gene deletion polymorphisms is not useful in predicting the efficacy of MTX in JIA patients. However, the possible influence of GSTM1 gene deletion polymorphism on bDMARD efficacy in JIA patients opens new horizons in investigations of GST gene polymorphisms and their influence on treatment outcomes in JIA and other rheumatic diseasGuseva 1 , E. Fedorov 2 , S. Salugina 2 , M. Krylov 1 , E. Samarkina 1 , M. Kaleda 2 1 Immunology and Molecular Biology of Rheumatic Diseases,S. Salugina Introduction: Autoinflammatory diseases (AIDs) are a group of rare, genetically determined diseases characterized by periodic events of inflammation, fever, and clinical symptoms that mimic rheumatic pathology. Laboratory serological markers of AIDs are C reactive protein (CRP) and serum amyloid A (SAA) protein. Serum IL-18 level is also elevated in AIDs. Objectives: We investigated whether SAA1 -13T/C (rs12218) and IL1B C3954T (rs1143634) gene polymorphisms may affect the susceptibility to pediatric patients (pts) with Familial Mediterranean Fever (FMF) and Cryopyrin associated periodic syndromes (CAPS). We also evaluated whether these polymorphisms can affect CRP, SAA and IL-18 serum levels. Methods: 44 FMF pts – 22 boys, 22 girls; age at onset – M (SD) 7.85 (5.64) years and 27 CAPS pts - 15 girls, 12 boys; age at onset – 4.68 (5.83) years, and 95 healthy individuals (controls) were included in this study. The diagnosis of FMF was based on Turkish paediatric criteria for the diagnosis of FMF and was confirmed by the detection of pathogenic mutations of the MEFV gene in the homozygous or compound-heterozygous states. The diagnose of CAPS was made on the basis of characteristic clinical signs and was confirmed by the detection of a pathogenic mutation in the NLRP3 gene. SAA1 (rs12218) and IL1B (rs1143634) gene polymorphisms were genotyped using allele-specific RT-PCR assay. CRP, SAA and IL-18 serum concentrations were measured in FMF and CAPS pts. Results: Table shows the genotypic frequencies of SSA1 and IL1B genes in FMF, CAPS and controls. There were no significant differences between FMF, CAPS pts and controls in the genotypic SSA1 and IL1B gene polymorphisms (p>0,05). The SAA1 (rs12218) gene polymorphism did not correlate with CRP, SAA and IL-18 levels in FMF and CAPS pts. The IL1B gene polymorphism also did not correlate with the levels of CRP and SAA in FMF and CAPS pts, but was significantly associated with the level of IL-18 in FMF pts. In carriers of at least one mutant allele T (CT+TT) the level of IL-18 was significantly higher compared to carriers of the CC genotype (2256.74 ± 1532.28 and 231.19 ± 218.80 respectively, p=0.003). Conclusion: Our preliminary study in small groups of pediatric FMF and CAPS pts revealed that SAA1 (rs12218) and IL1B (rs1143634) gene polymorphisms are not associated with susceptibility to FMF and CAPS. The influence of IL1B (rs1143634) gene polymorphism on proinflammatory cytokine IL-18 serum levels in FMF pts is also shown. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
M. Tordoff 1 , S. L. Smith 1 , G. Rice 2 , L. R. Wedderburn 3,4,5 , K. L. Hyrich 6,7 , A. Morris 1 , T. A. Briggs 8 , W. Thomson 1,7 , S. Eyre 1,7 , J. Bowes 1,7 on behalf of The CLUSTER Consortium 1 Centre for musculoskeletal research, Centre for genetics and Genomics versus arthritis, 2 Division of Evolution Infection and Genomics, University of Manchester, Manchester, 3 Infection, immunity and inflammation research and teaching department, UCL, great ormond street institute of child health, 4 Centre for Adolescent Rheumatology Versus Arthritis at UCL, UCL Hospital and Great Ormond Street hospitall, 5 NIHR Biomedical Research Centre at Great Ormond Street Hospital, Great Ormond Street Hospital , London, 6 Centre for Epidemiology Versus Arthritis, The university of manchester, 7 NIHR Manchester Biomedical Research Centre, Manchester Academic Health Science Centre, 8 Division of Evolution Infection and Genomics, The university of manchester, Manchester, United Kingdom Correspondence: M. Tordoff Introduction: Juvenile idiopathic arthritis (JIA) is a childhood rheumatic disease that can result in long term disability. The goal of treatment is remission. The absence of a response to multiple standard disease therapies is referred to as refractory JIA. Objectives: Investigate the role of rare genetic risk factors in a cohort of children with refractory JIA using whole exome sequencing (WES). Methods: WES of 99 children with JIA was performed with the Agilent SureSelect Human All ExonV6 kit. Individuals were defined as refractory if they had failed treatment to methotrexate and at least one further biologic drug. All quality control (QC), variant filtering and annotation was performed in Varseq (version 2.2.1). Variants with a read depth <30 and genotype quality <80 were removed. Rarity and pathogenicity filters were applied to remove variants with an allele frequency >1% (based on ExAC, gnomAD, gnomAD exome, NHLBI and 1KGp phase 3), classified as benign or likely benign on ClinVar, with a CADD score <15 and a REVEL score >0.7. Variants were annotated if they appeared in a gene from the primary immunodeficiency PanelApp (Martin et al., 2019), in a gene associated with an arthritis phenotype or in a gene that appeared on a paediatric monogenic gene list. The variants were then classified using American College of Medical Genetics (ACMG) guidelines (Richards et al., 2015). Benign, or likely benign, classified variants were removed. Results: A total of 470 variants passed VarSeq analysis. Of these variants, 24 were within genes of interest to JIA or paediatric inflammatory disease. Three gene regions will be discussed as examples. Within KIR2DL3 , the heterozygous variant p.Gly12Ala was detected in six individuals. This variant has not been reported on gnomAD. KIR2DL3 is as an inhibitory receptor to HLA-C molecules. This region has been linked to psoriatic arthritis previously. Two further variants, p.Trp20Gly and p.Arg162Thr, were also detected in this region. Two heterozygous variants, p.Asp127Asn and p.Ala265Val, were detected in NOD2 , which is a susceptibility locus for Blau syndrome and has a role in immune homeostasis. Blau syndrome is characterised by arthritis, uveitis and dermatitis. Variants p.Asp127Asn and p.Ala265Val had a gnomAD Exomes frequency of 6.6x10 -4 and 1.3x10 -4 . One final example is the region UNC13D . Three heterozygous variants were detected in this region. Variant p.Ille848Leu was detected in two individuals in this cohort and has recently been detected in a compound heterozygous state in two patients in a study of JIA. Variant p.Ille848Leu had a gnomAD Exomes frequency of 1.0x10 -4 . Conclusion: WES analysis of 99 children with refractory JIA revealed that individuals carry rare genetic variants within gene regions of interest to JIA and that have been implicated in inflammatory paediatric monogenic diseases. Further analysis is needed to determine if these variants are present in a cohort of JIA patients who do not have refractory diseases. These results highlight the need for further research into the genetic risk factors of refractory disease and that genetic analysis can provide information to improve treatment outcomeP255. Cyclic amp response element modulator α governs PD-1 expression on CD4+ T cells in psoriasis across age groups P256. Exploratory immunophenotype of the rare disease juvenile sjögren’s syndrome reveals a dysregulation of B and T memory cell frequencies 7. Preliminary results of the inflammatory biomarkers measurement in patients with fibrodysplasia ossificans progressiva in compare to systemic juvenile idiopathic arthritis P258. Partial usp18 deficiency leads to early onset childhood inflammation P259. New safest approach to study fibrodisplasya ossificans progressiva by using urinary stem cells E. Carlsson 1 , A. L. Carvalho 1 , S. Hofmann 2 , L. J. McCann 3 , P. J. Ferguson 4 , B. Thompson 5 , T. Liloglou 6 , S. Abraham 7 , A. E. Surace 1 , S. Northey 1 , S. Russ 2 , M. W. Beresford 1ediatrics, University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany, 3 Alder Hey Children’s NHS Foundation Trust, Liverpool, United Kingdom, 4 Pediatrics Department, University of Iowa Carver College of Medicine, Iowa City, Iowa, United States, 5 Department of Dermatology, Alder Hey Children’s NHS Foundation Trust, 6 University of Liverpool, Liverpool, United Kingdom, 7 Department of Dermatology, University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany Correspondence: E. Carlsson Introduction: The pathophysiology of psoriasis, psoriatic arthritis (PsA) and psoriatic juvenile idiopathic arthritis (PsJIA) are poorly understood. In psoriasis, effector CD4 + T lymphocytes contribute to inflammation and tissue damage. The inhibitory surface coreceptor Programmed Death (PD)-1 plays a key role in the control of effector T cell function, and its therapeutic inhibition in cancer associates with the development of psoriasis and other autoimmune phenomena. Increased expression of the transcription factor cAMP response element modulator (CREM)α is a hallmark of T cell mediated autoimmune diseases. Objectives: This study investigated PD-1 regulation and surface expression in CD4 + T cells from psoriasis, PsA and PsJIA patients to identify molecular pathomechanisms that may be used as biomarkers and/or treatment targets. Methods: Peripheral blood naïve and effector T cell populations (CD45RA, CCR7 surface expression) were quantified in controls, psoriasis, PsA and PsJIA. CD4 + T cells were isolated using FACS and characterised based on PD-1 expression. Cells were stimulated (anti-CD3/CD28) and cytokine expression and release was measured (LUMINEX, Meso Scale Discovery). Jurkat CD4 + T cells deficient (CRISPR/Cas9) or over-expressing (lentiviral) CREMα were generated to investigate underlying molecular mechanisms. Luciferase reporter assays were used to test trans -regulatory events mediated by CREMα. Chromatin immunoprecipitation was performed using antibody against CREMα and DNA methylation in the PDCD1 (encoding PD-1) promoter region was assessed by bisulfite pyrosequencing using primary cells and genetically modified model Jurkat cell lines deficient in CREMα. Results: CD4 + T cells from psoriasis, PsA and PsJIA patients exhibit reduced PD-1 surface expression when compared with matched controls (p<0.05). This associates with increased expression of CREMα and imbalanced secretion of pro-inflammatory IL-17A and immune-regulatory IL-2 in psoriasis and PsA. CREMα trans -represses the PDCD1 promoter and mediates DNA methylation through co-recruitment of the de novo DNA methyltransferase DNMT3a. Conclusion: This study for the first time links increased expression of CREMα in CD4 + T cells from psoriasis and PsA patients with altered PD-1 expression and effector cytokine expression. The CREMα:PD-1 network promises potential as disease biomarker and/or treatment target. Disclosure of Interest : None declared
L. Martin-Gutierrez 1 , H. Peckham 2 , A. Radziszewska 2 , J. Peng 2 , O. Nettey 2 , E. Jury 1 , C. Ciurtin 2 1 Centre for Rheumatology Research, 2 Centre for Adolescent Rheumatology Versus Arthritis, University College of London, London, United Kingdom Correspondence: L. Martin-Gutierrez Introduction: Sjögren’s syndrome (SS) is an autoimmune rheumatic disease characterised by dryness resulting from chronic lymphocytic infiltration of the exocrine glands. Patients also present with other extraglandular manifestations such as arthritis, anemia and fatigue or various organ and systems involvement. The disease is more frequent in women aged 30-50. However, in rare cases, the disease starts in childhood and is known as juvenile SS (JSS) or childhood SS. Children have different clinical manifestations compared to adults, with dryness being less common, making the diagnosis very challenging. Objectives: To investigate in depth the immune cell profile of patients with JSS for better understanding of disease pathogenesis. Methods: Peripheral blood was collected from a cohort of patients with JSS while attending appointments at UCLH clinics. None had received B-cell depletion therapy. Immune-phenotyping of 29 immune-cell subsets, including B and T cells, in peripheral blood from patients with JSS (n=10) and age and sex-matched healthy controls (n=10) was performed using flow cytometry as we have performed previously for patients with adult onset SS. Data were analysed using multiple t-tests and compared with the adult SS immune phenotype. Results: Patients with JSS had an average age of 18 years (range 16-21) with an average age of disease onset at 14 years (range 12-18). Up to 60% of patients presented Anti-Ro autoantibodies while 50% presented Anti-La autoantibodies. Patients with JSS had an altered immune profile compared to age matched healthy controls (average of 18 years, range 15-25). In the B cell compartment, JSS patients had higher frequencies of Total CD19+ B cells (p=0.0044), Naïve B cells (CD19+IgD+CD27-) (p=0.0183) and bm2 (CD19+IgD+CD38+) (p=0.0490) whereas memory B cell subsets such as early bm5 (CD19+IgD-CD38+) and late bm5 (CD19+IgD-CD38-) were significantly reduced (p=0.0249, and p=0.0117 respectively), similar to the profile seen in patients with adult-SS. Interestingly, in the CD4+ T cell compartment, central memory (CD4+CD27+CD45RA-) T cells were significantly reduced (p=<0,0001) but effector memory (CD4+CD27-CD45-) and effector memory-re-expressing-CD45RA (EMRA, CD4+CD27-CD45RA+) T-cell subsets were significantly elevated (p=0.0171 and p=0.0002 respectively). These changes were not identified in adult-SS patients. Finally, unlike our observations in patients with adult-onset SS there was no widespread deregulation of CD8+ T cell subsets in JSS patients; only a significant increase in CD8+CD25-CD127+ responders T cells (p=0.0392) was observed in JSS patients versus healthy. Conclusion: This is the first pilot study investigating the immunophenotype profile of patients with JSS. Our preliminary findings suggest altered immune phenotypes in both B-cell and T cell compartments and for B cells are in concordance with previous immunophenotyping studies in adult SS (predominance of naïve and lower frequencies of memory B cells), suggesting an immunological rationale for the use of similar therapies. Further studies, comparing the adult with the juvenile phenotype could help stratify patients for targetted therapies and improve treatment in this rare disease in children for which no evidence-based recommnedations exisMatkava 1 , I. Nikishina 1 , S. Arsenyeva 1 , M. Kaleda 1 , E. Samarkina 2 , M. Cherkasova 3 , E. Fedorov 1 , A. Arefieva 1 1 Pediatric Department, 2 Laboratory of Immunology and Molecular Biology of Rheumatic Diseases, 3 Laboratory of thromboinflammation, V. A. NASONOVA RESEARCH INSTITUTE OF RHEUMATOLOGY, Moscow, Russian Federation Correspondence: V. Matkava Introduction: Fibrodysplasia ossificans progressiva (FOP) is an autosomal-dominant rare genetic disease which provokes many inflammatory flare-ups with formation of heterotopic ossifications due to uncontrolled osteogenesis. It is one of the most disability condition which caused by mutation de novo in ACVR1 gene. Some data suggests that FOP may belong to the spectrum of autoinflammatory rheumatic disease. Objectives: To describe the first experience of measurement some serum biomarkers levels received from patients (pts) with FOP compared with Systemic juvenile idiopathic arthritis (sJIA) pts. Methods: Since 2018 we collected biobank of blood samples from 5 pts with FOP, 52 pts with sJIA and 5 samples from healthy children (as a control group). Serum levels of IL-18, IL1RA, IL1b, IL-6, TNFR1, TNFR2, ferritin were measured using standard commercial enzyme-linked immuno-sorbent assay (ELISA). CRP was determined by commercial nephelometric method. Results: The pts were divided into 2 groups: group I - with sJIA (52 pts), group II – with FOP (5 pts), control group – 5 pts. The ratio of boys and girls was 1:1,5 in pts with FOP and 1:1,7 in pts with sJIA. Median age of pts with FOP at the examination time was 12.5 years [5; 17], with sJIA – 11.4 years [7; 13], control group – 12.8 [7; 17]. CRP and ferritin levels were in normal range in all pts with FOP and control group and were significant higher in sJIA pts. Among of spectrum of investigated biomarkers levels of the IL-6, TNFR1 were in normal range. Level of the IL1b in pts with FOP was the same (0.001 pg/ml) with control group but significantly higher in sJIA pts (median 0,9 [0,002; 2,4]). However high levels for IL-1RA (reference range 78-970 pg/ml) were detected in both groups: sJIA (Me 1267,43 pg/ml [interquartile range (IQR) 936,75; 2292,75]) and FOP (Me 1342,36 pg/ml [IQR 927,0; 1811,6]) and values of FOP pts were higher than in 44 sJIA pts without macrophage activation syndrome (MAS) (Me 1165,2 pg/ml [IQR 868,0; 1743,8]) and lower than in 8 sJIA pts with MAS (Me 2654,2 pg/ml [IQR 1432,2; 6252,2]). In control group IL-1RA was in normal range (Me 404,77 pg/ml [IQR 255,3; 506,08]). Also high levels of TNFR2 (ref. range 3.4-10,8 ng/ml) were presented only in pts with FOP (Me 14,99 ng/ml [IQR 5,49; 23,12]) compared to sJIA (Me 9,47 ng/ml [IQR 4,46; 29,49]) and control group (Me 0,35 ng/ml [IQR 0,243; 3,496]). It was expected, that the level of IL-18 (ref. range 0-732,7 pg/ml) was significantly higher in pts with sJIA (Me 690,14 pg/ml [IQR 361,18; 3607]) than in FOP (Me 0,01 pg/ml [IQR 0,008; 0,05]) pts and control group (Me 0,0194 pg/ml [IQR 0,0032; 14,926]). Conclusion: Our preliminary data confirmed, that immunopathogenesis of FOP can be considered as autoinflammatory disease. High level of IL-1RA and TNFR2 was detected in pts with FOP. It seems important that of IL-1RA was higher than sJIA without MAS, but lower than in sJIA with MAS. This evidence confirmed our decision to treat FOP pts with targeted therapy (JAK-kinase inhibitors), which was successfully used in 15 pts. We are obtaining more blood samples for further investigations and identification more correlations between biomarkers and FOP. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
A. Munk s, Icahn School of Medicine at Mount Sinai, New York, United States Correspondence: A. Munk Introduction: Type I interferonopathies are characterized by an overabundance of IFN-I, which cause a broad spectrum of clinical presentation. Ubiquitin Specific Peptidase 18 (USP18) plays an important role in regulating the IFN-I response by dampening the IFN-I signaling pathway. Autosomal recessive USP18 deficiency results in severe systemic inflammation and neurological anomalies, which is fatal in the perinatal period. In our study, we sought to understand the etiology of disease in three different patients with genetic variations in USP18 who exhibited early onset autoinflammatory clinical features. Objectives: Assess USP18 variants expressed by patients, and characterize the function of individual USP18 proteins. Methods: HEK293T cells were transiently transfected with USP18 variants, and expression of USP18 mRNA and protein were measured using qPCR and western blot, respectively. Cells were transiently transfected and stimulated with IFN-I and levels of pSTAT1 and pSTAT2 were assessed using western blotting and flow cytometry. USP18 -/- dermal fibroblasts were transduced with USP18 variants, and IFN-related genes were measured using qPCR. Results: USP18 variants did not impair USP18 protein or mRNA expression. In contrast, we found that unlike WT USP18, the three distinct USP18 variants failed to prevent phosphorylation of STAT1 and STAT2 when stimulated with IFN-I. Conclusion: Assessed genetic variations in USP18 variants are hypomorphic in its ability to prevent type I interferon signaling, and are likely causative of patient’s autoinflammatory clinical features. Patient Consent: No, I have not receive consent Disclosure of Interest : None declared
I. Nikishina 1 , A. Borovikov 2 , V. Matkava 1 , S. Arsenyeva 1 , V. Tabakov 2 , M. Sharova 2 , I. Sermyagina 2 1 Pediatric Department, V. A. Nasonova Research Institute of Rheumatology, 2 Research Centre for Medical Genetics, Moscow, Russian Federation Correspondence: I. Nikishina Introduction: Fibrodysplasia ossificans progressiva (FOP) is an ultra-rare monogenic disease caused by a de novo pathogenic mutation in the ACVR1 gene with population frequency 1:2000000. FOP characterized by catastrophic progression a number of flare-up episodes that resolved to heterotopic ossification. Non-invasive methods for obtaining culture from these patients could help in understanding of «new bone formation» mechanism. Objectives: To analyze available approaches for future research to explore osteogenesis obtaining urinary stem cells (USC) from patients with FOP. Methods: From 24-year period (1998 – 2022) we observed 41 patients (pts) with FOP. DNA sequencing coding regions of the ACVR1 gene were performed by the Sanger method in most of patients. Fresh urine samples were taken from 9 pts. USC were cultivated by protocol described in Falzarano and Ferlini work [1]. Results: Fresh urine samples were taken from 9 patients with FOP (age 3 to 19; 4 female, 5 male). Among them 7 pts have «typical» heterozygous missense substitution c.617G>A (p.Arg206His), ultra-rare heterozygous missense substitution in the ACVR1 gene were presented in 2 pts (1 –p.Gly356Asp, 1 - p.Gly328Glu). We obtain and depone USC lines from 3 (33%) patients for future investigations. In other cases, we couldn’t grow USC lines due to low cell’s number and/or poor adhesion (4, 44%), bacterial contamination (2, 22%). In the group where it failed, we can repeat obtaining USC lines in a next hospitalization without additional risk of complications. Conclusion: The obtained USC lines from patients with FOP-specific mutations in the gene ACVR1 are a good model for studying the role of BMP in heterotopic ossification, which is important not only for understanding the nature of FOP, but also the universal processes of neo-osteogenesis in other rheumatic and non-rheumatic diseases. Obtaining primary cultures from patients with FOP by the usage invasive manipulations (puncture or tissue biopsy) always come with additional life-threatening risks. We suggest that USC can be a good non-invasive approach to learning and understanding a mechanism of abnormal ossification «in vitro». References. 1. Falzarano M. S., Ferlini A. Urinary stem cells as tools to study genetic disease: overview of the literature. Journal of clinical medicine. 2019. Т. 8. № 5. С. 627. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
P260. Juvenile dermatomyositis with subclinical cardiac manifestations in a 10 year-old Libyan boy: a rare case P261. Juvenile dermatomyosirtis as a paraneoplastic phenomenon in a preadolescent boy ( case report) 63. Withdrawn P264. Monocentric study of juvenile dermatomyositis P265. The evaluation of nailfold capillaroscopy findings and its association with disease activity in juvenile dermatomyositis patients P266. Clinical presentation and laboratory findings in patients with anti-mda 5 positive juvenile dermatomyositis-a single- center case series 7. Severe vasculopathy of a case of juvenile dermatomyositis associated with anti-MDA5 antibody without pulmonary involvement P268. Assessing the impact of a family day for young people and their families living with juvenile dermatomyositis (JDM) in the Northwest of England, UK 9. Myopathy with anti-3-Hydroxy-3-Methylgltaryl-coenzime a redutase antibodies in an adolescent P270. Mandibuloacral dysplasia -a genetic mimic of juvenile dermatomyositisP271. Pres juvenile dermatomyositis working group (JDM WG) achievements, September 2021-September 2022 72. Clinical presentation, risk factors and prognosis of MDA5-positive JDM; clinical diversity and red flags P273. Clinical spectrum of anti MDA-5 autoantibody associated juvenile dermatomyositis from a tertiary-care centre in Northern India P274. A pediatric case of intractable immune-mediated necrotizing myopathy treated with multi-targeted therapy P275. Myositis specific autoantibodies in juvenile idiopathic inflammatory myositis: our experience from a tertiary care Centre in North India P276. Defining criteria for disease activity States in Juvenile dermatomyositis based on the juvenile dermatomyositis activity index 7. Comparison between electromyography and whole-body mri in the assessment of disease activity in juvenile dermatomyositis 278. Early-onset juvenile dermatomyositis: a tertiary referral center experience and review of the literature P279. Features of myositis-specific and myositis-associated antibodies and antinuclear antibody patterns in patients with juvenile dermatomyositis 80. Interstitial lung disease in juvenile dermatomyositis– our experience from Northern India P281. There are significant delays in diagnosis of juvenile dermatomyositis- 30 years of clinical experience at a tertiary care centre in north india R. Tyagi, S. Basu, P. Vignesh, R. K. Pilania, D. Suri, A. Rawat, S. SinghTyagi Introduction: Juvenile dermatomyositis (JDM) is the most common type of inflammatory myopathy seen in children and comprises 1.7 % of all patients registered in Pediatric Rheumatological Clinic (PRC) at our institute. We report herein our clinical experience on the initial presentation of JDM at our centre. Objectives: To assess the association of delay in diagnosis of juvenile dermatomyositis with various clinical parameters Methods: We analysed patients registered in PRC at Advanced Pediatrics Centre, PGIMER, Chandigarh during the period of January 1992 to April 2022. Of the 8200 patients registered during this period, 140 had been diagnosed to have JDM. Diagnosis was based on Modified Bohan and Peter criteria. Time interval between onset of symptoms and presentation to our unit was noted and patients were classified as follows: i) Group 1- Time interval between 0-3 months ii) Group 2- Time interval between 3-6 months, iii) Group 3- Time interval between 6 -12 months, iv) Group 4- Time interval >12 months. Additional clinical parameters that were noted were age at onset, calcinosis, interstitial lung disease (ILD), mortality, lipodystrophy, gastrointestinal (GI) vasculitis, respiratory weakness. Statistical analysis was done using the SPSS 22 software Results: Of the 140 patients with JDM, 43 (30.7%) patients had a delay in diagnosis of more than 12 months (group iv). Of the latter, calcinosis was seen in 18 (41.8%) patients, ILD was seen in 6 (11.6%) patients and lipodystrophy was seen in 10 (23.2%) patients. ConclusionTrial registration identifying number: Our data show that 30.7% of patients with JDM at our centre have significant delays (>12 months) in diagnosis. We noted that calcinosis, ILD and lipodystrophy were more common in this subgroup. Our findings would impact therapeutic decision making in this condition. Disclosure of Interest : None declared
A. A. Abushhaiwia 1 , H. S.Alrabte 2 , Y. Elfawires 3 1 paediatric Rheumatology , Faculty of medicine, University of Tripoli, Tripoli Children’s Hospital, 2 Paediatric cardiology, faculty of Medicine, Tripoli University Tripoli Children Hospital, Libya, 3 Paediatric Rheumatology, Faculty of Medicine,Tripoli university , Tripoli Children’s Hospital, Tripoli , Libya Correspondence: A. A. Abushhaiwia Introduction: Juvenile dermatomyositis (JDM) is a rare autoimmune disease in the paediatric population, that typically presents with symmetric proximal muscular weakness and classic dermatologic findings. Cardiac abnormalities are known to occur in JDM but are less common than other clinical features. Cardiac involvement is usually subclinical, including conduction abnormalities. A previously healthy 10-year-old boy is described who presented with SVT by holter ECG secondary to newly diagnosed JDM and all symptoms resolved following treatment with oral corticosteroids, IVIG and MTX SC Objectives: To highlight the importance of early diagnosis and recognition of the index of suspicion of subclinical cardiac manifestations in patients with JDM Methods: Based on the clinical, biochemical and imaging findings, a definite diagnosis of JDM was made by using. Results: A previously healthy 10-year-old boy born to a non- consanguineous marriage and family history was not significant. He presented to P\ Rheumatology clinic with a 2-month history of gradual tiredness and weakness. Later on he had palpitation, breathlessness. Initially was diagnosed by cardiologist as a case of SVT by holter ECG. He not being able to sit, stand or ascend stairs or take turns in bed and severe myalgia since 2 months. After 4 weeks he developed a faint red rash on the upper half of his face, along with skin-colored papules on his hands and just below his right elbow. During symptom progression, there was dysphasia but no fever, weight loss, vision change, vomiting, or diarrhea. On physical examination his weight was 36kg (50th-75thpercentile) and his height 142cm (50th -75th percentile). His vital signs were normal included BP 100/65mmHg, HR 80 beats and RR 20 breaths. He was profoundly weak, with head lag. A faintly erythematous rash over the malar prominences, and hyperpigmented papules over the dorsal metacarpophalangeal and proximal interphalangeal joints he had (Gorton’s papules). Bilateral Flexion contractures involving almost every joint of upper and lower extremities were present; therefore, power in the extremities 4\5. Power of neck flexor and extensor was 2/5. Superficial reflexes, plantar response and sensory examination when performed were found to be normal. Other systemic examinations were normal. CMAS SCORING SHEET; WAS 8\52.Investigations performed showed WBC 8.3X103, HGB 11.5g and platelets375X103. ESR was 14mm, and CRP level was 3.4mg/dl was negative. Antinuclear Antibodies (ANA) was Positive high titer 1:640, dsDNA-abs was negative,u1RNP, Sm, SSA, SSB, SCL70, jo1 all were negative and RF was negative. CPK 3036u, LDH was 719 U/l ALT=144U/L, AST=268U/L.Chest X-ray, ECG and ECHO were normal, but holter ECG showed SVT, NCV&EMG were consistent with active myopathy and polymyositis. Based on clinical features, laboratory investigation and EMG the diagnosis of JDM was made. Patient was given oral steroids (1mg/kg/day) accompanied by methotrexate 15mg2 sc, IVIG 2g for 6 months, supplemental vitamin D, and calcium. Following this treatment, improvement continued with normalisation of the holter ECG, disappearance of the skin rash and normalisation of CPK and LDH within 2 months no any symptoms of weakness, or rash, and had 5/5 strength in the muscle groups of both upper and lower extremities. Conclusion: cardiovascular complications should be considered in any child with JDM. Undergo a routine cardiovascular risk assessment at the onset of diagnosis and the potential value of corticosteroids and IVIG for its treatment. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
M. A. Alessi 1 , N. Alquorain 2 , J. M. Alqahtani 3 , A. A. Alrahim 4 , R. A. Alrumaih 5 , N. S. Alsaif 5 1 Saudi Arabia , Dammam , Department of Pediatric , King Fahad Hospital of the University, 2 Dermatology, King Fahad Hospital of the University, 3 Dermatology, King Fahad Hospital of University , College of Medicine, Imam Abdulrahman bin Faisal University, Dammam, Saudi Arabia., 4 Dermatology, King Fahad Hospital of the University , College of Medicine, Imam Abdulrahman bin Faisal University, Dammam, Saudi Arabia., 5 Dermatology, King Fahad Hospital of the University, College of Medicine, Imam Abdulrahman bin Faisal University, Dammam, Saudi Arabia., Dammam, Saudi Arabia Correspondence: M. A. Alessi Introduction: We report a rare case of Juvenile Dermatomyositis (JDM ),to highlight ultimate need for multidisciplinary team approach to make an early diagnosis of occult malignancy in a preadolescence . Also, to report the success of initiation of intravenous (IV) pulse steroids and IVIG in a parallel with Chemo radiotherapy as a treatment modality of our case as malignancy-associated JDM. Objectives: A13 y/o Saudi boy was admitted due to skin rash , fatigue and Arthralgia for few weeks. He had progressing fatigue to difficulties on doing his usual activities . He reported difficulty of swallowing started 1 month ago. In addition he had progressive sense of nasal obstruction over 2 months time associated with mild bilateral epistaxis. There is weight loss around 4 kg over 1 month ,No fever. He had Gottron’s papules , heliotrope rash, itchy extensive rash over Arms , genitalia , back. Nail capillaroscopy showed dilated and capillaries with areas of dropouts. He had positive Gower’s signs and proximal muscle weakness . Investigations : normal CBC, Ferritin 336 ng/ml , ESR 43 mm/h, CRP 1.5 mg/dl , (CPK) 4195 U/L, (LDH) 810, SGOT 186 U/L , SGPT 92 U/L , (ANA) was highly positive 1280, (SSA) was highly positive 841 , Myositis Specific Antibodies panel revealed high titer of NXP-2AB >100 , and other Autoimmune serology were negative .Pelvic girdle MRI revealed inflammatory myositis. Based on the clinical and laboratory findings we made a diagnosis of JDM, his initial Childhood Myositis Assessment Scale was 31/52.Videofluoroscopic showed no abnormality. So our patient fulfilled the Bohan and Peter criteria1 for JDM. Due to dysphagia ENT evaluation done including nasopharyngoscopy that showed nasopharyngeal mass . CT head and neck showed right sided nasopharyngeal mass with foci of calcification and heterogenous no-vascular enhancement. Biopsy was taken and histopathology reports undifferentiated nasopharyngeal carcinoma. Multidisplaniray team meeting done between (Haemato-Oncology , Pediatric and adult Rheumatology and Dermatology services ) Methods: The presence of progressive nasal obstruction and epistaxis over short period in male pre-adolescent warrants need for detailed ENT assessment that showed the presence of nasopharyngeal mass. The presence of cutaneous necrosis is highly associated with malignancy in adult dermatomyositis which was noticed in our patient clinically and histologically.(1). Many studies in adult population reported that cutaneous vasculitis is suggestive of associated malignancy which is in general a distinctive cutaneous features of JDM.(2,3). Therefore, cutaneous vasculitis might have no predicting value for malignancy in JDM, however some dermatological findings in DM patients are highly suggestive of underlying malignancy, including all signs of severe vasculitis(4). Early pick up of these patients is crucial before steroid therapy as it can suppress the tumor growth and delay establishing the right diagnosis. Our patient went in complete remission after radiochemotherapy and JDM managment. Results: Malignancy and DM have been identified as paraneoplastic phenomenon in adult population . But this phenomenon is rare in children , Hence paediatric patients are not routinely assessed for occult malignancy, so there is need of study aimed to analyze the clinical features of patients with JDM in order to identify predictors of malignancies in these patients. Conclusion: Paediatric Rheumatologists should look for occult malignancy in adolescent age group who presented with atypical rash and JDM featuresK. Bouayed, Y. Mahdar, A. Sakhi CHU Ibn Rochd, casablanca, Morocco Correspondence: K. Bouayed Introduction: Although rare, juvenile dermatomyositis is the most common pediatric inflammatory myopathy. Studies concerning this condition in our country are almost non-existent. Objectives: The objective was to describe the epidemiological, clinical, paraclinical, therapeutic and evolutionary profile of juvenile dermatomyositis. Methods: This monocentric retrospective study, conducted between 2010 and 2020, enrolled 16 patients followed for juvenile dermatomyositis in the Department of Pediatric Rheumatology of the Ibn Rochd University Hospital in Casablanca. Results: The mean age at diagnosis was 8 years (1-14 years); the sex ratio M/F was 0.7. The average diagnostic delay was 228 days; the disease was revealed by a typical skin involvement (75%); amyopathic forms were described (25%); joint involvement was found (93.75%); calcinosis was the most frequent complication (43.75%), followed by diffuse interstitial lung disease (37.5%). Muscle and liver function tests were disturbed in 81.25% and 62.5% respectively. Dermatomyositis DOT was performed systematically, showing the presence of anti-MDA5 antibodies in 50% of cases. Juvenile dermatomyositis was confirmed according to the criteria of Bohan and Peter (56.3%). Corticosteroid therapy was systematically used for muscle damage (87.5%). Methotrexate was the main background treatment 87.5% followed by cyclosporine 43.8%; hydroxychloroquine and/or mycophenolate mofetil were prescribed for severe skin involvement 18.75% respectively; cyclophosphamide for visceral complications 25%; immunoglobulins 37.5% and Rituximab 18.75% for refractory forms. Complete clinical and paraclinical remission was reported in 43.75% of cases. An osteoarticular tuberculosis occured in a patient with ciclosporin treatment. Conclusion: Our series illustrates the phenotypic diversity of juvenile dermatomyositis as well as its severity. Joint involvement is predominant. Methotrexate seems to be ineffective in severe muscular forms. Early therapeutic intensification in refractory visceral and muscular forms improves the prognosis. Immunosuppressive drugs expose patients to infections, particularly tuberculosis in endemic areas. The diagnosis delay is long due to the lack of knowledge of this condition, which deserves the awareness of practitionersS. Caglayan 1 , F. çakmak 2 , R. İşgüder 3 , K. Ulu 1 , E. Ünsal 3 , N. A. Ayaz 2 , B. Sözeri 1 1 Pediatric Rheumatology, Umraniye Training and Research HospitaL, 2 Pediatric Rheumatology, Istanbul University, Faculty of Medicine, Istanbul, 3 pediatric rheumatology, Dokuz Eylül University, Faculty of Medicine, Izmir, Turkey Correspondence: S. Caglayan Introduction: Juvenile dermatomyositis (JDM) is a rare autoimmune vasculopathy that primarily affects the skin and muscle. It is characterized by skin findings such as heliotrope rash, Gottron papules, and proximal muscle weakness. Nailfold capillaroscopy (NFC) to evaluate microvascular involvement is a frequently used method in JDM patients Objectives: This study aims to evaluate the NFC findings in patients with JDM and analyze its relationship with disease activity Methods: This is a cross-sectional study involving pediatric patients who were diagnosed with JDM between 2014-2021, from three pediatric rheumatology centers in Turkey. Simultaneously at the date of NFC, patients were evaluated for disease activity with the physician visual analog scale (VAS), patient VAS, and childhood myositis assessment scale (CMAS). NFC was performed on eight fingers and at least four images were obtained from one finger. Capillary density, arterial width, venous width, apical loop, capillary morphology, presence of meandering capillary, microhemorrhage, avascular area, neoangiogenesis, and capillary ramification were evaluated from the images. Results: 16 JDM and 25 control patients were included in the study. There were 11 females and 5 males in the patient group, and 13 females and 12 males in the control group and there was no statistically significant difference. The mean age of the patients was 10.4 ± 3.4 years in the patient group and 12.6 ± 3.6 years in the control group, and there was no statistically significant difference. The mean age at diagnosis was 7.1±4 years, and the mean follow-up period was 40.8±29 months. Initially, 6 (37.5%) of the patients had severe disease activity, 6 (37,5%) had moderate, and 4 (25%) had mild disease activity. During the follow-up, 6 patients (37.5%) showed monocyclic, 6 patients (37.5%) chronic persistent and 4 patients (25%) polycyclic course. The most common clinical findings were proximal muscle weakness (100%), myalgia (93.3%), heliotropic rash (93%), malar rash (93%), fatigue (87%), Gottron papule (81%), Gowers sign (80%), periorbital edema (60%) arthralgia (53.3%), calcinosis (25%), and arthritis (20%). respectively. In patient group had an abnormality in at least one finding on NFC. Capillaryscopy findings of the patient and control groups are given in Table 1. Tortuosity and crossing capillary were non-specific changes that can be seen in the healthy control group, and there was no statistical difference between the two groups. There was no correlation between the CMAS and apical loop width, the presence of bizarre capillary, bushy capillary, microhemorrhage, avascular area, and neovascularization. Between the long time to diagnosis and the presence of apical loop width and bizarre capillary statistically significant difference was found (p=0,004, p=0,003 respectively). Avascular area and neoangiogenesis scores were higher in patients with high initial disease activity. There were nine patients in remission at the final follow-up. Even though they were in remission, 90% of the patients had dilated capillaries. Conclusion: Performing NFC findings have critical importance at any stage of the disease in the evaluation of JDM patients. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
A. Čengić 1 , V. Selmanović 1 , F. Hadzagić Ćatibušić 2 , S. Užičanin 2 , E. Vukas 2 , Z. Huseinbegović 2 , D. Pokrajac 3 , S. Hasanbegović 4 , V. Mišanović 5 , D. Anić 5 , Z. Begić 6 , A. Kadić 6 , M. Halimić 6 , S. Dizdar 6 , S. Kapić 6 , A. Džananović 7 , I. Pašić 7 , M. Bukvić 7 , A. Džinović 8 , A. Mustajbegović 3 1 Allergology, Rheumatology and Clinical Immunology, 2 Pedijatric Neurology, 3 Pediatric nephrology, 4 Endocrinology, 5 intensive care unit, 6 Cardiology, Pediatric Clinic, Clinical Centre University of Sarajevo, 7 Pediatric radiology, Radiology clinic, Clinical university centre Sarajevo , 8 Pulmology, Pediatric Clinic, Clinical Centre University OF Sarajevo, Sarajevo, Bosnia and Herzegovina Correspondence: A. Čengić Introduction: Juvenile dermatomyositis ( JDM) with positive anti-melanoma differentiation-associated gene 5 (anti-MDA5) antibody have been associated with distinct clinical phenotype which includes characteristic mucocutaneous features, small hand joint arthritis, mucus membrane and skin ulcers, palmar papules, less muscle involvement and severe interstitial lung disease (ILD). The clinical presentation of anti-MDA5 JDM differs substantially from the other forms of JDM. Objectives: To describe the main presenting clinical and laboratory features of children with anti-MDA5 positive JDM followed in our Pediatric Rheumatology Unit . Methods: Retrospective review of the clinical records of all patients with anti-MDA5 positive JDM who were treated in our center since January 2018 until May 2022. We followed fever, skin rashes that is characteristic for JDM, oral aphthae, palmar and plantar pustules, arthralgias, arthritis, weight loss, muscle weakness, CT verified ILD, C reactive protein, Sedimentation rate, aspartate aminotransferase(AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), creatine kinase (CK), ANA, ANTI- Ro52 Results: Three patients were included in case series two of them were males. The median age at diagnosis was 9 years (range 2– 9 years). The median diagnostic delay was 2.5 months (range 1 month – 4 months). All patients had JDM characteristic skin changes, aphthous stomatitis, arthralgia, arthritis and weight loss at presentation. Only 1/3 patient had palmo-plantar pustules and 2/3 CT verified ILD. None of the patients presented with cutaneous calcinosis nor dysphagia and dysphonia. All patients had slightly elevated serum AST ( median value 145, normal range 15-59 U/L ), ALT ( median value 151, normal range 9-72 U/l ) LDH ( median value 453, normal range 100-190 U/l) and all patient had normal levels of creatine kinase and CRP. Sedimentation rate was slightly elevated in all children (median 35). Only one child had positivity for antinuclear antibodies and 2/3 for antiRo52. Magnetic resonance imaging was performed in all three patients and the results showed muscle changes compatible with dermatomyositis. Conclusion: Anti-MDA5 JDM is a distinct subset of inflammatory myositis, with frequent skeletal and constitutional features such as intermittent fever, arthritis, weight loss, and less severe myositis, with strong association to ILD . Anti-MDA5 JDM is characterized by lower serum CK and slightly elevated other muscle enzymN. Choon Seong Selayang, Selayang, Malaysia Correspondence: N. Choon Seong Introduction: Anti-MDA5 antibody-positive juvenile dermatomyositis can have various subset of phenotype at presentation which could sometimes mimic other forms of autoimmune rheumatic disease. Objectives: Here, we report a rare manifestation of a patient with juvenile dermatomyositis. Methods: Case report. Results: A 15 years old girl, previously well, presented initially as articular symptom (oligoarthritis of knees) following upper respiratory tract symptoms. The symptoms resolved with nonsteroidal antiinflammatory drug and antibiotic. Two months later, she was noted incidentally to have multiple cutaneous rashes typical of dermatomyositis but no other systemic manifestations as yet. However, three months later, she had altered sensorium followed by similar oligoarthritis of knees. It rapidly progressed to involve digital gangrenes and spontaneous thigh hematoma and mononeuritis multiplex. She also had polyarthralgia, fever and hyperkeratosis. ANA and dsDNA were negative with normal CK. Myositis panel showed positive anti-MDA5 associated with antiRo52. No interstitial lung disease was detected. No evidence of thrombotic microangiopathy. Cerebrospinal fluid was clear of infection. Brain MRI revealed posterior reversible leukoencephalopathy. CTA both upper and lower limbs did not reveal any aneurysm or thrombosis. She was given pulse steroid followed by pulse cyclophosphamide. Her symptoms improved thereafter. Conclusion: It is important to recognize such severe systemic phenotype of anti-MDA5 patients to allow early intervention and better survival ofV. Cuthbert 1 , N. Woodend 2 1 Paediatric Rheumatology, Royal Manchester Children’s Hospital, 2 Paediatric Rheumatology, Royal Manchester Children;s Hosital & Versus Arthritis, Manchester, United Kingdom Correspondence: V. Cuthbert Introduction: JDM is a rare, autoimmune disease affecting primarily the muscles and skin and there are approximately 2-4 cases per million children and young people. JDM can have a huge impact on the lives of the young people, their siblings and families and many young people and families have never met others with the condition. Objectives: To organise the first family day for young people and families living with JDM in the Northwest of England, UK and to assess the impact of the day. For Young people and their families to feel less isolated living with JDM For Young people and siblings get to take part in an activity to build their confidence For Young people and their families receive information about JDM and living with the condition Methods: Invitations were sent out to young people and families with JDM who attended Alder hey Children’s Hospital, Manchester Royal Children’s Hospital, Sheffield Children’s Hospital and Leeds children’s Hospital in the UK. The family day was organised outside of a hospital setting at the Chill Factor in Manchester. The young people were invited to enjoy snow activities, a science workshop, arts and crafts and soft play, as well as enjoy a fabulous lunch! A programme of speakers was organised and included a specialist nurse from Great Ormond Street Children’s hospital, discussing her current JDM research; a Manchester patient talked about the lived experience of having JDM and entering adulthood and the transition experience into adult healthcare. A Question and answer session was organised for young people and families to ask questions to a multi-professional panel. Results: Feedback from questionnaires filled out on the day and email responses suggest the young people and families found the day beneficial and benefited from meeting up with other families and sharing experiences. The feedback suggested they found the education sessions and tips gained helpful and would aid management of the condition. Feedback suggested that the young people and their siblings had fun! Meeting others with the same condition was frequently mentioned in the feedback as very beneficial and helped to reduce feelings of isolation. Feedback suggests that the majority of young people with JDM and families would like to keep in touch with other JDM families they met. Conclusion: There is evidence from the feedback gained from the family day for JDM patients and families that the families found the day beneficial: Meeting others with the same condition in the same region of the UK helped to reduce feelings of isolation and there was enthusiasm for keeping in touch and for further similar evLima 1 , S. Alves 1,2 , M. Santos 3 , C. Zilhão 1,2 1 Pediatric Department, 2 Pediatric Rheumatology Unit, 3 Pediatric Neurology Unit, Centro Materno Infantil do Norte, Centro Hospitalar Universitário do Porto, Porto, Portugal Correspondence: J. Lima Introduction: Idiopathic Inflammatory Myopathies (IIM) may present with different clinical phenotypes, although the finding of myositis specific antibodies often leads to similar clinical manifestations, response to therapy and prognosis. Antibodies against 3-hydroxy-3-mehylglutaryl-coenzime A reductase (HMGCR) have been recently associated with Immune-mediated Necrotizing Myopathy (IMNM) in pediatric patients, accounting for 1% of all pediatric IIM. It is classically characterized by progressive proximal weakness, highly elevated serum creatine kinase (CK) levels, typical histopathological features of an IMNM, and poor response to therapy. Although cutaneous involvement is exceptional in adults, juvenile dermatomyositis (JDM)-like manifestations can develop in children. Objectives: To highlight IMNM as a rare but relevant differential diagnosis to JDM, through the report of an adolescent with anti-HMGCR myopathy. Methods: Case report. Results: We report a 16-year-old male patient with a 5-month history of progressive symmetrical proximal limb weakness, more noticeable in the morning, hindering dressing, climbing stairs, and getting out of bed. He denied any other symptoms including joint swelling, fever, dysphagia, respiratory or gastrointestinal manifestations. Physical exam was remarkable for Gottron sign on the knees and pronounced symmetrical proximal weakness (Childhood Myositis Assessment Scale (CMAS) of 11/52 points) with severe limb muscle atrophy. Cervical involvement was less pronounced. Laboratory evaluation revealed a markedly elevated CK (13469 U/L), AST (319 U/L), ALT (416 U/L) with normal inflammatory markers. Myositis autoantibody panel revealed positive anti-Ku. Thigh MRI was compatible with myositis and electromyography consistent with a myopathy. Electrocardiogram and echocardiogram were normal. Muscle biopsy showed predominant perifascicular atrophy with scattered fiber necrosis and upregulation of MHC-1, findings consistent with JDM. Treatment with oral prednisolone (1mg/Kg/day) and subcutaneous methotrexate (MTX) (20mg/wk) was started, in parallel with physical rehabilitation. One month later, only slight clinical improvement was achieved (CMAS 18 points; CK 5246 U/L), and intravenous immunoglobulins (0,8g/Kg/month) was started. Throughout the subsequent 6 months, clinical and MRI improvement ensued, reaching a CMAS score of 52/52. Though asymptomatic, muscular enzymes persisted elevated (lower CK level of 685 U/L), without the possibility of weaning prednisolone under 12,5mg/day, whereby it was decided to switch from MTX to azathioprine. As clinical and analytical worsening followed, extended antibodies panel was performed revealing anti-HMGCR (>200U/mL; reference value <20). Attending to the previous clinical response and expert recommendations, treatment with MTX was restarted, and we ponder treatment with rituximab in case of clinical worsening following new corticosteroid weaning. Conclusion: Anti-HMGCR antibodies are not always included in the commercial myositis panel, and this possibility should be considered in pediatric IIM patients with an indolent course, severe presentation or treatment refractoriness, even when cutaneous findings are present. We report a challenging case of a progressive myopathy with extremely high CK levels, that persisted beyond clinical improvement, with therapeutic implications. Recent studies have shown that increased CK levels do not always correspond to clinical aggravation in anti-HMGCR myopathy, making the decision of whenever to change or escalate therapy challenging. Disclosure of Interest : None declared
Mandibuloacral dysplasia Type A (MAD A; OMIM 248370), is a form of progeroid laminopathy syndrome. It is characterized by growth retardation, craniofacial anomalies, lipodystrophy with progressive osteoporosis and localised osteolysis. Pigmentary skin changes in addition to metabolic dysfunction are the other complications noted. A homozygous or compound heterozygous mutation in the gene encoding lamin A/C (LMNA) or ZMPSTE24 (FACE1) gene, causes type A or type B MAD, respectively. Juvenile dermatomyositis (JDM) is an autoimmune condition characterized by muscle weakness and skin changes. At least 10% of these patients have acquired lipodystrophy. Objectives: Laminopathies like MADA with a phenotype of generalised lipodystrophy with pigmentary changes can mimic JDM. We report a case with a novel mutation in the LMNA gene mimicking JDM. Methods: A 3-year-old Indian girl, born of 3 rd degree consanguinity, was referred to our rheumatology clinic as a suspected JDM. She had failure to thrive [weight: 8.6kg (<1 st centile); height 82.5cm (3 rd - 50 th centile)], pigmentary skin changes and proximal muscle weakness. From two years of age, there was history of progressive generalised skin hyperpigmentation with multiple hyperkeratotic flat maculopapular lesions over of the bony prominences in both upper and lower limbs. Hypopigmented scaly lesions on the buttocks, subtle heliotrope rash around the eyes and sparing of the oral mucosa and the arm pits with no signs of erythema or pruritus were noted. Craniofacial dysmorphism in the form of mandibular dysplasia with facies looking advanced for age were present. She had prominent eyes, a thin nose, crowded teeth, sparse hair on the scalp, with hypotrichosis over the extremities. The nails appeared dystrophic and the terminal phalanges were small with bulbous ends. On musculoskeletal examination, the muscle bulk and tone appeared normal. There was diffuse muscle weakness in the lower limbs with difficulty in rising from sitting position and generalised loss of subcutaneous adipose tissue of the with acral lipodystrophy. Nervous system examination was normal and development milestones were appropriate for age. Results: Lab investigations revealed normal hemogram and sedimentation rate (10mm/hr), and deranged muscle enzymes with creatinine phosphokinase 867U/L, lactate dehydrogenase 405U/L, aspartate aminotransferase 62U/L and alanine aminotransferase 64U/L. Antinuclear antibody (by immunofluorescence) and myositis antibody profile were negative. Lipid profile and HbA1C were deranged (Cholesterol 170mg/dL, HDL 51 mg/dL, LDL 85 mg/dL, VLDL 33.6 mg/dL, Triglycerides 168 mg/dL, HbA1C 6.4). MRI quadriceps was normal while roentgenogram showed acro-osteolysis of distal phalynges with wormian bones in the lambdoid sutures and mandibular hypoplasia. There was no cardiomyopathy on echocardiography. A provisional diagnosis of atypical JDM was made. But while initiating steroids and methotrexate we were mindful of the atypical features. Suspecting a JDM mimic such as cwhole exome sequencing was done. It showed a homozygous missense mutation in exon 9 of the LMNA gene (c.1583C>T) (p.Thr528Met) on chromosome 1q22 with sanger sequencing confirming both parents to be carriers. Immunomodulatory therapy was withdrawn, and the family counselled. Conclusion: On reverse phenotype match, our case had the classic features of MAD. However, the raised muscle enzymes with proximal muscle weakness, and skin lesions could mimic JDM leading to an unwarranted initiation of immunomodulatory therapy. Therefore genetic testing and counselling acquires relevance in establishing accurate diagnosis and preventing mishaps in subsequent pregnancM. G. L. Wilkinson 1 , C. Papadopoulou 1 , S. R. Veldkamp 2 , R. Campanilho-Marques 3 , S. Röstlund 4 , M. Nørgaard 5 , J. H. K. Swan 6 , O. Kul Cinar 7 , H. Sanner 8 , L. J. McCann 9 1 UCL Great Ormond Street Institute of Child Health, London, United Kingdom, 2 Center for Translational Immunology, University Medical Center Utrecht, Utrecht, Netherlands, 3 Pediatric Rheumatology Unit -Lisbon, Portugal, 4 Women´s Health and Allied Health Professionals Theme, Medical Unit Occupational therapy and Physiotherapy, Karolinska University Hospital, Solna, Sweden, 5 Department of Physiotherapy (Pediatrics), Aarhus University Hospital, Aarhus, Denmark, 6 Parent and patient representatives, Dundee, 7 UCL Great Ormond Street Institute of Child Health, London, United Kingdom, 8 Department of Rheumatology, Oslo University Hospital , Oslo, Norway, 9 Alderhey Children’s NHS Foundation Trust, Liverpool, United Kingdom Correspondence: L. J. McCann Introduction: The PReS Juvenile Dermatomyositis (JDM) working group (WG) brings together clinicians and researchers to improve knowledge, promote good practice and facilitate research in JDM. Objectives: To describe the workings of the group from September 2021-September 2022. Methods: The PReS JDM working party has 241 active members. A core group of elected / co-opted representatives meet 6x year. Meetings for all members are held 3x year; including one during the PReS annual conference. Results: Since the PReS conference 2021, work has been on-going in line with PReS pillars (Table 1). ACROSS ALL PILLARS: INPUT FROM ALLIED HEALTH PROFESSIONAL (AHP) & PARENT/PATIENT REPRESENTATIVES. Conclusion: The PReS JDM WP has been active in collaborative projects to enhance clinical care, translational research and education / training on an international platform. The working group benefits from inclusion of co-opted patient/parent and Allied Health Professional (AHP) representatives to ensure a holistic ethos to improve outcomes for children and young people with JDMMclellan, M. Al-Obaidi, S. Compeyrot-Lacassagne, C. Pilkington, E. Moraitis, C. Papadopoulou 1 Paediatric Rheumatology, Great Ormond Street Hospital, London, London, United Kingdom Correspondence: K. Mclellan Introduction: MDA5-positive juvenile dermatomyositis (JDM) represents a distinct clinical phenotype associated with skin and oral ulceration, milder muscle involvement and a higher incidence of interstitial lung disease (ILD) and severe rapidly-progressive ILD (RP-ILD). Objectives: To describe the presenting clinical characteristics of patients with MDA5-positive JDM, the incidence of ILD and risk factors associated with development of RP-ILD. Methods: Retrospective clinical notes review of patients with MDA5-positive JDM managed at Great Ormond Street Hospital (GOSH) over a 24-year period. Results: Demographics: Twenty-five patients were managed at our centre with MDA5-positive JDM. One additional patient was excluded as no notes were available. Sixty-four % were females. In terms of ethnicity, 60% White British, 20% Black African, 8% Mixed, 8% Asian, 8% European and 4% South Asian. Median age of presentation was 10.0 years (IQR 6.9, 12.3). Diagnostic delay was significant amongst this cohort with a median 14 weeks (8, 26) between initial symptoms and diagnosis. Twenty-four% of this cohort were also Ro-52 positive. Presentation: All patients had skin involvement; 52% had ulcerating skin disease, 64% had heliotrope rash and 100% had Gottrons papules on initial presentation. Forty-four % had peri-ungal changes, 35% had digital pitting or infarcts and 64% had nailfold changes. Forty % had peri-orbital oedema. Muscle involvement was present in all, with median CMAS 40/52 (36,44) and MMT8 57/80 (51,62). Eighty-eight % had arthritis, of whom an initial active joint count was available for 16 patients with a median of 5.5 joints (3,16). Forty % had gastrointestinal symptoms; fever, weight loss and mouth ulcers were present in 52%, 52% and 60% respectively. Twenty-four % had respiratory symptoms at diagnosis. Interstitial lung disease: Fifty-two % were diagnosed with ILD and a further 20% had abnormal pulmonary CT scans not thought to be diagnostic of ILD. 13% (2 patients) had RP-ILD; both of whom died despite immunosuppression and extracorporeal membrane oxygenation. There were no other deaths amongst this cohort. No respiratory involvement was identified in 28%. Of 19 patients who had a CT chest at diagnosis, 84% were abnormal. Only 1 patient developed respiratory involvement during the disease course whilst on treatment; the other 93% with ILD had evidence of ILD at diagnosis. Four risk factors were found to be associated with development of RP-ILD which reached statistical significance, a further 3 factors were significantly protective, as is shown in Table 1, out of 58 putative variables. 2 patients had pneumocystis pneumonia (PCP) , both of whom required admission to PICU and one associated with death. Table 1 shows risk factors at presentation which were found to be significantly associated with RP-ILD Conclusion: Skin and muscle involvement were identified in all patients, with the majority also presenting with ulcerating skin disease, oral ulceration and arthritis. The majority of this cohort had lung involvement and 2 patients died of RP-ILD. Four risk factors were found to be predictive of RP-ILD and a further 3 protective factors were identified. Rapid deterioration of respiratory symptoms was associated with PCP, while ILD was unlikely to develop whilst on treatment if not present at diagnosis. These findings need to be validated in a larger cohort of patients. Disclosure of Interest : None declared
P. L. Nadig, P. Vignesh, S. Basu, R. Tyagi, M. Sudhakar, S. Sharma, M. Dhaliwal, A. Rawat, S. Singh ediatrics, Post Graduate Institute of Medical Education and Research, Chandigarh, Chandigarh, India Correspondence: P. L. Nadig Introduction: With the recognition of myositis specific autoantibodies, distinct clinical phenotypes of juvenile dermatomyositis (JDM) now been identified. Anti–MDA-5 autoantibody associated juvenile dermatomyositis is associated with clinically amyopathic form of disease and rapidly progressive interstitial lung disease (ILD). However, ethnic variations in disease manifestations have been noted. Objectives: We describe a pediatric cohort of anti-MDA5 associated JDM from a tertiary care centre in North India. Methods: We retrieved the medical records of Pediatric Rheumatology Clinic from January 1992 to April 2022 and did a retrospective analysis of 8 children with anti-MDA-5 antibody positive JDM. Clinical features including age at presentation, cutaneous manifestations, muscle weakness, presence of ILD, and other manifestations were noted. Myositis associated autoantibodies were assay: Of 53 children with JDMS who underwent testing for MSA, 8 had positivity for anti-MDA5 (15%). The latter were analyzed in detail. Mean age at onset of clinical manifestations was 8.4 years and mean age at diagnosis was 9.6 years. F:M ratio was 1:3. Mean follow-up period was 16.5 months. Cutaneous manifestations included photosensitivity (n=8), malar rash (n=8), shawl sign (n=2), heliotrope rash (n=7), and Gottron papules (n=8), inverse Gottron papules (n= 5) and skin ulceration ( n=5). Muscle weakness was observed in 5 patients - 3 had severe truncal weakness and 2 had pharyngeal involvement. Arthralgia and arthritis were present in 4 and 5 patients respectively with involvement of both large and small joints. Two had Raynaud’s phenomena, and 1 had generalized adenopathy. ANA positivity was noted in 3 patients, 1 amongst these had anti-dsDNA positivity. Anti-Ro 52 positivity was noted in 2 patients. Of 6 patients who underwent HRCT chest, 3 patients had ILD; however, none amongst the latter had progressive disease on follow up. Treatment included oral corticosteroids (n=8), methotrexate (n=8), cyclophosphamide (n=2) and mycofenolate mofetil (n=2). Remission was seen in 5 out of 8 patients within a mean duration of 6 months after initiation of treatment. Three patients had relapses. Conclusion: We report 3 significant findings from our cohort of anti-MDA5 JDM: Rapidly progressive ILD as commonly noted with adult-onset anti-MDA5 associated JDM in South East Asian population, was not seen. Involvement of large joints (arthralgia/ arthritis) was a common finding Severe truncal weakness and pharyngeal involvement was noted Patient Consent: Yes, I received consent Disclosure of Interest : None declared
N. Okamoto 1,2 , Y. Sugita 1 , Y. Ozeki 1 , K. Shabana 1 , A. Ashida 1 1 Department of Pediatrics, School of Medicine, Osaka Medical and Pharmaceutical University, Takatsuki-City, 2 Department of Pediatrics, Osaka Rosai Hospital, Sakai-City, Japan Correspondence: N. Okamoto Introduction: We report the case of intractable Immune-mediated necrotizing myopathy treated with multi-targeted therapy. Objectives: Immune-mediated necrotizing myopathy (IMNM) is first defined in 2003 by European Neuromuscular Center classification and is characterized with significant muscle weakness, muscle atrophy and hyper serum creatine kinase (CPK). Invasion of inflammatory cell in muscle tissue is modest and patients are often misdiagnosed as muscular dystrophy. The specific autoantibodies, anti-single recognition particle antibody and anti-3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) antibody, are identified. The continuous treatment including glucocorticoid, immunosuppressant, immunoglobulin and rituximab are necessary to control disease activity and prevent the deterioration of muscle atrophy. Methods: A-5-year-old girl visited former hospital complaining acute muscle weakness and significant hyper serum CPK (35000 U/L) was revealed. She was transferred to our hospital and was diagnosed as IMNM by muscle biopsy and positivity of anti-HMGCR antibody (2.7 IU/ml). Blood test results; WBC 6210/μl, CPK 24635 U/L, aldorase 362U/L, AST 770 U/L, ALT 1199 U/L, LD 3491 U/L, ferritin 57.9ng/mL. Induction therapy was performed with methyl-prednisolone pulse therapy (30mg/kg/day, 3days in a week) followed by oral prednisolone and oral methotrexate 5mg/W (8mg/m 2 ). CPK decreased to 3000-4000 U/L and muscle weakness improved, however the Gowers’s phenomenon was positive and she needed help for daily activity. Additional single intravenous immunoglobulin therapy (IVIG, 500mg/kg/day, 5 days) was effective for activity improvement and CPK decrease that was still high up to 900 U/L. Monthly intravenous cyclophosphamide (IVCYC 500mg/m 2 /day, 1 day/month) and mycophenolate mofetil (MMF 600mg/m 2 ) were started due to disease activity and intolerance to methotrexate, however she complaint muscle weakness and CPK increased to 5000 U/L again. Instead of IVCYC, tacrolimus (TAC) 0.5mg/day was added and IVIG every 3 months was started, then CPK decreased to 1500-3000 U/L. Because muscle weakness appeared 1 month before each IVIG, rituximab (RTX) was performed. Since then, her disease is well controlled. Results: Conclusion: Multi-targeted therapy including MMF, TAC, IVIG and RTX was effective for intractable pediatric case of IMNM with anti-HMGCR antibody positivity. The written informed consent for the publication was obtained from her mother. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
V. Pandiarajan 1 , A. Rawat 1 , P. Nadig 1 , R. Kumrah 1 , A. Dod 1 , R. Garg 1 , R. Pilania 1 , A. Jindal 1 , D. Suri 1 , S. Singh 1 1 Allergy Immunology Unit,V. Pandiarajan Introduction: Distinct clinical phenotypes of juvenile idiopathic inflammatory myositis (JIIM) have been identified based on the myositis specific autoantibodies (MSAs). However, the spectrum of MSAs and clinical phenotypes within the particular category of MSA can vary amongst different population groups. Objectives: To analyse the spectrum of MSAs in children with JIIM and compare the clinical phenotypes in a cohort of JIIM from North India. Methods: We retrieved the records of 53 children with JIIM who underwent testing for MSA and had been followed up in the Pediatric Rheumatology Clinic, Advanced Pediatric Centre, Post Graduate Institute of Medical Education and Research, Chandigarh, India during the period January 2019- April 2022. Laboratory assay for MSAs was performed using a 16-antigen kit immunodot assay (Euroline Autoimmune Inflammatory Myopathies 16 Ag, Euroimmun, Lübeck, Germany). Results that were 2+ or more were considered positivity for MSA. Results: Of the 53 children with JIIM who underwent testing for MSA, 41 (77.3%) had positivity for MSA. Positivity for anti-TIF-gamma, anti-NXP2, anti-MDA5, and anti-SAE were noted in 5 (9.4%), 13 (24.5%), 8 (15%), and 4 (7.5%) children, respectively. Anti-synthetase syndrome with anti-Jo positivity was noted in 1 child. Positivity for anti-PM-Scl was noted in 5 children. Anti-SRP positivity was noted in a child with polymyositis. Of the anti-NXP2 positivity group, 2 had calcinosis-predominant presentation of juvenile dermatomyositis (JDM) with minimal muscle weakness and complete paucity of other cutaneous features of JDM. Amongst the MDA5 positivity group, clinically amyopathic form of JDM and rapidly progressive interstitial lung disease (ILD) was not seen. However, involvement of large joints, severe truncal weakness and pharyngeal involvement were noted in majority of them. Cutaneous relapses were commonly noted in the anti-TIF-gamma positivity group. Of the 4 children with anti-SAE positivity, only 1 had clinically amyopathic form of disease. Conclusion: Anti-NXP2 is the most common MSA noted in our cohort of JIIM from North India, followed by anti-MDA5, anti-PM-Scl, and anti-TIF-gamma. Anti-MDA5 positivity in our cohort is not associated with clinically amyopathic JDM or rapidly progressive ILD. Anti-SAE positivity is noted at a higher proportion (7.5%) in our cohort compared to studies reported from other regions. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
S. Rosina 1 , A. Consolaro 1,2 , A. Pistorio 3 , A. Rebollo-Giménez 1 , N. Ruperto 1 , A. Ravelli 3 1 UOC Clinica Pediatrica e Reumatologia, IRCCS Istituto Giannina Gaslini, 2 Dipartimento di Neuroscienze Riabilitazione Oftalmologia Genetica e Scienze Materno-Infantili, Università degli studi di Genova, 3 Direzione Scientifica, IRCCS Istituto Giannina Gaslini, Genova, Italy Correspondence: S. Rosina Introduction: The Juvenile DermatoMyositis Activity Index (JDMAI) is the first composite disease activity score validated for the measurement of muscle and skin involvement in juvenile dermatomyositis (JDM). Six preliminary versions (JDMAI1 to 6) have been proposed. Both JDMAI1 and JDMAI2 include the physician’s global assessment of overall disease activity (PhGA) on a visual analogue scale (VAS) (where 0 = no activity and 10 = maximum activity), the parent’s global assessment of child’s overall wellbeing (PaGA) on a VAS (where 0 = best and 10 = worst), and the hybrid MMT8/CMAS (hMC) expressed in deciles (0 = best to 10 = worst). To estimate the activity of skin disease, the JDMAI1 includes the skin activity VAS (where 0 = no activity and 10 = maximum activity), whereas the JDMAI2 includes the skin component of the Disease Activity Score (DAS) (score range 0 = no activity to 9 = maximum activity). The theoretical range is, therefore, 0 to 40 for the JDMAI1 and 0 to 39 for the JDMAI2. Objectives: Cutoffs for the states of inactive disease (ID), low disease activity (LDA), moderate disease activity (MDA) and high disease activity (HDA) are necessary to interpret the JDMAI scores. The aim of the study was to develop and validate these cutoffs for JDMAI1 and JDMAI2. Methods: The cutoffs definition cohort was composed of 129 patients included in the PRINTO JDM trial and evaluated at 6 months from baseline. Optimal cutoff values were determined against external criteria by calculating the 10 th and 25 th percentile (for ID), the 30 th and 40 th percentile (for LDA), and the 75 th and 90 th percentile (for HDA) of cumulative score distribution and through receiver operating characteristic curve analysis. External criteria included the modified PRINTO criteria for clinically inactive disease (for ID) and the PRINTO/ACR/EULAR level of improvement (for LDA and HDA). MDA cutoffs were defined as the score interval between LDA and HDA cutoffs. The choice of final cutoffs was based on clinical and statistical grounds. The validation cohort included 213 patients followed up in standard clinical care at 13 international paediatric rheumatology centres, and 275 patients enrolled in a study aimed to validate prospectively the provisional PRINTO/ACR/EULAR disease activity core set for the evaluation of response to therapy in JDM. Cutoff validation was conducted by assessing discriminative ability. Results: The selected JDMAI1 cutoffs were <=2.4 for ID, <=6.6 for LDA, 6.7-11 for MDA, and >11 for HDA. The selected JDMAI2 cutoffs were <=5.2 for ID, <=8.5 for LDA, 8.6-11.3 for MDA, and >11.3 for HDA. In cross-validation analyses, the cutoffs showed strong ability to discriminate among disease activity states defined subjectively by physicians and parents, parents’ satisfaction/dissatisfaction with illness outcome, levels of child’s pain and fatigue, and presence/absence of functional impairment and disease damage. Conclusion: Cutoff values for classifying various disease activity states in JDM using the JDMAI1 and JDMAI2 were developed. The cutoffs revealed good metrologic properties in both definition and validation samples, and are therefore suitable for application in clinical practice and researchssano, F. Baldo, F. Chironi, G. B. Beretta, G. Rogani, I. Borzani, F. S. Minoia, A. M. Cappellari, G. Filocamo Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy Correspondence: M. Rossano Introduction: Juvenile dermatomyositis (JDM) is a multisystem inflammatory disease involving primarily the skin and proximal muscles. One of the main challenges in the clinical management of JDM is accurate assessing of disease activity to optimize treatment and reduce disease morbidity. Objectives: The aim of this preliminary study is to understand the role in assessing disease activity of a validated electromyography (EMG) scoring protocol and a whole body MRI score (WB-MRI), by comparison with the clinical standardized evaluation as gold standard. Methods: A total of 17 patients with JDM seen at our hospital between Oct. 2018 and Dec. 2021 were enrolled. Each patient underwent concomitantly clinical evaluation based on hybrid MMT/CMAS (hMC) score, laboratory exams including creatinine kinase (CK), aspartate aminotransferase (AST) and lactic dehydrogenase (LDH) levels, EMG and WB-MRI. For the purpose of the analysis the EMG score was calculated as sum of two subsets: acute denervation signs (fibrillation potentials) with score ranging from 0 (maximal inflammation) to 8 (no inflammation) and reinnervation signs (motor unit remodeling) with a score ranging from 0 (no reinnervation) to 8 (maximal reinnervation). WB-MRI was scored in 42 muscular groups using a 0-2 point scale for each group; myofascial and subcutaneous tissue inflammation were assessed on the upper and lower extremities using a 0-1 point scale. A muscular visual analog scale (VAS) was defined by an expert rheumatologist: the disease was considered clinically inactive in case of VAS ≤ 0,5. Due to the choice to focus our study mainly on muscular involvement, our definition of inactive disease did not take into account PRINTO definition which includes also skin involvement. Correlations were assessed by Spearman’s rank order correlation coefficient (rs). Comparison of quantitative variables in the analysis of discriminant validity was made by Mann–Whitney U Test. Cohen’s kappa was used to evaluate the agreement between physician muscular VAS and EMG and MRI scores. Results: Seventeen patients (82.3% female) were enrolled for a total of 27 examinations considered in the study (7 at disease onset, and 20 follow-up visits). The median age at JDM diagnosis was 6.59 years (IQR 3.49-10.1), and the median duration of follow-up was 3.56 years (IQR 1.43-6.89). In 15 patients JDM satisfied Bohan and Peter’s criteria, while 2 patients were classified as amyopathic JDM. The hMC revealed a high correlation with the EMG score (rs=0.63), compared to the moderate correlation observed with MRI muscle score (rs =-0.47) and MRI myofascial score (rs =-0.43), and to the poor correlation with MRI subcutaneous score (rs =-0.11). The EMG score, and its single subsets (fibrillar potentials and remodeling), as well as the MRI muscle score differed significantly between patients with active or inactive disease according to the muscular VAS (p= 0.015, p=0.001, p=0.015, p = 0.004), proving a good discriminative ability. Overall agreement was substantial between EMG fibrillar score and muscular VAS (k= 0.701), and moderate between MRI muscle score and muscular VAS (k= 0.482). Conclusion: This preliminary data suggest a good association between the EMG score and clinical evaluation of muscular disease activity, endorsing the role of EMG in the management of JDM. Although at present standardized clinical evaluation remains the gold standard, EMG and WB-MRI were proved to have a fair discriminative power compared to clinical assessment of disease activity. Further prospective analysis on larger samples may contribute to better define the role of both EMG and WB-MRI in evaluating muscular disease activSener 1 , O. Basaran 1 , E. D. Batu 1 , E. Sag 1 , S. Oz 2 , S. Sari 2 , Y. Bilginer 1 , G. Haliloglu 2 , S. Ozen 1 1 Pediatric Rheumatology, 2 Pediatric Neurology, Hacettepe University Faculty of Medicine, Ankara, Turkey Correspondence: S. Sener Introduction: Juvenile dermatomyositis (JDM), the most common idiopathic inflammatory myopathy of childhood, is quite heterogeneous in terms of disease course. Objectives: The aim of our study was twofold: To evaluate the presentation, diagnosis, clinical course and management of JDM in children less than three years of age and to compare with the late-onset patients, and to review the literature for early-onset JDM. Methods: Nine patients with the early-onset disease and 63 patients with the late-onset disease with JDM followed between December 2010 and April 2022 were included in the study. We also reviewed the literature in terms of early-onset JDM from the inceptions of the PubMed/MEDLINE and Scopus databases up to April 1 st , 2022. Results: The median delay time between the symptom onset and diagnosis of the early- and late-onset JDM patients was 5.5 (2-31) months and 2.1 (0.2-25), respectively (p=0.05). While calcinosis was seen at a higher rate in the early-onset group (p=0.060), skin ulcers were also more common in this group, although not statistically significant (22.2% versus 3.1%, p=0.074). There was no difference in median serum creatinine kinase (CK) levels between the two groups (p=0.940). Anti-NXP2 was more common in the early-onset group (p=0.049). Muscle biopsy was frequently preferred in the diagnosis of early-onset JDM patients (77.7% versus 50.8%). In the treatment, corticosteroid and methotrexate (MTX) were frequently used in patients in both groups (p=1.000). In the early-onset group, intravenous immunoglobulin (p=0.001), cyclophosphamide (p=0.011) and biological agents (p=0.016) were used more frequently as a second and third-line therapy. Although there was no difference between the partial and complete remission rates, the relapse rate was significantly higher in the early-onset group (p=0.001). There was no mortality in either group during the follow-up. Of note, our early-onset JDM patients visited approximately 3 centers before the definite diagnosis of JDM. More than half of these patients had further investigations to rule out genetic, metabolic, and infectious myopathies. Our literature search revealed 32 articles reporting 75 patients with JDM who were younger than 3 years of age. The median diagnostic time window was 5 (1-30) months. Skin ulcers were seen in 12.6% of patients, while calcinosis was present in 29.5%. Twenty-three of the 44 patients (52.3%) had a muscle biopsy for diagnostic purposes. Corticosteroids (98.7%) and MTX (91.1%) were main agents used in the treatment. Forty-one patients (64.1%) received treatment other than MTX/corticosteroids. Complete remission was achieved in almost half of these patients (48.9%), but relapse was observed in 75%. The mortality rate was 10.2%. Conclusion: Diagnosis can be challenging and delayed in early-onset JDM patients. Furthermore, compared to later-onset JDM patients, this group had a higher relapse rate with a considerable requirement for intensive immunosuppressive treatment. Increasing the knowledge about clinical phenotype of early-onset JDM is crucial for timely introduction of disease-modifying treatmS. Sener 1 , E. D. Batu 1 , M. Kasap Cuceoglu 1 , Z. Balik 1 , E. Aliyev 1 , Y. Bayindir 1 , O. Basaran 1 , Z. Saribas 2 , Y. Bilginer 1 , S. Ozen 1 , B. Sener 2 1 Pediatric Rheumatology, 2 Medical Microbiology, Hacettepe University, ANKARA, Turkey Correspondence: M. Kasap Cuceoglu Introduction: Myositis-specific and myositis-associated antibodies (MSA and MAA) are highly specific for classifying patients with juvenile dermatomyositis (JDM) according to their different features. Objectives: We investigated the reflection of MSA and MAA on clinical features and their relationship with antinuclear antibody (ANA) patterns in JDM patients. Methods: We examined the presence of MSA/MAA and ANA by EUROLINE Autoimmune Inflammatory Myopathies Profile and EUROPLUS ANA Mosaics (EUROIMMUN, Germany) in 70 JDM patients, followed between January 2014 and March 2022. Results: MSA and MAA were positive in 41 JDM patients (58.5%) (Table 1). Muscle involvement and elevated CK levels were more frequent among patients with anti-NXP2, anti-TIF-1γ, anti-Mi-2 and anti-Ku antibodies (85.9%). Although skin involvement was prevalent in all patients, the frequency of calcinosis was highest in patients with anti-TIF-1γ antibodies (50%), and lung involvement was more common in patients with anti-MDA5 antibodies (40%). Calcinosis was more frequent among patients with positive myositis autoantibodies compared to the ones negative for MSA/MSA (p=0.016). ANA was positive in 34 JDM patients (48.5%). The most common ANA pattern was anti-cell (AC) 4 and 5. While AC4 pattern was present in all patients who were positive for anti-Mi-2, anti-Ku, anti-Pm-Scl75, or anti-Pm-Scl100 antibodies; AC5 pattern was detected in patients with anti-Mi-2, anti-Ku, or anti-Pm-Scl75 antibodies. Conclusion: In our JDM cohort, anti-MDA5 autoantibody was associated with pulmonary involvement, while calcinosis was most frequent among patients with anti-TIF-1γ autoantibody. Analyzing the association of MSA/MAA with JDM clinical phenotype could pave the way for using personalized medicine strategies in the management of these patients. Trial registration identifying number: not applicable Patient Consent: Yes, I received consent Disclosure of Interest : None declared
P. Vignesh 2 , M. Arora 2 , S. K. Loganathan 2 , M. Dhaliwal 2 , S. Sharma 2 , A. Rawat 1 , S. Singh 1 1 Pediatric, PGIMER, 2 pediatricsInterstitial lung disease (ILD) is one of the important complications of Juvenile dermatomyositis (JDMS) and is seen in ~20% of patients with (JDMS). ILD is predominantly associated with specific myositis antibodies like anti-aminoacyl-tRNA synthetase antibody, anti-MDA5, anti-Ku, anti-PM-Scl, anti-SRP, and anti-Mi2. We herein describe our experience of patients with JDMS with ILD from North India. Objectives: To analyse the clinical, immunological profile, treatment protocols and outcomes of children with JDMS and ILD. Methods: We analysed case records of all children diagnosed to have JDMS in the Pediatric Rheumatology Clinic during the period January 1992 to April 2022. The diagnosis of JDMS was based on modified Bohan and Peter criteria. Results: Of the 140 patients with JDMS, 16 (11.4%) had features of ILD. Male to female ratio was 1:1.2. Mean age at disease onset was 9.6 years (range 4- 14 years). Clinical features included fever in 8 (50%), heliotrope rash in 8 (50%), Gottron papules in 13 (81.2%), muscle weakness in 15 (93%) and arthritis in 7 (43.75%) patients. One patient had a clinically amyopathic form and this was associated with anti-Ku, anti-PM-Scl, and anti-Ro 52 antibodies. Additional features of scleroderma overlap were seen in 4 patients. Nail fold capillaroscopy was carried out in 7 patients and all showed abnormalities (i.e., capillary loss, dilated tortuous loops, micro bleeding). Of the 16 patients with ILD, myositis associated antibodies were tested in 8 patients , 3 amongst those had anti- MDA5 antibodies. One patient had anti-Jo 1 and anti-Ro 52. Anti-PM-Scl was found in 1 patient who was also positive for anti-Ku and anti-Ro 52. Anti-SAE was positive in 1 patient. Nine patients had ANA positivity (56%). High-resolution computed tomography (HRCT) was carried out in 11 patients. Predominant findings included ground-glass opacities (n=6), septal and intra-lobar thickening (n=3), reticular pattern (n=4), atelectasis (n=2), consolidations (n=2), and honeycombing (n=2). Lower lobe involvement was seen in 7 patients. Pulmonary function tests were carried out in 7 patients and all of them showed restrictive pattern. Pulmonary artery hypertension on 2D Echocardiography was noted in 4 patients. All patients received oral steroids and methotrexate. In addition, 12 patients received intravenous pulse methyl prednisolone (followed by tapering); 5 patients received monthly intravenous pulse cyclophosphamide for 6 months followed by maintenance azathioprine in 4; one patient required rituximab but continued to have progressive respiratory distress syndrome and succumbed. On follow-up, 12 patients were stable with immunosuppressants and had no worsening in ILD; 4 patients were lost to follow up. Conclusion: In our cohort, we noted that ILD was present in 11.4% of patients with JDMS. Children with JDMS-ILD need long term follow-up, however, the prognosis remains guarded. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
P282. The first case of Systemic Lupus Erythematosus (SLE) with Chronic Inflammatory Demyelinating Polyneuropathy (CIDP): in a libyan female child P283. A different perspective on the interpretation of neurocognitive functions in childhood-onset systemic lupus erythematosus (psle) patients without neuropsychiatric involvement: Functional Magnetic Resonance Imaging (FMRI) P285. Intima-media thickness and disease activity over time in childhood systemic lupus erythematosus: preliminary results from a perspective study P286. Pediatric neuropsychiatric systemic lupus erythematosus: case series from a tertiary care center in Eastern India P287. A child with chronic headache, fever and diffuse meningeal enhancement P288. Clinico pathological profile of pediatric lupus nephritis : ICH Kolkata experience P289. Monogenic systemic lupus erythematosus (SLE) in Northern Israel P290. Diet, gut microbiota, and intestinal permeability in systemic lupus erythematosus P291. Primary antiphospholipid syndrome – clinical review of three pediatric patients P292. High expression of spata5l1 protein in lupus nephritis P293. Potential role for oxysterol receptor gpr183 in b cell-driven pathology in juvenile-onset systemic lupus erythematosus P294. Pulmonary involvement in juvenile-onset systemic lupus erythematosus: data from the Uk JSLE cohort study P295. Pediatric systemic lupus: the experience of the french overseas departments of America P296. Instrument for nursing consultation for patients with juvenile systemic lupus erythematosus 97. Lupus manifestations in first degree consanguineous relatives of a single family in Nicaragua P298. Safety and efficacy of rituximab in pediatric lupus nephritis: retrospective cohort data of 25 patients P299. Turkish translation and adaptation of the pediatric automated neuropsychological assessment metrics (pedanam) for childhood-onset systemic lupus erythematosus: initial findings P300. Vitamin D levels in patients with juvenile systemic lupus erythematosus 301. Creation of a core dataset for international registry-based research on childhood-onset systemic lupus erythematosus P302. Myocarditis as an initial manifestation of pediatric systemic lupus erythematosus and associated with SARS COV-2 P303. Mofetil mycophenolate versus cyclophosphamide in juvenile systemic lupus erythematous: a long-term follow-up study P304. Hematological manifestations in the presence of antiphospholipid antibodies in a pediatric cohort 305. Different patterns of longitudinal changes in antinuclear antibodies titres in children with systemic lupus erythematosus P306. Multi-parametric interrogation of the Systemic Lupus Erythematosus (SLE) immunome reveals multiple derangements P307. Eosinophilic fasciitis associated with inaugural juvenile systemic lupus erythematosus P308. Levamisole and autoimmunity- wondering about wonder drugs P309. Reduced CD8+ T Cell and CD56+ natural killer cell cytotoxic capacity in patients with juvenile systemic lupus erythematosus 10. Lupus nephritis - study from a single centre in South India P311. Prevalence and predictors of damage in childhood-onset systemic lupus erythematosus: preliminary data from an international cohort of 1096 patients P312. Results of whole exome sequencing in a large cohort of patients with childhood-onset systemic lupus erythematosus - multiple case study P313. Clinical and epidemiological analysis of systemic lupus erytheis according to the moscow register of children with rheumatic diseases P314. Clinical profile of pediatric systemic lupus erythematosus from a hilly state in north-india: with an uncommon complication P315. Defining childhood-onset systemic lupus erythematosus, lupus nephritis, and end-stage kidney disease within a us administrative claims database P316. Association of sociodemographic factors with differential diagnostic coding patterns in childhood-onset lupus nephritisP317. Anti-SARS-COV2 vaccination in juvenile systemic lupus erythematosus: tolerability and impact on disease activity 8. Epstein–BARR virus encephalitis-associated hemophagocytic lymphohistiocytosis in a patient with childhood-onset systemic lupus erythematosus: a case report P319. Juvenile systemic lupus erythematosus manifesting as isolated hemiparesis in a seven year old girl P320. Distinct biological phenotypes stratify patients in groups with similar disease activity states; a multi-omic approach in childhood-onset SLE P321. DDX58 and MX1 gene expression discriminates jsle from healthy children and children with other inflammatory or infectious diseases 2 , A. Ravelli 3 1 Paediatric Rheumatology , Faculty of Medicine, Tripoli university, Tripoli Children’s Hospital , 2 Paediatric Rheumatology, Faculty of Medicine, Tripoli university , Tripoli Children’s hospital , Tripoli , Libya, 3 Head division of Paediatric Rheumatology, Giannina Gaslini Institute, Genova , Italy Correspondence: A. A. Abushhaiwia Introduction: chronic inflammatory demyelinating polyneuropathy (CIDP) is an uncommon manifestation of systemic lupus erythematosus (SLE). CIDP is characterized by muscular weakness with or without sensory loss in the extremities and can have a chronic progressive course with remission and repeated relapses. To our knowledge, few cases of CIDP and SLE have been previously reported .We herein report first Libyan female a child with JSLE complicated withwith Chronic demyelinating sensori-motor polyradioculoneuropathy (CIDP). Objectives: We report a case of SLE presenting as CIDP and discuss the diagnosis, management, and prognosis of CIDP. Methods: A case report Results: A 15th year-old Arab Libyan female child born from non-consanguineous healthy parents. There was no family history of neither autoimmune nor metabolic diseases. She developed bilateral LL muscular weakness with decreased deep tendon reflexes. At that point, the patient experienced paresthesia on both feet and sever epigastric pain since 2015 that requires her multiple admissions to hospital and required to use wheel chair. Electroneuromyography was compatible with chronic demyelinating peripheral polyneuropathy, in both lower limbs, brain MRI: showed acute pansinusitis with moderate left orbital proptosis. MRI spine:was normal. Diagnosis made by Neurologists as a case of chronic demyelinating peripheral polyneuropathy, she was treated with IVIG and with IV steroids and oral steroids but the patient stopped the follow-up appointments and treatment since May 2020. She was referred to paediatric rheumatology clinic last August 2021 with of a 7 year history of progressive bilateral leg weakness, Paraparesis and lost ability to walk. Accompanied by intermittent high grade fever reached 39oC fatigue, joint pain,severe epigastric pain associated with vomiting sometimes poor appetite , lost weight, mouth ulcer and Malar rash. Physical examination showed a grading for motor strength of LL 2\5, decreased tone, absent deep reflexes of LL, intact both superficial & deep sensation. She was under weight 24 kgm (<2nd percentile) with sever muscle wasting, contractures of both elbow and knee joints. Laboratorial exams showed anemia (Hb 9.7 g/dL), leukopenia1.42X10 3 , thrombocytopenia 132X10 3 , normal complete metabolic and panel thyroid profile, ESR 85mm, Protein ratio 0.01%, panel of autoimmune serology showed ANA was positive1: 800,Anti-ds DNA antibodies 76 IU, Anti SM antibodies was positive >300 U, anti-RNP was positive >200 U , Anti-SSA-52, SSA-60 (Ro) was positive 14 U and C4 was low 0.13 g/L.Antiphospholipid Ab were negative .The diagnosis of JSLE was made at this point and treatment with CIDP, Pulse therapy with methylprednisolone, and oral prednisone 40 mg/day, IV cyclophosphamide (750 mg/m 2 /month) for 6th cycles, azathioprine, hydroxychloroquine sulfate 200 mg/day and attended weekly sessions of physical therapy. Following treatment with cyclophosphamide IV and prednisone, the patient showed marked clinical improvement and regained her ability to walk with minor assistance. Case was discussed regarding case was discussed regarding the need further cycle of cyclophosphamide during in PReS sister initiative hospital with prof. Raville who’s suggested that steroids, IVIG, plasmapheresis therapy should be considered if there is relapse or disease flare up. Conclusion: Peripheral polyneuropathy has been rarely reported in association with JSLE,Also the treatment of JSLE can be challenging .As this case report has shown, late clinical diagnosis of CIDP but it demonstrates efficacy and safety of cyclophosphamide in the JSLE with Chronic demyelinating sensorimotor polyradiculoneuropathy (CIDP)E. Aliyev 1 , E. S. Akbaş Aliyev 2 , H. Daşgın 3 , K. Karlı Oğuz 3,4 , B. Anlar 5 , M. Kasap Cüceoğlu 1 , S. Demir 6 , H. T. Çak Esen 2 , E. Çengel Kültür 2 , S. Özen 1 , Y. Bilginer 1 1 Pediatric Rheumatology, 2 Child and Adolescent Psychiatry, Hacettepe University, 3 Aysel Sabuncu Brain Research Center (UMRAM), Bilkent University National Magnetic Resonance Research Center, 4 Radiology, 5 Pediatric Neurology , Hacettepe University, Ankara, 6 Pediatric Rheumatology, Erzurum Training and Research Hospital, Erzurum, Turkey Correspondence: E. Aliyev Introduction: SLE is an autoimmune disease characterized by multi-organ involvement, including neuropsychiatric (NP) involvement. NPSLE patients are easily overlooked, so early identification of risky cases for NPSLE is essential in practice. Objectives: We aimed to show neurocognitive effects with neuroimaging methods, especially fMRI before NP findings develop in SLE. Methods: Wechsler Intelligence Scale for Children-IV (WISC-IV) and resting-state fMRI (TR=2000ms; slice thickness=3mm, 180 volume) were performed on pSLE patients included in the study and on healthy controls (HCs). Data were analyzed with the general linear model. The t-test for the global BOLD signal variation within the group was compared with the two-sample t-test for the difference and the Spearman correlation test was between the groups. Spearman correlation test was used to evaluate the correlation. The cluster size p-FDR threshold correction is p<0.05 between SLE and HCs. Neurological and psychiatric clinical evaluation was performed on all participants, and patients with clinical neurological or psychiatric symptoms were excluded. Results: The mean age of 17 SLE patients (11 girls) included in the study was 16.4±2.4, and the mean age of 8 HCs (4 girls) was 17.4±1.7. There was no significant difference between age, gender, depression, anxiety, and other psychiatric symptoms. When the two groups were compared neurocognitively, significant differences were found in the WISC-IV total score, verbal comprehension, perceptual reasoning scores, patterns with cubes, number sequence, and vocabulary subtests. BOLD signal activation on fMRI was decreased in the left pallidum, putamen, and thalamus in the SLE group compared to healthy controls (voxel p<0.001, cluster (k) p< 0.05-FDR corrected). In addition, precisely the same brain regions showed significantly higher functional signal activation in HCs than in pSLE. The right and left insular and frontal operculum cortex and putamen positively correlated with ‘WISC-IV Total Score’ in SLE patients compared to the HCs (the p-FDR corrected for right and left brain regions according are 0.0075 and 0.0496). Brain regions (right insular and frontal operculum cortex and putamen) were a positive correlation with ‘WISC-IV Working Memory Score’ in between groups (p-FDR corrected are 0.0069, Stats (t) are 6.83). Therewithal left part of the brain, particularly the frontal operculum cortex, insular cortex, central opercular cortex, and putamen showed a positive correlation with ‘WISC-IV Working Memory Score’ in SLE patients compared to the HCs (p-FDR corrected are 0.0069, Stats (t) are 6,36). Finally right side of the brain regions: temporal pole, temporal fusiform cortex, anterior division, parahippocampal gyrus, and anterior division showed a negative correlation with ‘WISC-IV Working Memory Score’ between groups (p-FDR corrected are 0.0188, Stats (t) are -5,44). Conclusion: We showed that lupus could cause neurocognitive dysfunction even before neuropsychiatric involvement in children. With the prospective follow-up of SLE patients with impaired neurocognitive functions, whether they will develop neuropsychiatric SLE can be evaluated. Those may be useful in identifying the patient population at risk for neuropsychiatric involvement of autoimmune diseases if fMRI is used routinely in the future. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
F. Baldo, M. Rossano, E. A. Conti, F. Chironi, S. Testa, F. Lucioni, A. Petaccia, G. Filocamo o, Italy Correspondence: F. Baldo Introduction: Childhood systemic lupus erythematosus has been studied as a possible cause of early atherosclerosis, since the relationship between chronic systemic inflammation, atherosclerosis and, subsequently, cardiovascular disease has been clearly demonstrated in adults. However, due to the rarity of cardiovascular events in youngster, surrogate markers of atherosclerosis are needed. Carotid intima-media thickness is a feasible, noninvasive method to assess the risk of early subclinical atherosclerosis in children with cSLE. Objectives: To assess cIMT at baseline in a cohort of patients, to investigate its possible relationship with disease-related variables, as well as to register cIMT variations with over time. Methods: Outpatient with cSLE in a single pediatric rheumatology tertiary center center were included in the study. A clinical evaluation was performed the same day of the IMT measurement. IMT of the distal 10mm of the far wall of common carotid artery was determined by ultrasonography with qIMT technology, a semi-automated method, by a highly experienced single sonographer. A second measurement was done 36 months after the first visit. Clinical information at baseline was collected from clinical records, other information was collected prospectively. Correlation between variable was assessed with Spearman’s Rho, association between variables was studied with Mann-Whitney U-test. Results: To date, 21 patients have been enrolled in the study. Clinical data are shown in table 1. At baseline, mean age was 15,8 years (SD 3,5), and mean disease duration was 3,7 years (SD 3,4) 14 patients (67%) were on mycophenolate, 16 patients (76%) on hydroxychloroquine, all were on oral steroid. Eighteen patients (86%) showed renal involvement, 3 patients (14%) CNS involvement and 8 patients (38%) were positive to antiphospholipid antibodies, 5 patients experienced macrophage activation syndrome. We found a negative, statistically significant correlation (rs = -0.49392, p (2-tailed) = 0.019) between disease duration and IMT at baseline, while no statistically significant correlation was found between IMT at baseline and SLEDAI2k, BMI or current corticosteroid dose. No clear association was found between antiphospholipid antibodies, anti ds-DNA positivity, renal involvement, hypertension or Macrophage Activation Syndrome (MAS) and higher IMT values. AT follow up, mean IMT variation was +28,9 micrometers (SD 44,9). A statistically significant correlation was observed between the variation of SLEDAI2K between baseline and follow up visit (rs = -0.62945, p (2-tailed) = 0.038. Conclusion: Our study shows that cIMT in lupus can be increased. Unexpectedly, shorter disease seems to be linked to higher IMT values. This could be due to the burden of uncontrolled inflammation at disease onset, to high-dose induction steroid therapy or both. The negative correlation between variation of SLEDAI2K at a 36 months follow up is likely due to age bias, since IMT increases with age, while higher SLEDAI2k score at onset decreased over time. Further observations in a larger cohort of patients may better explore the relevance of IMT as an early marker of cardiovascular risk in cSLE affected children and show correlation with disease related parameters. Disclosure of Interest : None declared
J. N. Bathia, P. Pal Institute of Child Health, Kolkata, India Correspondence: J. N. Bathia Introduction: Neuropsychiatric systemic lupus erythematosus (NPSLE) is a serious manifestation of childhood onset SLE. In majority of the cases the central nervous system (CNS) involvement occurs during the first year of disease onset. We present a case series of NPSLE patients encountered during last 7 years in a tertiary pediatric care center in eastern India. Objectives: To evaluate the demographic, clinical, laboratory, radiological features of patients diagnosed as NPSLE as per the ACR 1999 classification and to evaluate the treatment given and response to treatment. Methods: It is a retrospective observational study of data of patients diagnosed as having NPSLE, admitted between January 2015 to April 2022 in the Department of Pediatrics, at Institute of Child Health, Kolkata. NP-SLE was defines as per the ACR 1999 classification. CNS manifestations due to secondary causes were ruled out. Results: Fifteen (n=15) patients were found to have NPSLE. 14 were females and 1 was male. The median age at onset of disease was 10 years (quartile 1: 9.5 yrs quartile 3: 12 yrs and IQR 2.5). 12 patients had NP manifestations at diagnosis of SLE, the rest manifested at 6, 12 and 14 months from diagnosis. Neuropsychiatric manifestatons was were variable. The most common manifestation, with 9 patients each,was cognitive dysfunction, psychosis and acute confusional state, anxiety and mood disorders followed by seizures (n=7), headache (n=3), cerebro vascular disease (n=3), aseptic meningitis (n=2), limb weakness (n=2), vertigo (n=1) and bulbar palsy with ptosis (n=1). amongst three patients has headche one had isolated headache as the only manifestation. Other system involvement were renal (n=6), musculoskeletol (=5), hematological (n=4), hepatitis (n=1) and pancreatitis (n=1). Anti-nuclear antibody (ANA) positivity was seen in all patients, anti double stranded DNA (anti dsDNA) was positive in seen in 10 and 2 patients were positive for antiphospholipid antibodies. MRI brain was done in all. 4 had normal MRI brain, 6 had diffuse brain atrophy, 2 had diffuse meningeal enhancement of which 1 also had cerebral venous sinus thrombosis, 1 had acute non hemorrhagic infract with beading in bilateral middle cerebral artery, 1 had ischaemic lesions and 1 had multifocal oedema. Depending on severity immunomodulator therapy was initiated with either pulse methyl prednisolone alone (n=2), cyclophosphamide alone (n=5), rituximab alone (n3) or a combination of Rituximab and cyclophosphamide (n=5). Maintenance was done with mycophenolate mofetil or azathioprine. Two APLA positive patients also received aspirin. One APLA negative patient received aspirin due to thrombotic lesion in brain. Steroids and hydroxychloroquine sulphate was given all. 4 patients had severe disease and were comatosed. All four recovered after 6 to 8 months of therapy with no residue. No patient had any further neuropsychiatric flare and there was no mortality. Conclusion: NPSLE is a serious manifestation of childhood onset SLE. Secondary causes of CNS manifestations should be ruled out especially infection. Prompt diagnosis and aggressive immunomodulator therapy results in good responP. Khatua 2 , M. GangulyIn juvenile onset systemic lupus erythematosus (SLE) neuropsychiatric manifestations are most commonly seen. Isolated lupus headache is a known clinical feature of Neuro Psychiatric - SLE (NP-SLE) however it is more common in adults. Objectives: To present a 10 years old girl who presented with chronic headache as the initial manifestation of NP-SLE. Methods: 10 years old female presented to us with recurrent headache for last 6 months. Initially she was treated as migraine. However, the headache persisted. Thereafter, she started having intermittent episodes of fever and there was left cervical lymphadenopathy. Lymph node biopsy was done outside which was suggestive of reactive lymphocytes with epitheloid granuloma. The tuberculin sensitivity test, chest X ray were non-contributory. Antitubercular therapy was started. She then started complaining of weakness of both lower limbs and had an episode of seizure and hence admitted Results: After admission the child was started on anti convulsants. On examination ther was pallor. Other systemic examination was normal. Laboratory findings were hemoglobin 8.5 g/dL, total leukocyte count 10.0 x 10 3 /μL, neutrophils 78%, lymphocyte 18%, platelets 90 x 10 3 /μL. C reactive protein 57 mg/L,albumin 3.7 g/dL. MRI brain with contrast showed diffuse meningeal enhancement, cerebral venous sinus thrombosis in superior sagittal and right transverse sinuses with mild to moderate dilatation of the ventricles. T2W sagittal screening of dorsal spine showed ill-defined signal abnormalities. Considering an infectious etiology, CSF analysis was done which was normal. Ophthalmoscopy showed retinal haemorrhages. Following reasonable exclusion of infectious causes, detailed evaluation of an underlying autoimmune pathology was undertaken. Antinuclear antibody (ANA) was positive (1:1000, homogenous pattern) with positive anti-dsDNA. Complement levels were, C3 49.7 mg/dL, C4 6.9mg/dL. Final diagnosis of neuro psychiatric systemic lupus erythematosus (NP-SLE) was made. APLA studies were negative. The anti-tubercular therapy was stopped and patient was initiated on systemic steroids, monthly cycles of cyclophosphamide, hydroxychloroquine sulphate, and aspirin . Patient responded well to the management protocol. At present she is asymptomatic on oral corticosteroids and monthly pulse cyclophosphamide. Conclusion: Isolated Lupus Headache in not uncommon however a high index of suspicion is required for appropriate diagnosis. Our patient also had diffuse meningeal enhancement in MRI brain. This case shows that not all diffuse meningeal enhancements have an infectious etiology. Disclosure of Interest : None declared
J. N. Bathia 1 , D. Biswas 2 , P. Pal 1 , R. Sinha 3 , D. DasguptaNephrology, Institute of Child Health, Kolkata, India Correspondence: J. N. Bathia Introduction: Lupus nephritis (LN) is one the most serious complications of systemic lupus erythematosus (SLE). With an overall incidence of about 70% in children, it is the most important determinant of long term prognosis Objectives: To determine the proportion of the various initial clinical features, the various patterns of biochemical parameters including immunological markers and to determine the proportion of children with the various histopathological staging and to determine the outcome of various immunosuppressive agents in induction and remission Methods: It is a single centre study of patients with LN attending the Lupus Clinic at the Institute of Child Health, Kolkata from January 2020 to October 2021. Results: A total of 60 patients were enrolled; the female:male ratio was 4:1. The median age at diagnosis was 10.6 +/- 2.4 years. The most common extra-renal manifestation was mucocutaneous (n=54) followed by musculoskeletal (n=26), hematological (n=29), cardiovascular (n=14), neurological (n=11), pulmonary (n=5) and endocrine (n=1). The various clinical features were fever (n=55), oedema (n=50), hypertension (n=31), oliguria (n=27) and gross hematuria (n=16). Most common biochemical parameter detected was proteinuria (95%); median value was 1.4 gm (IQR of 0.6 to 2.3), GFR less than 90 was seen in 72%. Mean eGFR was 72.2 +/- 26.9 ml/min/1.72 m 2 . 58.3% had microscopic hematuria. At presentation anti-Nuclear Antibody was positive in all; C3 was low in 92% and C4 in 85%. Anti-phospholipid antibody was positive in 18%, anti-double stranded DNA (anti ds DNA) was seen in 63%. On biopsy Class IV Lupus Nephritis was the most common (53%), Class III in 20%, class II in 5%, Class III/IV + V in 8%, class VI in 2% and class V in 12%. All children received steroids (pulse/oral). Mycophenolate Mofetil (MMF) was given as induction agent in 28 patients, cyclophosphamide in 19 and MMF in combination with cyclophosphamide was given in 2. Rituximab was used in 10 patients as a rescue agent and Tacrolimus was used in 1. At one year follow up, complete response was seen in 24 patients and partial response was seen in 28. The mean time to remission was 5.5 +/- 1.6 months. The incidence of flares was 0.18 per person year and the mean time to first flare was 19 +/- 6 months. Mortality rate was 10% (n=6); 2 had ESRD and 4 succumbed to serious infectons. The baseline eGFR in those who received cyclophosphamide was 61.3 +/- 19.9 ml/min/1.72 m 2 and in those who received MMF was 84.0 +/- 19.7 ml/min/1.72 m 2 . Binary logistic regression analysis was done to assess prediction of complete or partial response at 1 year. Considering eGFR and proteinuria there was marginal improvement in prediction compared to baseline (88.3% vs 86.7%, p value – 0.024) which accounts for 21% variability in outcome. The coefficients for eGFR was 0.023 (p = 0.268) and that for proteinuria was 0.001 (p = 0.04) Conclusion: Although a difficult disease to treat, early diagnosis and timely aggressive management leads to better response rates as seen in 86% of our patients. The efficacy of the use of Cyclophosphamide and Mycophenolate Mofetil needs a further elaborative study with a larger sample size. However, to summarize the findings of our study, we found that the use of MMF was more efficacious in inducing remission as compared to cyclophosphamide although not statistically significant Patient Consent: Yes, I received consent Disclosure of Interest : None declared
Y. Butbul Aviel 1 , T. Hershkovitz 2 , R. Zaid 3 , K. Weiss 3 , I. Spivak 1 1 Pediatric Rheumatology Service, 2 Rambam Medical Center Haifa Israel, Haifa, Israel, 3 Genetic Institute, Rambam Health Care Campus, Haifa, Israel., Rambam Medical Center Haifa Israel, Haifa, Israel Correspondence: Y. Butbul Aviel Introduction: Systemic Lupus erythematous (SLE) is a heterogenic clinical syndrome with a multifactorial etiology including diverse environmental, immunological and genetic causes and modifiers. Increasingly, utilizing next generation sequencing tooIs, monogenic forms of SLE have been identified. Objectives: The aim of our study was to identify monogenic causes of SLE in the unique pediatric population of Northern Israel. Methods: A retrospective and prospective study was carried out between 2010-2021 in a single tertiary pediatric medical center. Genetic testing including Whole exome sequencing (WES) was performed for select patients including family history of SLE, consanguinity and clinical findings suggestive of specific disorder. Results: 75 children were diagnosed with SLE. 18/75 (24%) had one or more relatives with SLE or suspected monogenic disorder, including a pedigree with 4 affected members. Mean age at presentation was 10.1±4.7years and 16/18 (89%) were females. A monogenic disorder was identified in total of 7/75 of pedigrees. Four patients were diagnosed with Prolidase deficiency, one patient with ADAR1 mutation related to Aicardi–Goutières syndrome and one pedigree with APC5 mutations. Candidate variants in genes related to immune system were identified in one proband and her father requiring further study. Additional WES results are pending. Conclusion: We detected monogenic causes of SLE in a select cohort of patient in Northern Israel. Identification of a genetic basis for disease has direct clinical implication for patients and families and can also enhance our understanding of the pathogenesis and disease mechanisms involved in the more common sporadic forms of SLE. Disclosure of Interest : None declared
M. Castro 1,2 , I. Almada-Correia 1,2 , C. M. Motta 3 , C. M. Sousa Guerreiro 4 , R. A. Moura 1 , N. Khmelinskii 1,5 , F. Oliveira-Ramos 1,5,6 , C. I. Mendes 7 , M. Ramirez 7 , J. E. Fonseca 1,5 , P. Costa-Reis 1,6 1 Rheumatology Research Lab, Instituto Medicina Molecular (iMM), 2 Faculdade de Ciências , 3 Instituto Nacional de Saúde Doutor Ricardo Jorge, 4 Laboratório de Nutrição, Faculdade de Medicina, Universidade de Lisboa, Centro Académico de Medicina de Lisboa, 5 Serviço de Reumatologia e Doenças Ósseas Metabólicas, 6 Unidade de Reumatologia Pediátrica, Hospital de Santa Maria, Centro Hospitalar Universitário Lisboa Norte, Centro Académico de Medicina de Lisboa, 7 Instituto Medicina Molecular (iMM), Lisboa, Portugal Correspondence: F. Oliveira-Ramos Introduction: The gut microbiota modulates mucosal immunity, gut permeability, and endotoxemia. In a systemic lupus erythematosus (SLE) murine model it was shown that the translocation of a gut pathobiont induces an autoimmune response and death, which is prevented by antibiotics and a vaccine. We hypothesize, therefore, that dysbiosis, impaired intestinal barrier integrity, and endotoxemia are crucial to the chronic activation of the immune system seen in SLE. Objectives: To study diet, physical activity, body composition, gut microbiota, and intestinal permeability in SLE patients. Methods: Evaluation of healthy controls (HC) and SLE patients (children and adults) who fulfil the 2019 EULAR/ACR SLE classification criteria. Individuals with inflammatory bowel disease, celiac disease, irritable bowel syndrome, diabetes, malignancy, or other immune-mediated diseases were excluded. Three-day diet recalls, PREDIMED, KIDMED and the International Physical Activity questionnaires were used to evaluate diet and physical activity. Body composition was analysed by whole-body air-displacement plethysmography. Gut microbiota was studied by Next Generation Sequencing, with amplicon sequencing-based 16S rRNA analysis. The intestinal permeability was assessed directly by the lactulose/mannitol test. Serum markers of gut permeability and inflammation (zonulin, claudin-3, sCD14) were measured by ELISA. The SLEDAI-2K score was used to evaluate disease activity. Results: We studied 16 HC (median age 35.5Y; 14-50Y; 88% females) and 36 SLE patients (11 children and 25 adults; median age 33.5 Y; 11-57Y; 89% females; median age at diagnosis 23.17Y; median disease duration 5.2 years (0.25-28.67Y); 66% had lupus nephritis; median SLEDAI-2k at time of sample collection 4). SLE patients had lower physical activity and higher sitting time, lower adherence to Mediterranean diet and higher fat mass and central obesity than HC (p<0.05). In addition, SLE patients had a lower intake of alpha-linolenic acid and manganese (p<0.05). A decreased a-diversity of gut microbiota (p=0.02) was identified in SLE patients, reflecting dysbiosis. Mediterranean diet, zonulin levels and SLE disease duration significantly impacted gut diversity in our cohort (p<0.05). Zonulin levels and the lactulose/mannitol ratio were elevated in SLE patients compared to HC (p<0.05), reflecting higher gut permeability. We also found significantly increased levels of sCD14 in SLE patients (p<0.05). There were no significant differences in claudin-3. No significant correlation was observed between any evaluated biomarker and SLEDAI-2k. Conclusion: Our data support the hypothesis that gut dysbiosis and higher intestinal permeability contribute to SLE pathogenesis, being two promising therapeutic targets in this disease. Disclosure of Interest : None declared
M. Rebelo 1 , A. I. Carvalho 1 , R. Maia 2 , C. Henriques 3 , S. Batalha 2 , P. Kjöllerström 2 , M. Conde 3 1 Pediatrics, 2 Pediatric Hematology, 3 Pediatric Rheumatology, Hospital Dona Estefânia - Centro Hospitalar Universitário Lisboa Central, Lisbon, Portugal, Lisbon, Portugal Correspondence: M. Conde Introduction: Primary antiphospholipid syndrome (Primary APS) is an acquired autoimmune thrombotic disease in which patients with persistently positive antiphospholipid antibodies (aPLs)- Lupus anticoagulant (LA), anti-Cardiolipin (aCL) and B2-glicoprotein I (B2GP1) antibodies) have a thrombotic event in the absence of another autoimmune disorder. Objectives: To describe cases of Primary APS diagnosed in a level III pediatric hospital over a 5-year period (2017 to 2022). Methods: Review of the medical records of patients < 18 years old diagnosed with APS, as per the revised Sapporo classification criteria. Patients with another rheumatic disease were excluded. Demographic, clinical, laboratory, treatment and follow-up data were reviewed. Results: Three cases of Primary APS were identified. All were diagnosed by a thrombotic event accompanied by persistent triple positive aPLs. None had a combination of clinical and/or serological work-up consistent with another autoimmune disease. No other clinically relevant pro-thrombotic risk factors were identified beyond the ones mentioned. Case 1: 15-year-old, previously healthy adolescent, maternal SLE and positive aPLs, presented with unilateral deep venous thrombosis (DVT) in the right lower limb. Blood tests showed mild thrombocytopenia and increased liver enzymes all of which resolved spontaneously. A prolonged aPTT (106s) led to the identification of persistent triple positive aPLs at high titres which have persisted to date. He had ANA 1/160 and transiently low C3. He was treated with enoxaparin and then switched to warfarin with complete resolution and no recurrence of thrombotic events. Case 2: 15-year-old obese adolescent boy, on oral isotretinoin for acne, presents with right popliteal DVT. Thrombophilia studies revealed persistent high titre triple positive aPL; ANA 1/80 and an isolated weak positive direct antiglobulin test (DAT) without anaemia or haemolysis. He was treated with enoxaparin. Three months later, he had another extensive DVT of his right lower limb. Enoxaparin dose was adjusted to weight and later switched to tinzaparin. Nine months later, he suffered a bilateral pulmonary thromboembolism (PTE) with a large thrombus in the inferior vena cava. The patient received enoxaparin, later switched to warfarin plus hydroxychloroquine (HCQ) and acetylsalicylic acid with satisfactory recovery. He presented mild thrombocytopenia since diagnosis that resolved after his last thrombotic event. Case 3: 12-year-old obese girl evaluated due to thrombocytopenia (platelets 28x10 9 /L), weak positive DAT (without anaemia or haemolysis) and prolonged aPTT (80,4s). She had persistent triple positive aPLs; a positive ANA (1/320) with otherwise repeatedly negative autoimmune work-up; heterozygous Factor V Leiden mutation in the thrombophilic work-up; and no other clinical or laboratorial findings. 6 months later she had a PTE with a small ipsilateral pleural effusion and worsening of thrombocytopenia. She was treated with enoxaparin. Immunoglobulin and high-dose steroid pulses were given to control thrombocytopenia and allow for anticoagulation. Prednisolone, azathioprine, HCQ and fluvastatin (dyslipidemia) were also initiated. Enoxaparin was maintained and prednisolone progressively reduced with no thrombotic recurrence. Conclusion: Pediatric Primary APS is rare and both primary and secondary prophylaxis are not well defined. Non-thrombotic APS manifestations overlap with other autoimmune diseases. Higher-risk patients presenting triple-positive aPLs require strict follow-up and may need combined anticoagulant therapy. Minimizing vascular risk factors is also essential along with anticoagulation while aPLs are present. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
P. P. Kahwage 1 , R. R. Moura 2 , G. R. Sousa 1 , K. B. Salomão 1 , F. P. Saggioro 3 , I. Facincani 1 , D. Q. Nascimento 4 , R. S. Costa 1 , R. G. P. Queiroz 1 , E. T. Valera 1 , L. Rodrigues 1 , V. P. L. Ferriani 1 , S. Crovella 5 , P. Sandrin-Garcia 4 , L. M. de Carvalho 1 1 Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, Brazil, 2 Advanced Diagnostics, Institute for Maternal and Child Health-IRCCS “Burlo Garofolo”, Trieste, Italy, 3 Pathology, Rede D’Or , São Paulo, 4 Genetics, Federal University of Pernambuco, Recife, Brazil, 5 Biological and Environmental Sciences, College of Arts and Sciences, Qatar University, Doha, Qatar Correspondence: L. M. de Carvalho Introduction: Systemic lupus erythematosus is a complex autoimmune disease with genetic and immunological factors involved in its triggering. The Spermatogenesis-associated protein 5-like protein 1 - SPATA5L1 is ubiquitously expressed in kidneys and other tissues and previously had been associated with indices of renal function and chronic kidney disease (CKD) (1) . In a previous study we reported a SPATA5L1 gene pathogenic variant associated with a high expression of the protein in full-house nephropathy (2) . Objectives: The aim of this study was to explore the expression of this protein in renal biopsies of lupus nephritis and normal kidney controls. Methods: This cross-sectional study was performed with 29 childhood-onset systemic lupus erythematosus patients – cSLE, followed in the Pediatric Rheumatology Clinic of the Clinical Hospital of Ribeirão Preto Medical School – São Paulo University, Brazil. All cSLE patients fulfilled the American College of Rheumatology classification criteria for SLE, onset of the disease occurred under 18 years and recruited up to 21 years of age. As controls, four healthy kidneys were obtained from patients submitted to an autopsy from our Pathology and Forensic Medicine Service. Sanger sequencing analysis was performed in the cSLE patients to identify a previously reported pathogenic variant (rs143453038) associated with high protein expression in full-house nephropathy. The evaluation of SPATA5L1 protein expression was performed in biopsy samples by immunohistochemistry of all patients and controls. This study was approved by the Research Ethics Committee. Results: The sequencing analysis did not find the SPATA5L1 gene variant previously reported (rs143453038) in cSLE patients. However, we observed strong positive immunostaining for SPATA5L1 in nephritis lupus renal biopsies (class IV and V), while no or weak SPATA5L1 immunostaining was observed in non-inflammatory renal tissues. Conclusion: The expression of SPATA5L1 protein is higher in lupus nephritis biopsies as compared to health kidneys. Our results suggested that the SPATA5L1 protein may be involved in lupus pathogenesis or may function as a renal biomarker of inflammatory activity in these patients. More studies are needed to clarify the role of SPATA5L1 protein in cSLE. References: 1.Köttgen A, Glazer NL, Dehghan A, Hwang SJ, Katz R, Li M, et al. Multiple loci associated with indices of renal function and chronic kidney disease. Nat Genet. 2009;41(6):712-7. 2.de Carvalho LM, de Sousa GR, Moura R, Saggioro F, Facincani I, Costa R, et al. Full-house nephropathy associated with high expression of SPATA5L1 due to a genetic pathogenic variant. Rheumatology (Oxford). 2022;61(4):e84-e6. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
N. M. De Gruijter 1,2 , B. Jebson 1 , A. Radziszewska 1,2 , H. Peckham 1,2 , O. Nettey 1,2 , M. Butt 1,2 , C. Ciurtin 1,2 , E. Rosser 1,2 1 Centre for Adolescent Rheumatology Versus ArthritisN. M. De Gruijter Introduction: Juvenile-onset systemic lupus erythematosus (JSLE) is a rare severe inflammatory disease that starts before the age of 18 and can affect any part of the body. JSLE is often accompanied by disruptions in the cholesterol metabolism and is associated with increased risk of developing cardiovascular disease. JSLE is also characterised by the production of autoantibodies, secreted by B cells, against nuclear components. To become antibody secreting cells or plasma cells, B cells must be activated. This occurs in B cell follicles of secondary lymphoid organs in a germinal centre (GC) reaction. B cells can also be activated outside the follicle into extrafollicular plasma cells. Heightened extrafollicular B cell activation is associated with the production of autoantibodies in multiple disorders, and the frequency of extrafollicularly activated B cells is increased in the peripheral blood (PB) of patients with adult SLE. In mice, oxidised cholesterol, or ‘oxysterol’ receptor GPR183 is required for appropriate B cell positioning in the lymphoid follicle. Objectives: To investigate whether pathological changes in GPR183-oxysterol interactions (due to altered cholesterol metabolism) contribute to heightened extrafollicular B cell responses and pathogenicity in (J)SLE. Methods: Multi-parameter flow cytometry was used for phenotyping PB B cells of 28 JSLE patients and 33 age-matched controls (HC). The parent into F1 mouse model of lupus was used; only female F1 animals were included due to known sex bias. To assess the effect of blocking GPR183-ligand interactions, following injection of parent lymphocytes, F1 mice were injected with small molecule GPR183-antagonist NIBR189 or vehicle as a control daily for two weeks, then twice weekly. Proteinuria was scored between 0-4 using urine dipsticks over 10 weeks after disease induction (NIBR189 n=4; control n=5). The frequency of GC (CD19 + GL7 + CD95 + ) B cells was assessed by flow cytometry at two weeks post-injection of parent lymphocytes (NIBR189 n=3; control n=5). Unpaired t-test was used to compare significance of differences in marker expression between groups. Two-way ANOVA was used to compare significance of differences in urine score between groups. All numbers are portrayed as mean, unless otherwise specified. Results: We found that GPR183 is co-expressed with B cell activation marker CD27 and identifies memory B cells. GPR183 + CD27 + B cells are reduced in JSLE compared to HC: 17.2% of PB B cells are GPR183 + CD27 + in JSLE, compared to 26.5% in HC ( p =0.0003). GPR183 + CD27 + B cells of JSLE patients have reduced expression of lymph node-homing and follicle-positioning receptor CXCR5 and gut-homing receptor CCR9: 56.6% and 47.87% in JSLE (n=12), vs. 80.0% and 62.8% in HC (n=14), respectively (CXCR5: p =0.0034; CCR9: p =0.0464). Treatment with GPR183 antagonist NIBR189 significantly suppressed experimental lupus severity, as measured by lower proteinuria score (3.2 in NIBR189-treated mice vs. 1.1 in control lupus mice, p =0.0006). The percentage of GC B cells was significantly increased in NIBR189-treated mice (2.63%) vs. control lupus mice (1.34%) ( p =0.0154) during disease initiation phase (2 weeks post-induction), suggesting reduced extrafollicular B cell differentiation. Conclusion: Our data suggest a possible role for GPR183-oxysterol interactions in driving aberrant B cell differentiation in (J)SLE and identify this pathway as potential therapeutic target in this disease. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
A. Faleye 1 , K. Mahmood 2 , M. Beresford 2,3 , C. Hedrich 2,3 , E. Smith 2,3 on behalf of UK JSLE cohort study 1 Paediatrics, Lagos State University Teaching Hospital, Lagos, Nigeria, 2 Paediatric Rheumatology, Alder Hey Children’s NHS Foundation Trust, 3 Institute of Life Course & Medical Sciences (Child Health) , University of Liverpool, Liverpool, United Kingdom Correspondence: A. Faleye Introduction: Pulmonary involvement in Juvenile Systemic Lupus Erythematosus (JSLE) has not been comprehensively described in the literature, particularly as compared to adult SLE. Objectives: To report the incidence, prevalence, demographic and clinical characteristics of pulmonary involvement in JSLE patients, assessing clinical and/or laboratory features associated with pulmonary involvement. Methods: Participants of the UK JSLE Cohort Study (2006-2021), <18 years at diagnosis, with 3 4 ACR-1997 classification criteria for SLE, were eligible for inclusion. Patients were grouped according to the presence or absence of pulmonary involvement (defined as a pBILAG pulmonary domain score A or B). Patients were assessed at diagnosis, 10-14, 22-26 and 58-62 months of follow-up, with demographic/clinical characteristics compared between those with/without pulmonary involvement. Results: 480 JSLE patients were included, median age was 12.93 years (interquartile range, IQR 10.5-14.7). 119/480 (24.8%) had pulmonary involvement, 109/480 (22.7%) at diagnosis. 30/157 (19.1%) displayed pulmonary involvement at 10-14 months, 22/128 (17.2%) at 22-26 months, and 11/49 (22.4%) at 58-62 months. Sub-types of pulmonary involvement are displayed in the Table. Pulmonary involvement was associated with higher ACR-1997 (median 6.0 [IQR 4-6] vs 5 [IQR 5-6], p<0.002) and pBILAG numerical disease activity scores (median 22 [IQR 11-33] vs 9 [IQR 3-16], p<0.001) at diagnosis, with no differences at subsequent time points. There were no differences in SLICC-SDI scores between those with/without pulmonary involvement (all p>0.05). PBILAG defined constitutional (48.3 vs 26.1%), musculoskeletal (49.1 vs 26.1%), gastrointestinal (10.3 vs 3.8%) and haematological (37.9 vs 20.6%) involvement was more common in patients with pulmonary involvement (all p<0.05). Differences were seen in haemoglobin levels, neutrophil counts, erythrocyte sedimentation rate, and C4 levels in those with/without pulmonary involvement (all p<0.05). Conclusion: Pulmonary disease is common in JSLE, with serositis the main form of involvement. It associates with wider organ involvement which suggests a need for close monitoring and aggressive treatment. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
elix 1 , F. Delion 2 , B. Suzon 3 , A. Ogrizek 4 , N. Elenga 5 , M. Dramé 6 , C. Hospice 7 , M. Mohamed Sahnoun 8 , B. Bader-Meunier 9 , C. Deligny 10 , Y. Hatchuel 7 1 Department of pediatrics, CHU de la Martinique, Fort de France, 2 Department of Pediatrics, CHU Guadeloupe, Pointe à pitre, 3 Department of internal medicine, 4 Department of pediatrics, CHU de la Martinique, Fort-De-France, France, 5 department of pediatrics, CH Andrée Rosemond , Cayenne, French Guiana, 6 CIC Antilles-Guyane, 7 Department of pediatrics, CHU de la Martinique, Fort de France, 8 Department of pediatrics, CH de l’ouest Guyanais, Saint Laurent du Maroni, 9 Department of Pediatric Rheumatology, CHU Necker enfants malades, Paris, 10 Department of Internal Medicine, CHU de la Martinique, Fort de France, France Correspondence: A. Felix Introduction: Systemic diseases of pediatric onset are more frequent in Afro-Caribbean population, especially Pediatric systemic lupus (pSLE). Objectives: Our work is a retrospective study of patients followed in French overseas departments of America for systemic lupus of pediatric onset. It describes their clinical and biological specificities at diagnosis, during childhood and early adulthood. Methods: A retrospective study was conducted between January 2000 and September 2021. Listings of patients with pediatric onset were obtained through multiple sources: computerized hospital archives, registry of referent pediatricians and adult specialists in internal medicine and French National Registry for rare diseases. The spectrum of diseases studied was systemic lupus according to international criteria with onset before the age of 18 years old. Results: Overall, 2148 patients were identified and 54 patients were included. Average follow-up was 8.3 years (range: 0.3 - 25 years). We found an increase of new diagnoses throughout the years. At onset, pSLE patients had median 10 SLICC criteria (range: 4-12) and median EULAR/ACR 2019 score was 38 (12 - 54). At onset, a third of patients had renal involvement, 15% neurolupus and 41% cardiac involvement. During childhood, 54% had renal involvement and 26% suffered from neurolupus. Patients suffered in median from 3 flares during childhood and 26% from more than 5 flares. Younger patients at onset had worst outcomes, they had more flares (median at 5 p = 0,02) and needed an average of 4 background therapies (p = 0,04). Conclusion: This is a large cohort of patients of Afro-Caribbean origin with a higher frequency of pSLE. The outcomes of Afro-Caribbean patients, where pSLE is more frequent, were similar to western countries with worse disease activity at onset. Further studies should be performed to identify the genetic and environmental factors in this population. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
J. C. O. A. Ferreira 1 , A. A. Costa 2 , H. C. S. Robba 2 , A. C. S. Monteiro 2 , J. R. Campana 2 1 Clinical Research Center, 2 Nursing Division, Instituto Da Criança Do HCFMUSP, São Paulo, Brazil Correspondence: J. C. O. A. Ferreira Introduction: The chronic nature of Juvenile systemic lupus erythematosus and the possible complications or limitations demands special nursing knowledge and attention to treat the adolescent patient and their family. The structural and functional maturation of the brain, besides disease activity, continuous use of medication, presence of comorbidities and pain can change body image and cause social isolation. Objectives: To develop an instrument for nursing care for patients with JSLE. Methods: It was developed an instrument for nursing consultation to patients with JSLE from a published study carried out on the care provided by Brazilian nurses to JSLE patients, approved by the local Ethics Committee number 2.089.884. Results: The instrument is composed by information from vital signs and anthropometric data, physical examination, clinical signs and manifestations of JSLE, aspects of care such as adherence, pain, sexuality, drug use, physical violence, suicidal ideation, bullying, vaccination, acceptance of disease. Conclusion: The instrument has already started to be used and allowed the diagnosis of problems such as pain, suicidal ideation and bullying. A multicenter study will be conducted to verify its reliability and reproducibilityV. Jirón Mendiola, M. Jarquín Jaime Reumatología Pediátrica, Hospital Infantil de Nicaragua Manuel de Jesús Rivera, Managua, Nicaragua Correspondence: K. V. Jirón Mendiola Introduction: Family studies have shown the genetic contribution to developing systemic lupus erythematosus, and a 20-fold increased risk of developing lupus has been found compared to the general population, in addition to the fact that 10% of first-degree relatives can also develop lupus. There are previous studies that described the aggregation of connective tissue diseases in families; thus, following a clinical observation, we studied an unusual case of Lupus with different manifestations in 4 siblings born to mothers with SLE, in addition, 5 cases of relatives in second and third degree of consanguinity were studied. Objectives: To describe an unusual case of lupus manifestations in first degree consanguinity members of a single family Methods: An observational retrospective study of a 45-year-old woman diagnosed with Lupus, mother of 4 children diagnosed with SLE, of which the clinical manifestations presented were described. Results: 9 cases were identified; all alive at the time of the study, of which clinical records and studies that conditioned the diagnostic criteria were available. A genealogical mapping was carried out with antecedents, finding 5 patients of first degree of consanguinity, 3 in second degree and 2 in third degree of consanguinity with a diagnosis of SLE, inhabitants of the same area of the country. Of the 9 cases reviewed, 5 were male and 4 female. The mean age at diagnosis was 22 years. The most dominant clinical presentation was photosensitive maculopapular rash, malar rash, aphthous ulcer in 8/9 (88.8%), arthritis in 7 of them, neurolupus with demyelinating syndrome in 4/9 (44%), migraine headache in 3/9. Cutaneous vasculitis was present in 2/9 (22.2%) Cardiopulmonary involvement with pleurisy and pericarditis was noted in 2/9 (22.2%), myelopathy in one patient, anemia in 2/9 (22.2%), and thrombocytopenia and leukopenia in one each . Autoantibodies were mainly ANA in all patients (100%) and anti-dsDNA in 9/9 (100%). The nine patients had received therapy without a diagnosis of SLE prior to its detection and integration of criteria, finally once the diagnosis was made, management with bolus glucocorticoids was indicated, two of them received rituximab and cyclophosphamide. Conclusion: The genetic predisposition to SLE is already described, however the extensive grouping of SLE in the same family is less described, especially when they present with similar manifestations and classification criteria for SLE, with a predominance of neurological manifestations in members of a single family. Our observational study is the first to describe the family grouping of SLE in Nicaragua, finding a considerable number of patients, which would provide a follow-up guideline to investigate genetic factors and/or racial risk factors in our population. Disclosure of Interest : None declared
E. Kalashnikova 1 , R. Rinat 1 , N. Lubimova 2 , K. Ekaterina 2 , E. Isupova 3 , E. Gaidar 3 , V. Masalova 3 , L. Snegireva 3 , T. Kornishina 3 , L. Sorokina 3 , M. Kaneva 3 , O. Kalashnikova 3 , V. Chasnyk 3 , M. Kostik 3 1 Pediatrics, Saint-Petersburg State Pediatric Medical University, Saint Petersburg, 2 Laboratory of autoimmune and autoinflammatory diseases, Almazov National Medical Research Centre, 3 Pediatrics,M. Kostik Introduction: Lupus nephritis (LN) is one of the severest manifestations of systemic lupus erythematosus (SLE), determining the outcomes of the disease. Objectives: to analyze the safety and efficacy of rituximab (RTX) in pediatric patients with lupus nephritis. Methods: in the retrospective cohort study were included 25 patients with LN (10 boys and 25 girls) with onset age 13 (12; 15 years). Diagnosis was made using Systemic Lupus International Collaborating Clinics (SLICC) classification criteria. RTX was prescribed in dosage 375 mg/m 2 every week (2-4 infusions) with repeated courses every 6-12 months (2-4 infusions) according disease activity, the degree of B-cell depletion and hypoIgG-emia. The indications for rituximab were severe LN, resistant to conventional non-biologic treatment or corticosteroid dependence/toxicity. The dynamics of clinical, laboratory data, activity of the disease by SLEDAI, corticosteroid doses were assessed in the onset, and during RTX trial. Results: The main patient’s characteristics were: Pre-RTX non-biologic conventional treatment includes: cyclophosphamide 15 (60%), MMF 8 (32%), azathyoprine 3 (12%), hydroxychlorquine (HCQ)12 (48%), pulse therapy of methylprednisolon with following oral methylprednisolon 25 (100%). Time before RTX was – 7.0 (3.0; 24.0) months, whole observation period was 7.0 (0; 24) months. Initial pre-RTX treatment (corticosteroids, HCQ, non-biologic DMARDS) slightly reduced SLEDAI levels and the proportion of the patients with LN, but following administration of the RTX realized in prominent reducing of SLEDAI, anti-dsDNA level, proteinuria, hematuria, C4 complement, ESR, and median corticosteroid dose by 80% from the initial, as well as the proportion of patients without corticosteroids. Two deaths were observed due to catastrophic SLE with MAS, accompanied severe infection (invasive aspergillosis, n=2). Three patients realized SAE: pneumonia (n=2), transient agranulocytosis (n=1) after 3rd RTX infusion and meningitis, caused by Lysteria monocytogenis, after 1st RTX infusion (further RTX treatment continued without adverse events), patella osteomyelitis (n=1). Eight patients received antibacterial treatment for different respiratory infections not required hospital admissions. Conclusion: RTX showed effectiveness in LN, where previous non-biologic treatment was insufficiently effective. Randomized controlled trials are required to evaluate the efficacy and safety of RTX and set the benefits compare to conventional SLE treatment. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
M. Kasap Cuceoglu 1 , S. Demir 2 , E. S. Akbas Aliyev 3 , P. Avar Aydin 4 , E. Aliyev 1 , E. Cengel Kultur 3 , S. Ozen 1 , T. Cak 3 , Y. Bilginer 1 1 Pediatric Rheumatology, Hacettepe University, Ankara, 2 Pediatric Rheumatology, Erzurum City Hospital, Erzurum, 3 Child and Adolescent Psychiatry, Hacettepe University, 4 Pediatric Rheumatology, Ankara University, Ankara, Turkey Correspondence: M. Kasap Cuceoglu Introduction: Childhood-onset SLE is associated with major organ involvement, including neuropsychiatric disease (NPSLE) is easy to overlook unless it’s obvious. The Pediatric Automated Neuropsychological Assessment Metrics (Ped-ANAM) is a computerized battery that measures cognitive ability, mental processing speed, and memory. Objectives: We aimed to study the psychometric properties of the Turkish version of the the Ped-ANAM and compare to a formal cognitive testing tool in SLE patients. Methods: We performed Ped-ANAM, and the Wechsler Intelligence Scale for Children-IV (WISC-IV) as a formal neuropsychological testing tool on 30 cSLE, synchronously at Hacettepe University Ankara. All participants were assessed by a child and adolescent psychiatrist with Schedule for Affective Disorders and Schizophrenia for School-Age Children Present and Lifetime Version, DSM-5- (K- SADS-PL-DSM-5-T) to rule out current psychiatric disorders. PedANAM is an automatic battery that only requires a computer without the need for other specialized equipment; a psychometric specialist consists of 10 subtests and four performance parameters. The WISC-IV measures intellectual performance as a multidimensional construct. The WISC-IV contains 10 core subtests and 4 indexes namely, Verbal Comprehension Index, Perceptual Reasoning Index, Working Memory Index, and Processing Speed Index. SLE patients were divided into two groups as present/absent neurocognitive dysfunction (NCD). NCD definition: At least one of the WISC-IV was below -2 standard deviations, or at least two were between -1 and -2 standard deviations. Results: Thirty patients with cSLE (male, 30%) were recruited for the study. The median age of cSLE patients was 16.6 (11-20) years, and the median follow-up period for cSLE was 59 months (6-180). When the SLEDAI-K activity indexes of the patients were evaluated, there was no severe disease activity in both groups. 12 (40%) of 30 cSLE patients in the study met the criteria for NCD. There was no significant difference according to age, gender, SLEDAI-K disease activity index, organ involvement, depression, anxiety, and other psychiatric symptoms between NCD and without NCD groups. When we compared the median (min_max) of PedANAM-CPS indices of the two groups; NCD SLE patients had lower CPSpca than without NCD SLE [-1.75 (min:-13.30, max:1.47) vs. 0.19 (min:-13.30 max:2.69), p = 0.087] and CPSmultiscore was higher in NCD SLE than without NCD SLE [8.50 (2.93 – 17.30) vs. 5.64 (1.65 – 15.22), p = 0.043]. There were statistically significant differences in simple reaction time (p=0.014), spatial processing (p=0.036), attention/processing speed (p=0.059), spatial processing/matching grids (p=0.053) in PED-ANAM subtests. Six Ped-ANAM subtests (simple reaction time, attention/proc. speed, learning, logical relations-reasoning, spatial processing, delayed memory) significantly correlated with WIS-C-IV subsets (vocabulary, coding measures, picture concepts, similarities, block design, perceptual reasoning, verbal comprehension, coding, symbol search) in overall two groups (Spearman’s correlation coefficient >0.4, P < 0.05; n=30). Conclusion: The findings of this study represent the results that the Turkish version of Ped-ANAM can also be used in the clinic for this purpose. The significant difference between groups in subtest scores indicates that Ped-ANAM supports this view. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
D. Klepárník, Z. Pytelová, K. Bouchalová Department of Paediatrics, Faculty of Medicine and Dentistry, Palacky University Olomouc and University Hospital, Olomouc, Czech Republic Correspondence: D. Klepárník Introduction: Juvenile Systemic lupus erythematosus (jSLE) is a heterogenous disease. The prevalence of hypovitaminosis D in patients with jSLE was determined to be between 36 and 96%(Rezaei E. 2020). According to the available literature, no study has yet looked at vitamin D levels in jSLE patients at the time of diagnosis. Double-blind randomized trial proved the effect of vitamin D supplementation in patients suffering from jSLE (Lima GL 2016). Objectives: Comparison of 25-OH vitamin D levels in patients with jSLE at the time of diagnosis and levels measured at the last follow-up. Methods: Twenty-five patients were included in this study. Data were collected from September 2021 to February 2022. Patients who suffered from jSLE, most met SLICC or ACR classification criteria. All patients were treated with standard jSLE therapy and substituted with vitamin D based on their baseline levels. The normal range for serum 25-hydroxyvitamin D is 75–250 nmol/L (Charoenngam N 2020). A comparison of vitamin D levels at the time of diagnosis and last follow-up with a reference value of 75 nmol/L was performed by a nonparametric Wilcoxon single-sample test. A comparison of concertation at the time of diagnosis level and last follow-up was performed using the Wilcoxon paired test. Results: The patients mean age was 15.71, SD ± 2.964. The mean age at the time of diagnosis was 14.49 years, SD ± 3.12 years. Vitamin D levels at the time of diagnosis were statistically significantly lower than 75 nmol/L ( p = 0.020 ). The level of vitamin D at the last control was statistically significantly higher than 75 nmol/L ( p = 0.023 ). Vitamin D levels at the time of jSLE diagnosis were significantly lower ( p <0.0001 ) than at the last follow-up. Conclusion: Initially, 18 patients had suboptimal levels of vitamin D. At the last follow-up, only 3 patients had suboptimal levels. Our results suggest a good effect of vitamin D supplementation. We demonstrated that low vitamin D levels were present in patients at the time of diagnosis of jSLE. To date, no published study has investigated the status of vitamin D levels at the time of jSLE diagnoB. Lewandowski 1 , R. E. Sadun 2 , J. C. Cooper 3 , A. Belot 4 , E. M. Smith 5 1 Pediatric Rheumatology, National Institutes of Health, Rockville, 2 Rheumatology, Duke University, Durham, 3 Pediatric Rheumatology, University of Colorado, Denver, United States, 4 Pediatric Rheumatology, Centre Hospitalier Universitaire de Lyon, Lyon, France, 5 Pediatric Rheumatology, University of Liverpool, Liverpool, United Kingdom Correspondence: R. Sadun Introductionis associated with more severe disease, often requires more aggressive treatment than adult-onset SLE, and has been under-researched compared to its adult counterpart. Regional cSLE registries exist in some countries to capture demographics, disease features, and outcomes. International, iter-registry collaborations could allow pooling of data across registries, with the potential to greatly advance cSLE outcomes research by improving patient numbers. However, differences in collection methodology and data fields have been major barriers. Objectives: To obtain international consensus around standardized data collection that enables high-quality research on patients with cSLE. Methods: To identify essential, “core” data elements for registry-based research in studies of children and adolescents with cSLE, 21 SLE experts (15 peds rheum, 4 med/peds or adult rheum, 2 peds nephrology) with international representation (10 countries; 5 continents) completed two sequential Delphi surveys to indentify areas of consensus and disagreement prior to a virtual consensus meeting. The virtual consensus meeting was conducted over two days in May 2022 and used adapted nominal group technique to discuss and vote on data elements to be included in the core dataset. Consensus required ≥ 80% agreement for inclusion. Results: Twenty (95%) experts responded to the first Delphi questionnaire and 21 (100%) to the second questionnaire. Twenty-one physicians and 4 patients/caregivers attended the consensus meeting, all with equal votes. The group achieved consensus for core data collection for cSLE registries in the following areas: demographics, SLE classification criteria, visit data, laboratory testing, kidney biopsy data, disease activity measurements, damage index, medications, and severe adverse events (Table 1). In addition, consensus was achieved regarding frequency of data collection (every 3 months + flare visits). Experts agreed that an additional follow-up meeting will be needed to clarify data elements pertaining to patient reported outcome measures. Conclusion: A core dataset for registry-based international childhood-onset SLE research was defined by global expert consensus. The core dataset considers unique aspects of children with childhood-onset SLE in a range of geographic and resourced settings. The core dataset will facilitate international collaborative research for children and adolescents with childhood-onset SLE worldwide. Trial registration identifying number: Not applicable Patient Consent: Not applicable (there are no patient data) Disclosure of Interest : L. Lewandowski Grant / Research Support with: CARRA-PReS Collaborative Award, R. Sadun Grant / Research Support with: CARRA-PReS Collaborative Award, J. Cooper Grant / Research Support with: CARRA-PReS Collaborative Award, A. Belot Grant / Research Support with: CARRA-PReS Collaborative Award, E. Smith Grant / Research Support with: CARRA-PReS Collaborative Award
H. F. F. Menchaca Aguayo 1 , P. P. Ramos Tiñini 2 , E. R. Mercedes-Perez 1 , E. Faugier 3 , A. Barba 1 , A. Guzman 4 , K. Primero 4 , H. Bermudez 1 1 Pediatric Rheumatology, Hospital Infantil de Mexico Federico Gomez, 2 Pediatric Rheumatology, Hospital Infantil de México Federico Gómez, 3 Pediatric Rheumatology, Hospital Infantil de Mexico Federico Federico, Ciudad de Mexico, 4 Reumatología Pediátrica, Hospital Infantil de México Federico Gomez, Ciudad de México, Mexico Correspondence: H. F. F. Menchaca Aguayo Introduction: Currently, after the onset of the COVID 19 pandemic, there has been an increase in cases of autoimmune diseases, this in relation to the viral pathophysiology that triggers an autoimmune disruption in a genetically predisposed individual (1). The incidence of myocarditis as a cardiovascular manifestation in pediatric Systemic Lupus Erythematosus Erythematosus (pSLE) is approximately 2% to 19% (2), however, myocarditis triggered by SARS-CoV-2 has been described 2 to 3 weeks after infection (3). Objectives: To describe the case of a patient with debut of pSLE with carditis and its association with SARS-CoV-2. Methods: Description of a clinical case and review of the literature. Results: A 16-year-old female with 6 months of evolution with constitutional syndrome, myalgias, arthralgias and hair loss; 3 days before, medium effort dyspnea was added. On admission she was tachycardic, polypneic, oximetry 80%, supplemental oxygen was administered. Physical examination: pale, frontal alopecia, bilateral cramps and left basal hypoventilation. Laboratory tests: normal chromic anemia, normal renal and hepatic function tests, normal renal and hepatic function tests. Proteinuria 12 hours 24.3 mg/m2/h, PCR SARS-CoV-2 negative, Ig G SARS-CoV-2 positive. Chest X-ray: left pleural effusion and cardiomegaly (CI 0.68). TTE: structurally healthy heart, trivial pericardial effusion, moderate LV systolic dysfunction, moderate TR and mild MI. SLE was diagnosed (ACR 1997 criteria) due to: serositis, proteinuria, positive lupus anticoagulant (1.61) and homogeneous ANA pattern 1: 640. One day after admission he presented respiratory and hemodynamic deterioration, requiring aminergic support and non-invasive mechanical ventilation and was admitted to the PICU. Pulses of methylprednisolone 30 mg/kg/day for 5 days and human immunoglobulin 2 g/kg/day were administered, as well as treatment for heart failure: carvedilol, lisinopril, spironolactone and furosemide. She presented clinical improvement and after 5 days she was discharged from PICU. Conclusion: Our patient presented carditis as the initial manifestation of SLE and positive Ig G serology for SARS CoV-2, so this infection could have been the trigger for the clinical manifestation and decompensation of the disease. In the context of COVID-19 pandemic and in the presence of patients with systemic clinical symptoms, autoimmune diseases should be considered and in the presence of cardiovascular manifestations it is necessary to perform specific inflammatory markers and cardiological screening evaluations. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
A. Meneghel 1 , A. Carriero 2 , G. Martini 2 , F. Tirelli 2 , L. Iaccarino 3 , F. Zulian 2 1 Pediatric Rheumatology Unit, Department for Women and Child Health, University Hospita of Padova, 2 Pediatric Rheumatology Unit, Department for Women and Child Health, 3 Rheumatology Unit, University Hospital of Padova, Padova, Italy Correspondence: A. Meneghel Introduction: In the last decades, the mortality rate of Juvenile Systemic Lupus Erythematosus (jSLE) has significantly decreased but the morbidity has increased due to both the severity of the disease and side effects of immunosuppressive therapy. Thus, while there is a growing interest towards the identification of treatment protocols that would allow a better control of the disease, data regarding the long-term prognosis of jSLE are limited so far. Objectives: To compare the long-term prognoses of two groups of jSLE patients treated with different therapeutic protocols: Cyclophosphamide (CYC) and Mofetil Mycophenolate (MMF). Methods: We performed a retrospective cohort study including jSLE patients, diagnosed according to ACR 1997 and/or EULAR/ACR 2019 criteria, followed in our Unit between 2003 and 2021. Patients were divided into two treatment groups: CYC and MMF. Clinical, demographic, laboratory variables, therapies, disease activity index (SLEDAI-2K), damage index (SLICC/SDI) and disease flares (SFI index) were collected at the time of diagnosis (T0), at treatment initiation (Tstart), at 6 months (T6) and annually after treatment initiation (T12, T24, T36, T48, T60). Patients were defined as “active disease” (AD), “disease in clinical remission on medication” (CRM) and “disease in clinical remission off medication” (CR) according to the most recent definitions. The groups were compared for clinical variables (by Fisher exact test and Mann–Whitney U test) and treatment efficacy over time (by mixed-design analysis of variance model) considering statistically significant a p-value <0.05. Results: 20 patients (15 F) were included: CYC=12 (9 F), MMF=8 (6 F). No statistically significant differences between the two groups were found for clinical and demographic characteristics at T0 (Table). During the follow-up, mean SLEDAI-2K showed a significant reduction (p<0.001) from Tstart (CYC=19.9±5.8; MMF=22.9±5.5) in both groups at T12 (CYC=6.9±5.0; MMF=6.3±5.3), T24 (CYC=9.1±10.0; MMF=4.3±2.7), T60 (CYC=11.7±5.0; MMF=7.7±7.6), showing no statistically significant difference between the 2 treatments. As for the doses of corticosteoids and SFI, no statistically significant differences were detected between the 2 groups at any timepoints. We found a statistically significant difference in SDI between the 2 groups at T6 (p=0.028), T 12 (p=0.015), T24 (p=0.04), T36 (CYC=5, MMF=0; p=0.029). Of interest, 1 patient in the MMF group achieved full clinical remission at both T48 and T60. Conclusion: CYC and MMF present comparable efficacy both in the short- and long-term follow up, independently from the use of corticosteroids. Patients treated with MMF showed a significantly lower cumulative damage as defined by SDI over time. Although with the limit of its retrospective nature and small size of the cohort, our study, confirms the role of MMF as first-line treatment for jSLE, also considering the higher risk of side effects on reproductive system reported with CYC. Future prospective studies on larger samples are needed to confirm these results. Disclosure of Interest : None declared
P. Morán Álvarez 1,2 , Á. Andreu 1 , L. Caballero 3 , S. Gassiot Riu 4 , R. Berrueco Moreno 4 , M. Vazquez Diaz 1 , J. Calzada Hernandez 4 , L. Giovannelli 5 , A. Boteanu 1 , J. Antón 4 , F. De Benedetti 2 , C. Bracaglia 2 1 Hospital Universitario Ramón y Cajal, Madrid, Spain, 2 Ospedale Pediatrico Bambino Gesù, Rome, Italy, 3 Hospital Universitario Gregorio Marañón, Madrid, 4 Hospital Sant Joan de Deu, Barcelona, 5 Ospedale Pediatrico Bambino Gesù, Rome, Spain Correspondence: P. Morán Álvarez Introduction: Antiphospholipid syndrome (APS) is an autoimmune disease characterized by thrombotic events (TEs) and/or pregnancy morbidity, in association with two consecutive positive determinations (at least 12 weeks apart) of antiphospholipid antibodies (aPLs). Several manifestations which are not considered clinical criteria of APS have been identified, including hematological disorders. Objectives: To identify the different variables associated with the development of hematological manifestations in the presence of aPLs in a pediatric cohort. Methods: Multicentric historical cohort of children £ 18 years from three Spanish and one Italian tertiary referral hospitals with at least 2 positive determinations ( 3 12 weeks apart) of IgG/IgM anticardiolipin (aCL), IgG/IgM anti β2-glycoprotein I (aβ2GP) and/or LA. Demographic, clinical and analytical data were collected. A bivariate and multivariate analysis were carried out. Multicollinearity was also explored to build the model. Results: One hundred and thirty-one children were included in the study. The median age was 10.9 5.1 years and 64.9% were female. Most of them were Caucasians (81.4.7%). Thirty-three (25.2%) children had a positive family history for autoimmune diseases and 34 (26%) a personal history for systemic lupus erythematosus (SLE). Fifty-five (42%) patients developed at least one hematological manifestation. Of them, we registered: 22.9% thrombocytopenia; 8.4% leukopenia; 5.3% hemolytic anemia; and 1.5% Evans syndrome. Related to thrombotic events (TE), 16 (12.2%) children had at least one TE. Significant differences between the groups (patients with hematological manifestations and patients without hematological manifestations) in terms of age, gender, race, TE and family history for autoimmune diseases were not identified. However, a higher prevalence of SLE was found among the children with hematological manifestations (36.4% vs 18.4%; p=0.021). Other clinical manifestations, immunological parameters and results of bivariant analysis are shown in Table 1. The multivariate analysis identified as independent risk factors to develop a hematological manifestation, children with: SLE diagnosis [OR 1.2, 95 CI (0.5-5.8), p= 0.015], cutaneous manifestations [OR 1.01, 95 CI (0.6 -2.8), p= 0.091], LA + [OR 0.8, 95 CI (0.4 -3.6), p= 0.058], and IgG aβ2GP + [OR 0.9, 95 CI (0.4 - 3.9), p= 0.048]. Sex, age, familiar history of AIDs or IgM B2GP+ did not show a higher risk. Conclusion: Non-criteria manifestations, especially hematological disorders, are the most frequent events in the presence of aPLs in our cohort. Cutaneous manifestations, a positive personal history of SLE, LA and IgG aβ2GP positivity were associated with a higher risk of developing hematological manifestations. Therefore, their inclusion as APS classification criteria, C. Bracaglia, V. Messia, I. Caiello, L. Giovannelli, F. De Benedetti, E. Marascon autoimmune disease characterized by the presence of antinuclear antibodies (ANA). Monitoring of anti-DNA antibody levels canmay decrease over time secondary to the natural history of lupus or to treatments. Objectives: To investigate the trend of ANA and anti-dsDNA autoantibodies titers over time in children with a diagnosis of SLE. Methods: We enrolled 15 children with a diagnosis of SLE, fulfilling the SLICC criteria. For all patients included in the study ANA and anti-dsDNA antibodies testing was carried out from diagnosis every 3-4 months for 2 years. ANA were defined as negative for titers lower than 1:80. Laboratory parameters, clinical and demographic data was retrieved and analyzed. Results: Following two years of follow-up, all patients had ANA titers significantly lower than at time of SLE diagnosis (Mann Whitney test, p=0.0002). After two years of follow-up, 11 patients (73%) still had positive ANA (group 1), while 4 patients (26%) had negative ANA (group 0). At time of diagnosis no significant differences in ANA titers (Mann Whitney test, p=0.74) nor in disease activity, as measured by SLEDAI, (Mann Whitney test, p=0.88) were observed (table 1). No significant differences in organ involvement were observed (table 1). Assessing the change over time in ANA titers, the 2 groups of patients showed two different patterns: in group 0, ANA titers quickly declined and disappeared in the first 6 months after diagnosis; in group 1, ANA titers declined more slowly, remaining positive at 2-year follow-up. Both C3 and C4 increased in the follow-up period, with no different patterns between the 2 groups. Similarly, anti-dsDNA antibodies titers declined over time with no clear different patterns between the 2 groups. Conclusion: Our analysis showed two different patterns in the reduction of ANA titers over time in patients with childhood onset SLE, with 26% of patients becoming ANA negative after 6 months from diagnosis and remaining persistently negative during follow-up. Our data have important implications, specifically for the recruitment of patients into clinical trials, where the latest classification criteria of SLE require ANA positivity as entry criterion. Moreover, a seronegative state may represent a different subcategory of patients with SLE with specific pathogenetic pathways involved, possibly independently from autoantibodies. Therefore, further studies are needed to confirm and expand our dataK. Nay Yaung 1,2 , J. G. Yeo 1,2,3 , M. Wasser 1 , P. Kumar 1 , S. P. Tang 4 , S. K. Ng 4 , S. L. Poh 1 , T. Arkachaisri 2,5 , S. Albani 1 1 Translational Immunology Institute, Singhealth/Duke-NUS Academic Medical Institute, 2 Duke-NUS Medical School, 3 KK Women’s and Children’s Hospital, Singapore, Singapore, 4 Paediatric Rheumatology Unit, Department of Paediatrics, Selayang Hospital, Kuala Lumpur, Malaysia, 5 Paediatric Rheumatology & Immunology, KK Women’s and Children’s Hospital, Singapore, Singapore Correspondence: K. Nay Yaung Introduction: Systemic lupus erythematosus (SLE) is a complex, systemic autoimmune disease that interferes with the balance between regulation and immunity, resulting in immune system dysfunction. Disease course is unpredictable due to alternating remissions and flares, impeding reliable patient assessments. Thus, mechanistic insights are required for better assessment. Current studies are mainly descriptive, and SLE is best interrogated with a multi-parametric, holistic approach such as mass cytometry (CyTOF). We hypothesize that significant differences exist between the immunomes of newly diagnosed SLE patients and healthy subjects. Objectives: The objectives of this study are as follows: (1) To characterise immune signatures of newly diagnosed SLE patients and in the process: Study the roles of B and T cells in SLE to gain a holistic understanding of the adaptive immune response, (2) To compare immunological profiles of newly diagnosed SLE patients with age-matched healthy controls and (3) Build a database of CyTOF data for paediatric SLE patients so that comparisons can be made with adult SLE data in the future. Methods: Peripheral blood mononuclear cells (PBMCs) of 5 SLE subjects (median age 125 months) were tested with CyTOF. Data was uploaded to an online analytical database, the Extended Polydimensional Immunome Characterization (EPIC) discovery tool, for comparison with 51 age-matched controls. Normalization and FlowSOM (Flow cytometry analysis by Self-Organising Maps) clustering to 50 nodes were performed with 37 functionally and phenotypically important immune markers. Mann-Whitney U test identified significantly different cluster frequencies. Results: Correspondence analysis comparing global differences in cluster frequencies showed segregation of SLE subjects away from healthy controls. Multiple significant differences were identified (p < 0.05). Notably, a memory CD4+CD152+PD1+ T cell subset (CD4+CD152+PD1+CD45RO+CD25-FoxP3-) was enriched in SLE (median: 2.17%, interquartile range: 1.66 to 7.74% of CD45+ PBMCs) versus control (1.34%, 1.06-1.58%; p = 0.00267). Expression of known checkpoint inhibitors (PD1, CD152) could be important contributors to SLE immunopathogenesis. Secondly, the innate lymphoid cell 2 (ILC2) subset (Lin-CD7+CD25+CD127+GATA3+) was markedly depressed in SLE (0.11%, 0.1-0.255%) versus control (0.41%, 0.25 - 0.55%; p = 0.0293). ILC2s protect epithelial integrity; a reduction suggests impaired protective roles in SLE. It would be interesting to study in further detail the changes in other protective and regulatory compartments. Supervised cell frequencies from bivariate analysis correlate strongly with unsupervised cell frequencies, validating these results (Pearson’s correlation coefficient r = 0.9926, p < 0.001 (CD4+CD152+PD1+CD45RO+CD25-FoxP3-); r = 0.8863, p < 0.05 (ILC2)). Conclusion: With a multi-parametric unbiased approach comparing SLE subjects to a database of age-matched healthy controls, we identified two immune subsets of potential immunopathogenic importance for further studies. This would guide the redesign of the CyTOF panel to include more functionally important organ-specific markers such as skin-homing or kidney-homing receptors. Data obtained from this study will also form the basis of the paediatric SLE database for comparison with adult SLE CyTOF data, which we have commenced work on. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
I. Pizarro Madureira Salgado Da Costa 1 , L. Fernandes 2 , M. P. Ramos 3 1 Unidade de Reumatologia Pediátrica, Centro Hospitalar Universitário Lisboa Central, 2 Unidade de Radiologia, Centro Hospitalar UniversitárioLisboa Central, 3 Unidade de Reumatologia Pediátrica, Centro Hospitalar Universitário Lisboa Central, EPE, Lisboa, Portugal Correspondence: I. Pizarro Madureira Salgado Da Costa Introduction: Eosinophilic fasciitis is a rare disorder presenting with skin edema and erythema, followed by collagenous thickening of the subcutaneous fascia. Its pathogenesis is poorly understood and been associated with multiple conditions, such as strenuous exercise, exposure to certain medications as well as hematological and autoimmune diseases. Objectives: Increase awareness for the diagnosis of eosinophilic fasciitis Methods: Report of a patient with eosinophilic fasciitis and juvenile systemic lupus erythematosus Results: A 17-year-old girl was referred to a tertiary hospital with a 9-month history of fatigue and migratory arthralgias. In the previous month, she also reported limb edema and a generalized photosensitive rash. Her medical history was unremarkable, with no regular medication. Her sister had been diagnosed with juvenile systemic lupus erythematosus. Physical examination revealed a disseminated maculopapular lupus rash with scaly appearance, as well as a malar rash. There was also bilateral erythema, edema and thickening of the skin of the four limbs and trunk, sparing the fingers and toes. Laboratory investigations showed non regenerative anemia, eosinophilia, elevated erythrocyte sedimentation rate, elevated transaminases and LDH; positive ANA, anti-double-stranded DNA, anti-nucleosome, antihistone, RF and CCP; low levels of C3 and functional CH50 complement. Direct Coombs test, creatine-kinase, creatinine, urea, electrolytes, urine analysis and thyroid hormones were within normal limits. Systemic sclerosis antibody panel and antiphospholipid antibodies were negative. Diffusing capacity for carbon monoxide, cardiac ultrasound and ECG were normal. Nailfold capillaroscopy revealed a normal pattern. Skin biopsy revealed an infiltrate of lymphocytes and plasma cells surrounding the superficial and deep vessels, the adipocytes and the skin derivatives in the dermis. In immunofluorescence, a dense linear pattern of IgM was found in the dermoepidermal junction. Whole-body MRI showed edema and thickening of the fascia and subcutaneous tissue, affecting the trunk and the limbs. The diagnosis of juvenile erythematous lupus and eosinophilic fasciitis was established and treatment with high dose steroids and hydroxychloroquine was started. During the following months, clinical stability was achieved and steroids were gradually tapered. However, skin edema recurred and she developed an irregular, peau d'orange texture and a “groove sign” of the arms. Methotrexate was added after nine months of the diagnosis. The patient responded favorably and steroids were suspended. During the last 2 years of follow-up there was no recurrence of the eosinophilic fasciitis. Conclusion: Eosinophilic fasciitis is a scleroderma-like syndrome predominantly affecting the extremities, sporadically seen in children. In contrast to systemic sclerosis, sclerodactyly, Raynaud’s phenomenon, internal organ involvement and nailfold capillaries abnormalities are uncommon. Early recognition and treatment are essential in order to prevent long-term disabling outcomes in eosinophilic fasciitis. Myalgias, fatigue and, less frequently, arthritis are commonly associated symptoms. Eosinophilia is a prominent laboratory finding in the early phase of this disease. MRI is an important tool for the diagnosis, particularly when the skin biopsy is inconclusive. Eosinophilic fasciitis has been described in association with collagen tissue diseases, such as Sjögren’s syndrome and systemic lupus erythematosus. To the best of our knowledge, this is the first reported case of juvenile eosinophilic fasciitis associated with inaugural systemic lupus erythematosus. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
A. Prabhudesai, A. Khan, N. P. Maldar, R. Khubchandani NH SRCC Children’s Hospital, Mumbai, India Prabhudesai Introduction: Drug induced lupus (DIL) has a clinical phenotype resembling that of idiopathic systemic lupus erythematosus (SLE) but usually without any major organ complications and which resolves after drug withdrawal. The drugs commonly implicated in DIL include hydralazine, procainamide, anti-tuberculous drugs like isoniazid and more recently reported tumor necrosis factor α (TNFα) inhibitors. Levamisole is a synthetic Imidathiazole derivative, originally used as an anthelminthic with potent immunomodulatory properties. This low-cost drug has been widely used in dermatology especially in developing countries for the management of various dermatoses including Alopecia areata. Although not a commonly recognized cause of DIL, Levamisole has been associated with widespread autoimmunity mainly in the form of cutaneous vasculitis and Antineutrophil cytoplasmic antibodies (ANCA) associated vasculitis with a few reports on lupus-like side effects. Objectives: To alert practitioners about Levamisole induced autoimmunity so as to enable early diagnosis and avoid an extensive diagnostic work up. Methods: A 7-year-old girl had developed patchy alopecia on the scalp for which she had been taking Levamisole 3mg/kg twice a week for 2 years. Results were satisfactory and she did well for about 1.5 years while continuing the drug. However, at this point in time she was referred to the rheumatology department for fever, arthralgia, mouth ulcers, increased hair fall and a positive Antinuclear antibody (ANA) (titre 1+). On examination, besides the patchy alopecia, she also had short stature (121cm; between 3 rd -10 th percentile), pallor and bilateral hallux valgus. Her spine Xray showed bullet shaped vertebrae and the labs suggested a Coombs positive hemolytic anemia, with normal complements. In light of these findings and the presence of consanguinity, albeit distant, we suspected a monogenic cause of lupus and a whole exome sequencing was advised. The genetic report showed a homozygous nonsense variation in exon 12 of the PAPSS2 gene (c.1666C>T) (Brachyolmia Type 4 with Mild Epiphyseal and Metaphyseal Changes (OMIM 612847)). This explained the skeletal findings, but it failed to explain the autoimmunity. Since hemolytic anemia and disseminated autoimmunity have been reported with Levamisole, she was advised to omit Levamisole and was kept under follow up. Results: Her symptoms improved rapidly without the need for steroids or immunomodulators. After 4 months her general condition improved. Fever and arthralgias subsided and hemoglobin improved while Coombs turned negative and the acute phase reactants normalised. We thus concluded it to be Levamisole induced autoimmune haemolytic anemia. Only the alopecia was persistent for which she was subsequently started on Methotrexate 12mg/m 2 subcutaneously. She showed dramatic improvement and now has full hair growth and no systemic complaints. Conclusion: Levamisole is a widely used immunomodulatory drug. Knowledge about its potential to induce autoantibodies should caution practitioners against its long-term use and enable early diagnosis of levamisole induced autoimmunity with less aggressiveRadziszewska 1,2 , H. Peckham 1,2 , N. M. de Gruijter 1,2 , O. Nettey 1,2, UCLH, GOSH, 2 Centre for Rheumatology Research, Division of Medicine, University College London, London, United Kingdom Correspondence: A. Radziszewska Introduction: CD8 + T cells and NK (natural killer) cells are cytotoxic lymphocytes which play a role in adaptive and innate immune responses to infection. They exert their effector functions by releasing toxic proteins such as perforin and granzymes to kill target cells. Recent evidence suggests that aberrations in cytotoxic cell function can contribute to tissue damage in autoimmunity. SLE (Systemic Lupus Erythematosus) is a multisystem autoimmune disease characterized by production of autoantibodies against nuclear antigens. While B cells and CD4 + T cells have been studied extensively in SLE, there is limited and at times contradictory evidence of how cytotoxic cells contribute to disease pathology, in particular, in the more severe, juvenile form of the disease (JSLE). Objectives: The aim of this study is to investigate the phenotype and function of cytotoxic lymphocytes in patients with JSLE compared to age/sex-matched healthy controls (HCs). Methods: Peripheral blood mononuclear cells (PBMCs) and serum were collected from JSLE patients (n=40, 15.6-29.8 years), and age/sex-matched healthy controls (n=51, 15.2-31.3 years). Ex vivo PBMCs and PBMCs cultured for 72hr with anti-CD3 were stained with anti-CD3, -CD8, -CD56, -CD45RO, -CD132, -perforin, -granzyme A, -granzyme B, -granulysin, -CD107a, -IFN-γ, -TNF-α, and -IL-2 antibodies and analysed by flow cytometry. Serum perforin levels were measured using a multiplex bead-based assay in 17 patients and 18 HCs. NK function was assessed by incubating isolated NK cells from JSLE patients or HCs together with CFSE labelled K562 target cells for 4hrs. After co-incubation cells were stained with DRAQ7 dye to identify non-viable cells via flow cytometry. Statistical significance was calculated using Mann-Whitney U tests or t-tests as appropriate. Results: Patients with JSLE had diminished frequencies of CD8 + T cells expressing perforin (p=0.028) and effector cytokines IFN-γ (p=0.029) and TNF-α (p=0.01) compared to HCs. These cytotoxic and pro-inflammatory molecules were in large part produced by memory CD8 + T cells and frequencies of effector memory and central memory CD8 + T cell subsets were reduced in peripheral blood in JSLE (p=0.0048 and p=0.0017, respectively). In addition, the proportions of NK cells as a whole (p=0.000012), as well as perforin (p=0.000006) and granzyme A (p=0.00013) expressing NK cells were markedly diminished in JSLE patients versus HCs. This was accompanied by a reduction in serum perforin concentration in JSLE (p=0.025). Reductions in cytotoxic capacity in JSLE were observed regardless of treatment regimen or patient disease activity. The expression of CD8 + T cell cytotoxic markers and intracellular cytokines was rescued by stimulation of the T cell receptor in vitro with anti-CD3, indicating that a mechanistic defect in cytotoxic mediator/cytokine production is unlikely in JSLE. NK functional killing assays revealed that NK cells from patients with JSLE were as effective at killing labelled targets as NK cells from HCs (mean % specific target death 12.27 vs 11.12, respectively, p=0.71). Conclusion: Cytotoxic capacity of CD8 + and NK cells as well as CD8 + memory T cell and NK cell frequencies were reduced in patients with JSLE. NK cell cytotoxic function appeared to be preserved in JSLE. Further work is necessary to establish if there is a functional defect in CD8 + T cells in JSLE and to ascertain if the reduction in cytotoxic cell frequencies is due to migration of these cells out of peripheral blood into affected tissuavindran 1 , D. Patro 2 , A. P. Rao 2,3 , J. Raghuram 3,4 1 Pediatrics, 2 Pediatric Rheumatology, Manipal Hospital, Ola Airport Road, Bangalore, 3 Pediatric Rheumatology, Indira Gandhi Institute of Child Health, 4 Pediatric Rheumatology, Aster Whitefield, Bangalore, India Correspondence: S. Ravindran Introduction: Systemic Lupus Erythematosus (SLE) is a chronic multiorgan autoimmune inflammatory disease and nephritis is a major risk factor for morbidity and mortality in SLE. Objectives: To analyze the clinical and laboratory profile of children with lupus nephritis in a cohort of SLE patients attending a pediatric rheumatology clinic in SouthIndia. Methods: We retrospectively reviewed 60 cases of biopsy proven lupus nephritis from a total of 134 patients with SLE over a period of 10 years (April 2011-March 2021) in the Pediatric Rheumatology Clinic in Manipal hospital, Bangalore, India. Results: The findings are summarized in the table below. Out of 134 children with SLE, 60 (44.7%) had biopsy-proven LN. 60% had renal involvement at the onset, 88.3% within 6 months, and 100% patients had presented within 2 years of onset of the disease. Class IV (41.7%) was most common followed by class III (25%). Conclusion: Majority of the children who develop LN, do so in the initial 2 years of the disease onset. Anti dsDNA antibody elevation was seen in majority of patients. Presence of anti dsDNA might indicate increased risk of developing LN and should be followed more closely. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
Sahin 1 , E. M. Smith 2,3 on behalf of the UK JSLE Study Group, A. Corley 2,3 on behalf of the UK JSLE Study Group, C. Ciurtin 4 , S. Demir 5 , D. Schonenberg-Meinema 6 , N. Kifer 7 , A. L. Rodríguez-Lozano 8,9 , E. Marasco 10 , C. Bracaglia 10 , A. Rodrigues Fonseca 11 , M. F. Rodrigues 11 , G. Kavrul Kayaalp 12 , N. Aktay Ayaz 12 , S. Tharwat 13 , E. Marrani 14 , K. Bouchalova 15 , D. Kleparnik 15 , S. K. Feitosa de Oliveira 16 , B. Kasap-Demir 17,18 , P. Costa-Reis 19 , T. Kallinich 20 , A. S. L. von Stuckrad 20 , K. Tenbrock 21 , K. Vollbach 21 , E. Demirkaya 22 , M. J. Wahadat 23,24 , H. C. Culpan 25 , M. Jelusic 7 , S. Ozen 5 , O. Kasapcopur 1 , M. W. Beresford 3,26 on behalf of the UK JSLE Study Group, S. Kamphuis 24 1 Department of Pediatric Rheumatology, Istanbul University Cerrahpasa, Istanbul, Turkey, 2 Department of Women’s & Children’s Health, Institute of Life Course and Medical Sciences, University of Liverpool, 3 Department of Paediatric Rheumatology, Alder Hey Children’s NHS Foundation Trust, Liverpool, 4 Centre for Adolescent Rheumatology, University College London, London, United Kingdom, 5 Department of Pediatrics, Division of Rheumatology, Hacettepe University Faculty of Medicine, Ankara, Turkey, 6 Department of Pediatric Immunology, Rheumatology and Infectious Diseases, Amsterdam UMC, University of Amsterdam, Emma Children’s Hospital, Amsterdam, Netherlands, 7 Department of Paediatrics, University of Zagreb School of Medicine, University Hospital Centre Zagreb, Zagreb, Croatia, 8 Servicio de Inmunología, Instituto Nacional de Pediatría, 9 Programa de Maestría y Doctorado en Ciencias Médicas, Odontológicas y de la Salud, Universidad Nacional Autónoma de México (UNAM), Mexico City, Mexico, 10 Division of Rheumatology, Bambino Gesù Children’s Hospital, IRCCS, Rome, Italy, 11 Pediatric Rheumatology Unit ,2 Department of Pediatric Rheumatology, Istanbul Faculty of Medicine, Istanbul University, Istanbul, Turkey, 13 Rheumatology & Immunology Unit, Department of Internal Medicine, Faculty of Medicine, Mansoura University, Mansoura, Egypt, 14 Pediatric Rheumatology Unit, Aou Meyer, Firenze, Italy, 15, 16 Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil, 17 Department of Pediatrics Division of Nephrology & Rheumatology, İzmir Katip Çelebi University, Izmir, 18 Department of Pediatrics, Health Sciences University İzmir Tepecik Training and Research Hospital, Izmır, Turkey, 19 Pediatric Rheumatology Unit, Hospital de Santa Maria, Faculdade de Medicina, Universidade de Lisboa, Lisbon, Portugal, 20 Department of Pediatric Respiratory Medicine, Immunology and Critical Care Medicine, Charité Universitätsmedizin Berlin, Berlin, 21 Department of Pediatrics, RWTH Aachen University Hospital, Aachen, Germany, 22 Department of Paediatrics, Division of Paediatric Rheumatology, Schulich School of Medicine & Dentistry, University of Western Ontario, London, ON, Canada, 23 Department of Immunology, Erasmus Medical Center, University Medical Centre Rotterdam, 24 Department of Paediatric Rheumatology, Erasmus MC Sophia Children’s Hospital, Rotterdam, Netherlands, 25 Department of Public Health, Istanbul University Cerrahpasa, Istanbul, Turkey, 26Liverpool, United Kingdom Correspondence: S. Sahin Introduction: A higher damage score is a strong predictor of increased mortality in lupus patients. Objectives: To describe the prevalence, predictors and organ-distribution pattern of damage in a large multinational cohort of cSLE patients. Methods: Detailed data on all items of PedSDI, ACR 1997, SLICC 2012 and EULAR/ACR 2019 were collected over the entire disease course for patients from 19 centers across Europe, Africa, Middle East, North and South America Results: Overall, 1096 cSLE patients were included; One-third (32.5%) of the participants were >18 years of age at last visit. Distribution of ethnicity was as follows: 62% White, 18% Asian, 11% Black (African, Afro-Caribbean etc.), 5 % Mixed and 4% Latin American. After a mean disease duration of 6 years, 35% of the patients had accrued damage (PedSDI damage score of ≥1) in at least one PedSDI domain; 20.2% of all study group had a PedSDI score of 1; 7.1% had a score of 2; 4.1% had a score of 3 and the remaining 3.6% had a score of ≥4. The mean PedSDI score at last visit was 0.62 ± 1.1 (range 0–7). Participants with early onset disease (<10 years) were more likely to have damage as compared to those with later onset disease (≥10 years) (p<0.05). Among all items of the 3 classification criteria, the presence of discoid rash (p<0.05), renal disease (p <0.01), neurological disorder/domain (p<0.001), chronic cutaneous lupus (p<0.05), anti-Sm positivity (p<0.05), lupus anticoagulant (p <0.01), anticardiolipin antibodies (p<0.05), or constitutional symptoms/signs (p<0.05) was associated with damage. Other damage predictors (not included in 3 classification criteria) were as follows: systemic hypertension (p<0.001), fulfillment of Sapporo classification criteria for antiphospholipid antibody syndrome (p<0.001), anti-RNP positivity (p<0.05), anti-SSA positivity (p<0.05), anti-SSB positivity (p<0.05), corticosteroid (p<0.001) and cyclophosphamide usage (p<0.001). No correlation was found between PedSDI and SLEDAI-2K scores neither at disease onset nor at last visit. Conclusion: This PReS Lupus Working Party initiative multicenter study, one of the largest multinational cohort of patients with cSLE to date, indicate that one-third of the cSLE patients develop damage after an average disease duration of 6 years and this was not correlated with SLEDAI-2K scores. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
M. Frkovic 1 , D. Grguric 1 , S. Barisic 1 , J. Ellyard 2 , M. Cook 2 , T. Arsov 3 , C. G. Vinuesa 4 , M. Jelusic 1 1 University of Zagreb School of Medicine, UHC Zagreb, Zagreb, Croatia, 2 Department of Immunology and Infectious Diseases, The John Curtin School of Medical Research, Australian National University, Canberra, Australia, 3 Institute of Immunobiology and Human Genetics, Faculty of Medicine, University in Skopje, Skopje, North Macedonia, 4 The Francis Crick Institute, London, United Kingdom Correspondence: M. Sestan Introduction: Childhood-onset systemic lupus erythematosus (cSLE) is a complex disease with not fully understood pathogenesis that may involve multiple interactions between genetic, epigenetic and environmental factors. Genes may play a more important role than environmental and hormonal factors in cSLE, as opposed to adult-onset SLE. Objectives: The purpose of the research was to point out new and rare gene variants using the whole exome sequencing (WES) in patients with cSLE that may be involved in the pathogenesis and to expand existing genetic databases. Methods: This multiple case study included patients with cSLE, based on the revised classification ACR-97 criteria and the 2012 SLICC criteria, reviewed in University Hospital Centre Zagreb in the period 1991 to 2019. We performed WES on selected 17 “trios” containing proband case with cSLE and parents, including other informative family members, with severe, atypical clinical features, syndromic characteristics, early onset of the disease, resistance to conventional therapy and/or family pattern of occurrence. Results: Overall, in 7 patients with cSLE the total number of 17 novel and/or rare variants that may contribute to disease was discovered. One variant was classified as pathogenic according to the American College of Medical Genetics (ACMG) classification guidelines. This was structural, frame-shift variant in exon 34 of KMT2D gene (NM_003482.3:c.8626delC; 55 reads C, 56 reads delC), predicted to truncate the protein (p.Gln2876Serfs*34), occurred de novo in patient with cSLE complicated with lupus nephritis, neurolupus, immunodeficiency and severe intestitial lung disease and previously unrecognized syndrome features consistent with Kabuki syndrome. In addition, we identified four likely pathogenic variants. Two of them were very rare heterozygous mutations: the first in ADAR1 gene (NM_001111.3:c.2815A>G; 13 reads A, 15 reads G), predicted to encode the protein (p.Ile939Val), and the second in SH2B3 gene (NM_005475.2:c.398G>A; 7 reads G, 6 reads A) predicted to encode (p.Cys133Thr). Both of them were present in a patient with syndromic features, skin pigmentation changes and cSLE, complicated with lupus nephritis, CNS involvement, immunodeficiency and coagulopathy. The third one was heterozygous mutation in BLK gene (NM_001715.2:c.211G>A) predicted to encode (p.Ala71Thr), with low prevalence in population databases, in a patient with cSLE complicated with myocarditis, nephritis and pancreatitis, inherited from the mother affected with SLE. The same patient and her mother carry rare frame-shift variant in CR1 gene (NM_000651.4:c.4052dup) predicted to encode the protein (p.Asp1351Glufs*23). We have also identified a number of variants classified as variants of uncertain significance (VUS). These are gene variants that encode proteins involved in various cellular signaling pathways, especially related to tyrosine phosphorylation; differentiation, survival, and proliferation of cells; V(D)J recombination in developing lymphocytes; regulation of apoptosis and Th1 and Th2 polarization. Conclusion: Although application of WES has improved the diagnostics and treatment of patients with autoimmune diseases, there is growing number of variants that we still cannot use in the clinical context. It is necessary to improve methods to properly categorize them and increase the amount of practicable information from WESV. Sevostyanov 1 , P. Lototskaya 2Systemic lupus erythematosus is a systemic autoimmune disease of unknown etiology, which is based on a genetically determined violation of immune regulation, which determines the formation of organ-specific antibodies to antigens of cell nuclei with the development of immune inflammation in the tissues of various organs. Objectives: Make a clinical and epidemiological analysis of systemic lupus erythematosus (SLE) according to the Moscow register of children with rheumatic diseases. Methods: An observational descriptive cross-sectional study was conducted, including 65 patients diagnosed with systemic lupus erythematosus, aged 7 to 17 years (inclusive), of which 74% (n=48) were girls and 26% (n=17) were boys. All patients live in Moscow and are included in the city register of children with rheumatic diseases. Results: The median age of patients was 16 years, the median age at the disease onset was 12.58 months. Half of the patients (n=33) had an initial diagnosis other than SLE. When verifying the diagnosis, from the clinical criteria of SLICC-2012, the most common patients had acute and subacute skin lesions - 48%, thrombocytopenia - 35%, arthritis - 32% and leukopenia - 31%. According to the nature of the course of SLE, an acute course was detected in 27.7% of patients, subacute - in 67.7%, and primary chronic - in 4.6% ofminimal (40.0%) or its absence was noted (30.8%). Basic anti-inflammatory therapy was received by 98.5% of patients, 87.5% of them in a combination of several drugs. Genetically engineered biological therapy is received by 23% of patients. In the structure of GIBT, rituximab predominates - 73.3%. Conclusion: SLE is one of the most complex diseases in childhood, to improve the prognosis of which requires timely diagnosis and early initiation of antirheumatic therapy. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
J. Sheekha 1 , A. Sharma 2 , S. Guleria 2Systemic Lupus Erythematosus (SLE) is a prototype of autoimmune diseases. It is characterized by multisystem inflammation due to exaggerated B- cell and T-cell responses and loss of immune tolerance against self-antigens. Pediatric SLE (pSLE) in comparison to adult onset SLE presents with more severe symptoms at the onset of disease and has a more aggressive clinical course. Avascular necrosis of the hip can occur as a complication of the disease itself or that of treatment. Objectives: To study the clinical profile and presence of osteonecrosis as a complication of pSLE patients from Dr Rajendra Prasad Government Medical College (DRPGMC), Kangra, Himachal Pradesh, India. Methods: Case records of children with systemic lupus erythematosus registered in Pediatric Rheumatology Clinic (PRC) at DRPGMC from July 2017 to Dec 2022 were reviewed for the demographic and clinical characteristics and associated complications, particularly osteonecrosis (ON). We here report this data and cases of pSLE who developed osteonecrosis of hip joints over course of their illness and treatment. Results: During the above-mentioned period, a total of 17 pSLE patients were registered in PRC. Out of which 15 were females (F:M=7.5:1). 24% patients were in the age group of 6-10 years, 35% between 11-15 years and 16-18 year age group comprised 41% of the total SLE patients. There were no patients below 5 years of age. Fever and arthralgia/arthritis were the most common manifestation at the time of presentation followed by rash and photosensitivity. Other clinical features noted were alopecia, headache, myalgia, Raynaud phenomenon and oral ulcers. 45% patients were positive for dsDNA. Hypocomplementemia was seen in 75% of patients at the time of diagnosis. Lupus nephritis was noted in 35% (6/17) of patients. 2 patients belonged to class III nephritis, one each to class II, IV and V. Renal biopsy was not done in one patient. All the patients were initiated on hydroxychloroquine. Two patients developed macrophage Activation Syndrome (MAS) during the clinical course. Interestingly, 2 female patients developed symptomatic osteonecrosis of bilateral hips diagnosed 7 years and 1 year after diagnosis and initiation of treatment. Both had received pulse methylprednisolone and were on tapering doses of oral gluco-corticoids. They were treated with core decompression and autologous bone marrow infiltration. Both were doing well on follow-up after 2 and 2.5 years of surgery. Conclusion: DISCUSSION Osteonecrosis (ON) or avascular necrosis (AVN) is, though uncommon, a known complication of pSLE. It can occur in patients with pSLE due to disease or as a complication of treatment (cortico-steroids). It has been reported to occur in these patients even without initiation of steroids and can occur early after initiation of cortico-steroids. CONCLUSION SLE is a multisystem disorder and has varied clinical presentations most common being fever, arthritis, and nephritis. Use of corticosteroids in the treatment of SLE makes it challenging to distinguish between the role of disease itself and that of treatment in the pathogenesis of AVN. Regular screening and preventive measures like the use of bisphosphonates along with vitamin D and calcium might helpE. Smitherman 1 , R. A. Chahine 1 , A. O. Hersh 2 , J. R. Curtis 1 1 University of Alabama at Birmingham, Birmingham, 2 University of Utah, Salt Lake City, United States Correspondence: E. Smitherman Introduction: Data is lacking regarding modern real-world practices and disease outcomes of individuals with childhood-onset systemic lupus erythematosus (cSLE) and lupus nephritis, especially across multiple centers. However, researchers also lack approaches to identify end-stage kidney disease (ESKD) in patients with cSLE utilizing claims-based algorithms. Objectives: To determine optimal definitions that can be used to identify patients with cSLE, lupus nephritis, and ESKD using a large, real-world administrative claims database. Methods: We utilized administrative claims data from IBM MarketScan Commercial and 8-State Medicaid Databases from 2006-2020. We adapted previously validated definitions to identify patients with cSLE: 1) ≥ 3 ambulatory or inpatient claims with ICD-9 code 710.0 or ICD-10-CM code M32.* (excluding M32.0 for drug-induced lupus) with at least 30 days between each code; 2) codes restricted to provider type pediatric rheumatology, rheumatology, pediatric nephrology, nephrology, dermatology, or acute care hospital; and 3) age ≥ 5 and <18 years at the time of the first SLE code. To define incident diagnoses, we selected patients with: 1) at least 182 days between insurance enrollment and the first SLE code; and 2) no evidence of prior anti-malarial or immunosuppressive use more than 182 days before the first SLE code. We then adapted previously validated definitions to select the subgroup of cSLE patients with evidence of lupus nephritis using ≥ 2 ambulatory or inpatient claims with ICD-9 codes 580.*-586.*, 791.0 or ICD-10-CM codes M32.14, M32.15 with at least 30 days between each code. Finally, we defined presence of ESKD using: 1) procedure and diagnosis codes for dialysis from any encounter; 2) procedure and diagnosis codes for kidney transplant from any encounter; or 3) ≥ 3 claims with diagnosis codes for ESKD from any encounter type. We considered each ESKD criteria separately and as a composite if any of the 3 criteria were met. We calculated the frequency of patients meeting each of these criteria. Results: From 2006-2020, we identified 2,590 individuals receiving care for cSLE in the IBM MarketScan Commercial and 8-State Medicaid Databases, of which 1,302 met criteria for an incident diagnosis. Definition for lupus nephritis was met in 580 (45%) of incident cSLE cases. Of individuals meeting the lupus nephritis definition, 166 (29%) had evidence of ESKD: 100 (17%) with dialysis, 83 (14%) with kidney transplant, and 69 (12%) with other ESKD diagnosis codes. Only 35 (4%) of 722 individuals with incident cSLE who did not meet lupus nephritis definition had evidence of ESKD: 24 (3%) with dialysis, 9 (1%) with kidney transplant, and 2 (0.3%) with other ESKD diagnosis codes (Table 1). Conclusion: This study of individuals with evidence of cSLE and lupus nephritis within a US administrative claims database revealed a relatively high frequency of meeting ESKD criteria among cSLE patients. We identified few individuals with evidence of ESKD who did not meet previously validated lupus nephritis definitions; nevertheless, ESKD care should be included in the definition of lupus nephritis when using administrative claims algorithms. Disclosure of Interest : None declared
isparities in long-term kidney outcomes have been documented in patients with childhood-onset systemic lupus erythematosus (cSLE) complicated by lupus nephritis (LN). However, there remains limited understanding of the impact of health care quality, including access to care, on disease outcomes. Objectives: To evaluate early care utilization patterns as a measure of access in individuals identified with cSLE and LN using a large, real-worldidentified individuals with cSLE using: 1) ≥3 ambulatory or inpatient claims with ICD-9 710.0 or ICD-10-CM M32.* (excluding M32.0) with ≥30 days between each code; 2)5 and <18 years at the time of the first SLE code. We then selected=“selected” patients with: 1) ≥182 days between insurance enrollment and the first SLE code; and 2) no evidence of anti-malarial or immunosuppressant use >182 days prior to the first SLE code to define incident cases. Finally, we identified cSLE patients with LN by ≥2 ambulatory or inpatient claims with ICD-9 580.*-586.*, 791.0 or ICD-10-CM M32.14, M32.15 with ≥30 days between. We abstracted sex, age at first SLE code, codes for dialysis, codes for kidney transplant, and time between first SLE and first LN code. For patients enrolled in commercial insurance, we abstracted US geographic region and population density. For those enrolled in Medicaid, we abstracted race and ethnicity. We divided patients by whether SLE codes or LN codes were used first. Descriptive statistics and bivariate analyses were conducted with significance levels set to 0.05. Results: We identified 580 individuals who met criteria for both incident cSLE and LN. Patients with LN were 82% female and had a mean(SD) age at first SLE code of 14.5(2.9) years. In patients with commercial insurance, the majority were from the US South (51%), followed by West (18%), Northeast (16%), and Midwest (15%). We noted that while there was a mean(SD) time between first SLE and LN diagnosis codes of 0.5(1.5) years, there was a range from -9.5 to 9.7 years. We grouped patients into those who received SLE codes first (77%) and those with LN codes first (23%). In the LN codes first group, we noted a statistically higher proportion of patients with Medicaid (43% vs 29%, p=0.002), living in a rural metropolitan statistical area (14% vs 9%, p=0.032), of Black or unknown race (Table 1), and of Hispanic ethnicity (28% vs 8%, p<0.001). Patients with LN codes first were also more likely to have evidence of kidney transplant. Conclusion: In this group of individuals identified with cSLE and LN, we noted two distinct patterns of whether patients first received SLE diagnosis codes or LN diagnosis codes. Patients who received LN codes before SLE codes were more likely to be publicly insured, live in a rural area, be Black or Hispanic, and have evidence of kidney transplant. Additional analyses are planned to better understand if these patterns reflect heterogeneous disease course or differential access to subspecialty careI. Suardi 1,2 , C. B. Chighizola 1,2 , M. Gattinara 3 , G. Carrea 1,2 , L. M. Argolini 1 , R. Caporali 1,2 , M. Gerosa 1,2 1 Unit of Clinical Rheumatology, ASST Pini – CTO, Milan, Italy, 2 University of Milan, 3 Unit of Pediatric Rheumatology, ASST Pini – CTO, Milan, Italy, Milan, Italy Correspondence: I. Suardi Introduction: In approximately 10-20% of cases, systemic lupus erythematosus (SLE) begins in pediatric age; juvenile SLE (jSLE) tends to have a severe presentation. SLE has a remitting course; infections and vaccinations have been proposed as potential triggers of disease flares. Therefore, the safety of anti-SARS-COV2 vaccination has been questioned in jSLE. Objectives: The aim of this study consists in evaluating side effects and disease flare after anti-SARS-COV2 vaccination in a monocentric cohort of patients with jLES. Methods: We retrospectively reviewed the clinical records of patients with jSLE diagnosed before 18 years of age regularly followed-up at our institution. The following data were collected: demographic details, clinical and serological features, ongoing pharmacological treatment, anti-SARS-COV2 vaccination, side effects and disease flare after vaccination. Low lupus disease activity state (LLDAS) was defined according to SLEDAI; disease flare was defined as a new BILAG A or B score in any system. Continuous data are expressed as median (interquartile range [IQR]) and categorical data as percentages. The association between categorical variables was assessed by chi-squared or Fisher exact tests. Univariate logistic regression analyses were drawn. Statistical analysis was performed using Stata v14. Results: Out of the 70 patients with jLES, 65 subjects (93%) received at least one dose of anti-SARS-COV2 vaccine (75% Pfizer-BioNtech, 25% Moderna, Table 1). A 3-dose immunization schedule was completed in 46 cases (46% Pfizer, 26% Moderna, 28% heterologous booster). Adverse events were registered in 28 cases (43%); 24 of the 46 patients completing the vaccination schedule (52%) had side effects after the third shot. The most common side effects were: fever (26%), fatigue (23%), pain at the injection site (21%) and arthromyalgias (23%). Ongoing treatment with immunosuppressors and belimumab was associated with an increased risk of adverse events (odds 2.88, p=0.04 and odds 5.5, p=0.03, respectively). Patients with previous musculoskeletal involvement had more commonly side effects to anti-SARS-COV2 vaccine (p=0.066). A disease flare occurred in 7 cases: 5 after the first and second shot (71%) and 2 after the third (29%), at a median time of 41 days and 27 days after vaccination, respectively. Flares consisted in lupus nephritis (71%) and arthritis (28.6%). 5 subjects experiencing a lupus flare had a moderate disease activity before starting the vaccination program, while 2 patients were in remission. Overall, patients with LLDAS before first vaccination dose displayed a lower rate of flare (p=0.059, χ2=5.25). Conclusion: Our data suggest that anti-SARS-COV2 vaccination is well tolerated among patients with jLES, even though patients receiving immunosuppressors and/or belimumab have an increased hazard of side effects. Disease flares after anti-SARS-COV2 vaccination occur rarely in patients with LLDACheawcharnprapan, T. Kanjanaphan, O. Lertkovit, O. Sirimongkolchaiyakul, S. Tangcheewinsirikul Paediatrics, Faculty of Medicine Vajira Hospital, Navamindradhiraj University, Bangkok, Thailand Correspondence: S. Tangcheewinsirikul Introduction: Epstein–Barr virus (EBV) encephalitis-associated hemophagocytic lymphohistiocytosis (HLH) syndrome causes high morbidity and mortality. Rarely reported in patients with systemic lupus erythematosus (SLE), EBV encephalitis-associated HLH is even rarer in patients with childhood-onset SLE (c-SLE). Objectives: Here, we report the unique case of a 12-year-old female patient with c-SLE who presented with neuropsychiatric (NP) manifestations. Methods: Diagnostic workup included basic and immunological blood tests, cerebrospinal fluid (CSF) analysis, brain imaging, and bone marrow assessment. Results: Two months before admission, the patient was diagnosed with c-SLE based on the presence of malar rash, polyarthritis in bilateral hands, hypocomplementemia, and antinuclear antibody positivity. Despite treatment with prednisolone and hydroxychloroquine for two months, she experienced a generalized tonic seizure and exhibited progressive altered mental status, visual and auditory hallucinations, and low-grade fever for several weeks. Physical examination at admission revealed fever, BP 118/84 mmHg with an inability to incorporate time/place/person, and hepatosplenomegaly. A neurological exam showed no cranial nerve involvement, normal muscle tone, and normal deep tendon reflexes. Other evaluations were unremarkable. Laboratory tests revealed the following: erythrocyte sedimentation rate, 86 mm/h (normal range, 0–20 mm/h); C-reactive protein, 8.9 mg/L (normal range, 0–5 mg/L); creatinine, 0.42 mg/dL; and blood urea nitrogen, 10 mg/dL. Additional laboratory tests to evaluate for HLH revealed markedly elevated levels of ferritin (3966 [normal range, 13–150] ng/mL) and fasting triglycerides 850 mg/dL. Complete blood count showed pancytopenia with urinalysis showed significant proteinuria based on a urine protein-creatinine ratio of 3.8. CSF analysis revealed 60/mm 3 WBCs, 110,000/mm 3 RBCs, 141 mg/dL glucose (blood–CSF glucose ratio of 0.7), and <4 mg/dL protein with negative gram stain and bacterial culture. Magnetic resonance imaging and magnetic resonance angiography of the brain did not reveal evidence of thromboembolic events or bleeding. Broad-spectrum antibiotics and acyclovir were initiated. Based on the lack of bacterial growth after 48 h in blood culture, pulse intravenous methylprednisolone (IVMP) (1000 mg; 30 mg/kg/day) for three days was initiated for the treatment of MAS. Bone marrow aspiration biopsy revealed the presence of histiocytes with active hemophagocytic activity, which led to the potential diagnosis of HLH or MAS secondary to active SLE. Despite the initial aggressive treatment with pulse IVMP, the patient had persistent altered mental status, pancytopenia, and hyperbilirubinemia. Therefore, other potential causes of secondary HLH, such as infection and thrombotic microangiopathy, were considered. Renal biopsy revealed class II lupus nephritis without evidence of thrombosis. The CSF analysis for viral encephalitides revealed EBV positivity using polymerase chain reaction. After a multidisciplinary team discussion, the HLH treatment protocol, including intravenous immunoglobulin, corticosteroids, and cyclosporin A, was initiated. After the treatment initiation, the patient did not have fever, seizures, or hallucinations. Her level of consciousness and laboratory parameters gradually improved. Conclusion: The current case illustrates the diagnostic approach for a patient with c-SLE who presented with NP symptom and refractory to the treatment of MAS with pulse IVMP. The need to consider other potential etiologies, the multidisciplinary care team’s approach and prompt management are recommended to achieve maximum benefit for immunosuppressivM. Tsinti, V. Dermentzoglou, E. TsitsamiAthens, Greece Correspondence: M. Tsinti Introduction: Neuropsychiatric manifestations are frequent among JSLE patients but most often correlate high polysystemic disease activity. Objectives: To report a case of diffuse NPSLE manifesting as left hemiparesis and diagnosed on the basis of imaging and CSF findings as well as autoantibody profile. Methods: Case presentation Results: A Seven year old girl initially treated by the neurology department for left hemiparesis predominantly of the arm is presented. Clinical findings were otherwise normal. Seizures were absent. Family history was notable for mother and paternal grandfather diagnosed with multiple sclerosis (MS). Brain MRI showed right lobe hemiatrophy, most prominent in the frontal lobe. Subcortical white matter lesions were detected in both hemispheres. MR perfusion images showed decreased cerebral blood flow in the right frontal lobe suggesting hypoperfusion. The imaging features were not consistent with MS or ischemic stroke but more suggestive of ischemia from vasculitis and vasculopathy. ECG was normal. Metabolic screening, renal and hepatic biochemistry and blood markers of inflammation were within normal limits. Evaluation of CSF revealed normal leukocyte count, protein, glucose and lactic levels, raised neopterins, and the presence of oligoclonal bands both in CSF and serum (type 3), findings compatible with brain inflammation in the context of systemic inflammatory disease. Cytokines suggestive of type I interferonopathy IL8=86 pg/ml {nv<5 } and IFNα=7 pg/ml {<5 } were detected. Autoantibodies for autoimmune encephalitis anti-: NMDAR, AMPA1R , AMPA2R, GABAβ1R, LGI1R, DPPX , CASPR TPO as well as serum anti-NMO IgG and anti-AQP4 were absent. Thyroid function was normal. ANA titer 1/1280, fine speckled with mitotic cells, normal c3 and c4 and IgG 693 mg/dl (nv 812-1698) were detected. Repeat evaluation 3 months later revealed unchanged clinical, CBC, inflammatory markers and MRI findings but raising ANA titer=1/2560 and the presence of multiple anti-Extractable Nuclear Antigens autoantibodies: anti-SM, anti-RNP, anti-SSA/Ro, anti-SSB/La and anti-cardiolipin. Whole Exome Sequencing was unrevealing. Mycophenolate mofetil and HCQ were initiated and physiotherapy continued. Three months later movement was significantly improved, ANA title was diminished (1/640) and anti-ENAs were negative. Conclusion: Isolated hemiparesis was the first manifestation of Diffuse NPSLE. The diagnosis was posed on the basis of MRI, CSF findings suggestive of intrathecal Immunoglobulin synthesis in the context of systemic inflammation and autoantibody profile with very high ANA titers and specific autoantibodS. J. van Tilburg 1 , Y. M. Mueller 1 , H. J. de Wit 1 , C. G. van Helden-Meeuwsen 1 , A. W. Langerak 1 , M. Gruijters 2 , A. Mubarak 2 , M. Verkaaik 2 , P. D. Katsikis 1 , S. Kamphuis 2 , M. A. Versnel 1 1 Department of Immunology, Erasmus MC, University Medical Center Rotterdam, 2 Department of Paediatric Rheumatology, Erasmus MC-Sophia Children’s hospital, University Medical Center Rotterdam, Rotterdam, Netherlands Correspondence: M. J. Wahadat Introduction: Childhood-onset systemic lupus erythematosus (cSLE) is a devastating relapsing remitting autoimmune disease characterized by significant heterogeneity between patients which hinders diagnosis, management and treatment choices. Patients with distinct clinical disease phenotypes may respond to the same medication and vice versa, underlining that solely using clinical phenotype to decide which treatment to start or taper is not enough. Therefore, defining tools that can stratify patients into groups paralleling disease severity can facilitate personalized treatment strategies. Objectives: To stratify patients into biological phenotypes via unsupervised hierarchical clustering of transcriptomic and proteomic data and study the immunological cellular landscape that characterizes these clusters. Methods: Gene expression and cytokine profiles were determined in childhood-onset SLE patients, pre-selected based on three disease activity states, namely at diagnosis, Low Lupus Disease Activity state (LLDAS) and flare. We used an unsupervised hierarchical clustering method, agnostic to disease severity, to identify clusters with distinct biological phenotypes. Disease activity was scored by the clinical SELENA-SLEDAI. Forty-color flow cytometry was used to identify immune cell subsets associated with the identified clusters. Results: This approach identified three unique clusters, each characterized by a set of differentially expressed genes and cytokines. When specifying the clinical phenotypes, the clusters were remarkably unique: in cluster 1 patients were mainly in LLDAS, cluster 2 contained mainly patients at diagnosis, and cluster 3 contained a mixed group of patients with disease flares, patients at diagnosis and patients in LLDAS. Remarkably, healthy controls clustered together with cSLE patients in cluster 1. Interestingly, the biological phenotypes did not reflect previous organ involvement. Lastly, some patients moved from one cluster to another over time, reflecting change of disease activity states. High-dimensional spectral flow cytometry identified specific immune cell subsets that differed between the clusters, including CD11c+B cells, conventional dendritic cells, plasmablasts and early effector CD4+T cells. Conclusion: This study shows that combining transcriptional and proteomic data is a reliable method to cluster patients into distinct biological phenotypes that are related to disease activity state but not to organ involvement. This may lead to a new concept where treatment choices are not based solely on organ involvement but also on measuring novel biological parameters. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
K. Webb 1,2 , T. Spracklen 3 , C. Butters 1 , H. Facey Thomas 1 , R. Stander 1 , M. Erasmus 4 , J. Day 1 , T. Scriba 4 , C. Scott 1 , S. Mendelsohn 4 1 Paediatric Rheumatology, University of Cape Town, Cape Town, South Africa, 2 Crick African Network, Francis Crick Institute, London, United Kingdom, 3 Cape Heart Institute, University of Cape Town, 4, Cape Town, South Africa Correspondence: K. Webb Introduction: Juvenile onset systemic lupus erythematosus (jSLE) is defined by a type 1 interferon (IFN) gene signature. It is not known if this signature is present in the pre-clinical phase of jSLE or whether it may act as an identifiable risk factor for the development of jSLE. There is no simple type 1 IFN assay validated for use as a simple, affordable research tool in large cohort studies to identify jSLE risk. Objectives: This study sought to identify individual type 1 IFN candidate genes whose expression might discriminate children with jSLE from healthy children and those with other inflammatory disorders. Methods: Children were recruited with consent, these included: children with jSLE, healthy children and children with other inflammatory disorders. RNA was extracted from whole blood and the expression of 10 type 1 IFN inducible genes was determined by multiplex Fluidigm real-time quantitative PCR ( DDX58, IRF7, IRF8, IFI6, IFNAR, MX1, MX2, OAS1, IFIT6, ISG15 )groups, adjusted by Bonferroni correction. Receiver operating characteristic (ROC) analysis was used to investigate the discriminatory capacity of each gene. Results: The cohort included multiple samples from 7 children with jSLE, 53 healthy children and 62 children with inflammatory diseases (33 MIS-C, 19 infections, 10 Kawasaki disease). When jSLE was compared to all controls, after correction for multiple testing, DDX 58 (p=0.01) and MX1 (p=0.03) were differentially expressed. DDX58 and MX1 discriminated children with jSLE from healthy children well: DDX58 area under the curve (AUC)= 0.846 (p=0.001) and MX1 AUC=0.774 (p=0.009). Out of all genes, DDX58 and MX1 also best discriminated jSLE from the whole mixed group of inflammatory controls: DDX58 AUC =0.810 (p=0.02) and MX1 AUC=0.798 (p=0.03). When MIS-C and Kawasaki disease were excluded, both genes still discriminated jSLE from the infective controls: DDX58 AUC=0.800 (p=0.01) and MX1 AUC=0.850 (p=0.003). Conclusion: In this small, preliminary cohort, DDX58 and MX1 were identified as potential genes which individually discriminate jSLE from healthy children, children with inflammatory diseases and children with infectious diseases. Further research is required to confirm this observation. A single PCR gene expression test might provide an easy, affordable measure of lupus-specific type 1 IFN expression as a tool in future studies to better understand the role of type 1 IFN in the pre-clinical phase of jSLE and identifying lupus risk. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
P322. PIMS through the waves of COVID 19: data from the JIR cohort P323. Pediatric IGG4-related disease in India - an experience of four children from a single pediatric rheumatology center R. Kechiche 1 , A. Schwartz 1 , F. Bajolle 2 , S. Poignant 3 , R. Basmaci 4 , C. Pajot 5 , U. Meinzer 6 , L. Morin 7 , V. Lambert 8 , P. Dusser 1 , N. Matsa 1 , M. Hofer 9 , I. Kone-Paut 1 , C. Galeotti 1 on behalf of French Covid-19 Paediatric Inflammation Consortium 1 Paediatric rheumatology, CHU Bicêtre, Kremlin-Bicêtre, 2 Paediatric cardiology, CHU Necker, Paris, 3 Paediatrics, CHU Nantes, Nantes, 4 Paediatrics, CHU Louis Mourier, Colombes, 5 Paediatrics, CHU Toulouse, Toulouse, 6 Paediatrics, CHU Robert Debré, Paris, 7 Paediatric intensive care unit, 8 Paediatric radiology, CHU Bicêtre, Kremlin-Bicêtre, France, 9 Paediatric rheumatology, CHUV, Lausanne, Switzerland Correspondence: A. Schvartz Introduction: Paediatric inflammatory multisystem Syndrome (PIMS) is a new systemic inflammatory disease linked to SARS-CoV2 that affects children. It was first reported in may 2020. Objectives: The objectives of this study were to describe patients with PIMS through the international JIR cohort registry and to compare the different profiles and treatments of these patients over the different waves. Methods: Study patients with international PIMS criteria were included from March 2020 to June 2021. Patients were identified in the JIR cohort, an international registry collecting demographic, clinical and paraclinical data on patients with pediatric inflammatory diseases. Two groups were distinguished: from March 2020 to July 2020 for patients in the first wave, from July 2020 to June 2021 for patients in the 2nd and 3rd waves. These two groups were compared using a Fischer test for categorical data and a Mann-Whitney test for quantitative data. Results: 136 patients meeting the PIMS criteria were included (64 patients in the 1st wave, 72 patients after). Patients had less frequent myocarditis (51 patients in wave 1 vs. 36 patients after, p=0,0003) and respiratory distress (34 patients vs 10 patients, p<0,0001). Corticosteroids were used more frequently in the second wave (32 patients in wave 1 vs. 67 patients after July 2020, p<0,0001). Intravenous immunoglobulins were used as much over the waves (58 patients in wave 1 vs 68 patients after, p=0.5). Antibiotics were less used since the second wave (53 patients received antibiotics before July 2020 vs 11 after, p<0,0001). The duration of hospitalization decreased significantly (p<0,0001) with a median duration of 9 days during the first wave (interquartile range, 7-12) and 7 days (interquartile range, 5-10) after the first wave. Conclusion: There was a decrease in the number of complications of PIMS, particularly cardiac and respiratory complications, and a decrease in the length of hospitalization over time. The treatment of PIMS has also evolved, with a clear increase in the use of corticosteroids and a decrease in the use of antibiotics. Disclosure of Interest : None declared
D. B. Pandya, M. Aggarwal, S. Sawhney 1IgG4-related disease (IgG4-RD) was first described in year 2003 as an inflammatory syndrome of unknown etiology characterized by tumorous swelling of the organs such as salivary glands, orbit, pancreas, biliary tree, retroperitoneum, lacrimal gland, kidney and thyroid. 1 In terms of ocular involvement, it usually presents with ocular adnexal masses which can involve the orbit, extra ocular muscles, lacrimal system, optic nerve, or sclera. 2 Unilateral or bilateral eyelid or periorbital swelling is the most common disease manifestation. 3 The three major histopathological features associated with IgG4-RD are, dense lymphoplasmacytic infiltrate, storiform fibrosis and obliterative phlebitis. Other supportive finding includes elevated IgG level more than 135 mg/dl, histopathological evidence of >10 IgG4+ cells/hpf and elevated IgG4-to-IgG ratio of>40 % is commonly accepted. 4, 5 Glucocorticoids are the first line of therapy. Azathioprine, methotrexate or mycophenolate mofetil have been used with limited success in patients who are resistant to or dependent on glucocorticoids. 6 Patients resistant to glucocorticoids may benefit with the use of rituximab. Children with IgG4 related diseases are not yet well described in literature. Objectives: We aim to reveal spectrum of IgG4-related diseases in four Indian Children at Our Center. Methods: This is a retrospective analysis of four children who were diagnosed with IgG4-related disease between January 2013 to January 2022 at our center. Our collected data included demographics , clinical presentation , management and follow up details. Results: This Table shows Characteristics of four Indian children with IgG4-related disease at our PRC All four patients are in clinical remission as per their last follow up. Conclusion: Eye involvement remains the most common manifestation in pediatric IgG-4 related disease in our cohort. MRI Orbit is an investigation of choice in such patients. One patient had renal involvement. All patient had elevated IgG4 level. All patients had transient improvement after steroids. Methotrexate was not effective in controlling inflammation in any of our patients. MMF and azathioprine was found to be effective in our cohort. Trial registration identifying number: 1. Stone JH, Zen Y, Deshpande V. IgG4-related disease. N Engl J Med. 2012; 366:539. 2. Wallace ZS, Khosroshahi A, Jakobiec FA, Deshpande V, Hatton MP, Ritter J, et al. IgG4-related systemic disease as a cause of “idiopathic” orbital inflammation, including orbital myositis, and trigeminal nerve involvement. Surd Ophthalmic. 2011;57:26. 3. Cheuk W, Chan JK. IgG4-related sclerosing disease: a critical appraisal of an evolving clinicopathologic entity. Adv Anat Pathol. 2010; 17:303. 4. Plaza JA, Garrity JA, Dogan A, Ananthamurthy A, Witzig TE, Salomão DR. Orbital inflammation with IgG4-positive plasma cells: manifestation of IgG4 systemic disease. Arch Ophthalmic. 2011; 129:421–8. 5. Umehara H, Okazaki K, Masaki Y, Kawano M, Yamamoto M, SaekiT, et al. Comprehensive diagnostic criteriafor IgG4-related disease (IgG4-RD), 2011. Mod Rheumatol. 2012; 22:21–30. 6. Kawano M, Yamada K. Treatment of IgG4-related disease. Curr Immunol Rev. 2011; 7: Patient Consent: Yes, I received consent Disclosure of Interest : None declared
P324. Factors affecting survival in juvenile versus adult onset systemic sclerosis P325. Profound hypocalcaemia following pamidronate administration in a child with Systemic Sclerosis (SSC) background P326. Pediatric mixed connective tissue disease versus other overlap syndromes: a multicenter study P327. pediatric sclerosis in a center of south of Spain P328. Diffuse juvenile systemic sclerosis patients show distinct organ involvement and have more severe disease in the largest JSSC cohort of the world. results from the the juvenile scleroderma inception cohort 29. Juvenile systemic sclerosis treatment practices in an international cohort and comparison to recent share consensus guidelinesP330. Occurrence of arthritis is in 37% of the patients without overlying skin involvement in juvenile localized scleroderma. summary of the extracutaneous involvement in a monocentric cohort P331. Clinical characteristics of juvenile onset systemic sclerosis patients from the juvenile scleroderma inception cohort compared to adult age juvenile-onset patients from eustar. are these differences suggesting risk for mortality? P332. Patient and physician reported outcomes of juvenile systemic sclerosis patients significantly improve over 12 months observation period in the juvenile systemicP333. How common is the coexistence of juvenile localized and systemic scleroderma? results of a multination survey P334. Is there a correlation between the distance spanned with six-minute walk test and disease progression in juvenile systemic sclerosis? P335. Juvenile localized scleroderma: a single center pilot study in the management of children in real clinical practice P336. Clinical and immunological profile in children with positive anti-rnp antibodies: single center experience P337. Clinical and epidemiological analysis of systemic sclerodermaP338. Connective tissue nevus misdiagnosed as juvenile localized scleroderma P339. Are there predictor variables for progression of interstitial lung disease in juvenile systemic sclerosis? A. Adrovic 1 , G. Karatemiz 2 , S. Sahin 3 , M. Yildiz 3 , F. Haslak 3 , A. Gunalp 3 , K. Barut 3 , G. Hatemi 4 , V. Hamuryudan 4 , O. Kasapcopur 3 , E. Seyahi 4 1 Pediatric Rheumatology, Koç University Hospital, 2 Rheumatology, Istanbul Sisli Hamidiye Etfal Training and Research Hospital, 3 Pediatric Rheumatology, 4 Rheumatology, Istanbul University-Cerrahpasa, Cerrahpasa Medical School, Istanbul, Turkey Correspondence: A. Adrovic Introduction: Systemic sclerosis (SSc) is a rare autoimmune disease characterized by progressive fibrosis of the skin, internal organs involvement and vasculopathy, which could lead to significant morbidity and mortality. Previous studies comparing adult (aSSc) and juvenile (jSSc) patients reported the lower frequency of vital organ involvement and better disease outcome in jSSc. Objectives: We aimed to compare survival and factors affecting mortality in jSSc and aSSc patients. Methods: Demographic characteristics, clinical features, autoantibody profiles, and treatment data were retrieved from the databases. The data presented were the cumulative clinical and serological manifestations throughout the follow up period. The chi-square test was used to compare categorical variables between groups. Univariate and multivariate logistic regression analyses were performed to the independent factors of mortality. The Kaplan-Meier method was used for survival analysis. Results: A total of 214 patients were included: 156 (72.8%) adults and 58 (27.1%) juvenile. The mean age at study was 52.3±15.7 vs. 16.3±4.4 years, at the disease onset 37±14.7 vs. 8.8±4.1 years and at diagnosis 42±15.2 vs. 10.4±3.8 years for adult and juvenile onset patients, respectively. The mean follow-up duration was 6.3±4.9 years for adult vs. 6.6±4.9 years for juvenile patients (p>0.05). The mortality was higher in adults with death seen in 24 (15.9%) adults comparing to 1 (3.33%) of juvenile patients (p=0.005). The frequency of interstitial lung disease and systemic hypertension was significantly higher among aSSc patients: 77 (50.9%) vs. 9 (30%) (p<0.001) and 27 (17.9%) vs. 0(0%) (p=0.009), respectively. The cardiac and renal involvement was more common among aSSc while musculoskeletal features were more frequent in jSSc, but with insignificant difference. The interstitial lung involvement (p=0.03) and cardiac insufficiency (p=0.05), represent independent risk factors of mortality in patients with SSc. Conclusion: The survival rate is better in jSSc patients comparing to adults. Vital organ involvement (lung, heart) represent main factors of mortality. Prospective studies with longer follow-up and higher patients’ number are required to confirm our findings. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
E. Sefi 1 , S. Lacassagne 2 , M. Al Obaidi 3 1 General Paediatric Department, The Whittington Hospital, 2 Paediatric Rheumatology, Great ormond Street Hospital, 3 Paediatric Rheumatology, Great Ormond Street Hospital For Children, London, United Kingdom Correspondence: M. Al Obaidi Introduction: We present the case of a 15 year old boy with severe hypocalcaemia following pamidronate administration for treatment of SSC related calcinosis. Objectives: To highlight the potential challenges facing the general paediatricians in managing children and young people with rare diseases and the side effect of medications they are not familiar with. Methods: A 15 year old boy of Afro-Caribbean origin with SSC was diagnosed at the age of eleven. He presented with rash, myositis, arthritis, digital ulcers with tissue loss. At the time of diagnosis he was vitamin D deficient and was treated with vitamin D. He initially had a good partial response to Methotrexate then Tocilizumab was added. On both medication, he remained in remission for months but went on to develop rapidly progressive calcinosis involving his wrists and fingers with local superinfection. At that time he was being treated with tocilizumab infusions, Methotrexate, amlodipine, hydroxycholoroquine and prednisolone. Results: He was treated acutely with IV antibiotics and a decision was made to give intravenous pamidronate. Prior to treatment his calcium level was normal at 2.23 mmol/L, phosphate 1.65mmol/L and alkaline phosphatase was normal at 301 iu/L and his vitamin D level was not available. One week following the infusion a routine blood test at the local district hospital where he receives shared care revealed a critically low calcium level of 1.44 mmol/L. He was called back from home for emergency treatment of this. He described feeling tired but was otherwise asymptomatic. He was admitted and monitored. Despite intravenous calcium infusions, his calcium level remained low. Subsequently his vitamin D level was found to be low at 18 nmol/L (normal >50 nmol/L) with parathyroid hormone level of 41.6 omol/L. Following vitamin D administration alongside calcium supplementation his calcium levels normalised over the course of a week. Conclusion: Discussion Pamidronate is a useful treatment of calcinosis but as we have described it can cause profound hypocalcaemia especially in the presence of pre-existing vitamin D deficiency. Pamidronate is not a common medication used in district general hospitals but it is important that paediatricians are aware of its potential side effects as these children are likely to present to their local hospitals for emergency treatment. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
E. D. Batu 1 , S. Sahin 2 , A. Gunalp 2 , S. Ozdel 3 , Z. Kizildag 4 , A. Pac Kisaarslan 5 , I. Bagrul 3 , M. Kasap Cuceoglu 1 , A. Tanatar 6 , H. E. Sonmez 7 , E. Sag 8 , S. Demir 9 , E. Celikel 10 , S. Caglayan 11 , B. Celikel Acar 10 , B. Sozeri 11 , N. Aktay Ayaz 6 , Y. Bilginer 1 , M. H. Poyrazoglu 5 , E. Unsal 4 , O. Kasapcopur 2 , S. Ozen 1 1 Hacettepe University Faculty of Medicine, Ankara, 2 Istanbul University, Cerrahpasa Faculty of Medicine, Istanbul, 3 Dr. Sami Ulus Maternity and Child Health and Diseases Research and Training Hospital, Ankara, 4 Dokuz Eylul University Faculty of Medicine, Izmir, 5 Erciyes University Faculty of Medicine, Kayseri, 6 Istanbul University Faculty of Medicine, Istanbul, 7 Kocaeli University Faculty of Medicine, Kocaeli, 8 Ankara Research and Training Hospital, Ankara, 9 Erzurum Regional Research and Training Hospital, Erzurum, 10 Ankara City Hospital, Ankara, 11 Umraniye Research and Training Hospital, Istanbul, Turkey Correspondence: E. D. Batu Introduction: Overlap autoimmune syndromes are very rare in childhood. Pediatric mixed connective tissue disease (MCTD) is a specific subgroup of overlap syndromes associated with anti-U1-RNP antibodies. Objectives: We aimed to analyze and compare the characteristics of patients with pediatric MCTD and other overlap syndromes. Methods: We included all patients with MCTD and other overlap syndromes who had the disease onset before 18 years of age. All MCTD patients met either Kasukawa or Alarcon-Segovia criteria. The patients with other overlap syndromes had the features of ≥2 different systemic autoimmune diseases and did not meet MCTD diagnostic criteria. Results: A total of 30 MCTD (F/M=14/1) and 30 patients (F/M=29/1) with other overlap syndromes were included from 11 centers. The median ages at disease onset were similar between two groups while the median age at diagnosis was slightly younger in the overlap group than MCTD group (11 vs. 11.4 years and 11.8 vs. 13.3. years, respectively). The duration of follow-up was 40.5 months in the whole group. The most prominent phenotype at disease onset and the last visit was SLE in the MCTD group and JIA in the overlap group. Systemic scleroderma phenotype at the last visit was more frequent among MCTD patients compared to overlap patients (60% vs. 33.3%; p=0.038). Weight loss (36.7% vs. 13.3%), digital ulcers (20% vs. 0), puffy hands (60% vs. 20%), Raynaud phenomenon (86.7% vs. 46.7%), hematologic involvement (79% vs. 26.7%), and anti-Sm (29% vs. 3.3%) were more common among MCTD patients than overlap patients (p<0.05 for all). Gottron papules, on the other hand, were more frequently observed in overlap patients compared to MCTD patients (40% vs. 16.7%; p=0.045). Renal involvement (30% vs. 10%) and elevated erythrocyte sedimentation rate during active disease (80% vs. 56.7) were more frequent in MCTD than overlap patients, but these differences were not statistically significant. Regarding treatment, around 90% and 85% of patients received hydroxychloroquine and corticosteroids, respectively in both groups. The duration of corticosteroid treatment was longer in MCTD patients than overlap patients (52.5 vs. 13.5 months; p=0.059). NSAID, mycophenolate mofetil, rituximab, IVIG, ACE inhibitors, calcium channel blockers, and aspirin were more frequently prescribed to MCTD patients while methotrexate was more commonly used by overlap patients. None of the patients died. And none achieved complete remission off drugs. A higher percentage of overlap patients achieved complete remission on drugs than MCTD patients (51.7% vs. 23.3.%; p=0.047). Conclusion: Although considered in the same group of diseases, both disease phenotype and outcome differs between pediatric MCTD patients and children with other overlap syndromes. Analyzing and addressing the differentiating features of these entities could pave the way for early diagnosis and effective treatment in these group of patients during childhood. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
A. Capilla Miranda, L. Fernandez Silveira, O. Neth, C. García Malagón, M. Camacho Lovillo 1 Servicio Inmunología, Reumatología e Infectología pediatrica, Hospital Universitario Virgen del Rocío, Sevilla, Spain Correspondence: M. Camacho Lovillo Introduction: Pediatric sclerodermia includes two groups of clinical entities, systemic sclerosis (SS) and localized scleroderma (LS). Objectives: To analyze the pattern of disease expression, laboratory data, treatments used and outcome in pediatric patients diagnosed with SS and LS, followed in a tertiary care Hospital in Spain. Methods: Medical charts of all pediatric patients younger than 16 years old, diagnosed with SS and LS between 1997 and 2021 and followed at the pediatric rheumatology clinic in our hospital were retrospectively reviewed. Results: We describe a cohort of 9 patients, 5 with SS and 4 with LS. Regarding SS patients, the mean age at disease onset was 11 ± 2.55 years (mean ± 1SD, range 7-13 years), all were female. Sclerodactyly and Raynaud’s phenomenon were the most prevalent clinical feature (5/5). Other sings of skin involvement included edema in 4/5, telangiectasia in 3/5, calcinosis, livedo and digital ulcer in 2 /5 patients each. Nailfold capillary changes were reported in 3/5 patients at the onset of the disease and in 5/5 during the overall disease course. Musculoskeletal symptoms, such as arthralgia (3/5), arthritis (3/5) and reduced muscle strength (1/5) were present in most children. One of our patients presented an overlap syndrome with a muscle biopsy compatible with dermatomyositis and increased serum muscle enzymes. Gastrointestinal or respiratory self-reported symptoms were exceptional, only one patient suffered from dysphagia. However, 2 patients had respiratory system involvement with HRCT or reduced FVC and in 3 patients, although mild, manometry showed esophageal dysmotility sings. None of our patients presented neurologic manifestations, cardiac or renal involvement. Antinuclear antibodies were present in all patients. Anti-topoisomerase I (Scl-70) was found positive in 1 patient, anti-ENA (unable to specify type) was detected in 1 patient and anti-Ro was found in 2 patients. One patient had positive RF. Antiphospholipid antibodies were negative in all patients. None of these patients had abnormal laboratory parameters included blood count, C-reactive protein or erythrocyte sedimentation rate. Metotrexate was selected as first line treatment in most of them (4/5), subsequently changed to mycophenolate in 2 due to intolerance and poor response. Three patients received systemic corticosteroid, cyclophosphamide in 1, tocilizumab in 1. Two patients received oral vasodilators and 4 received topical vasodilators (diltiazem) with good response. At the end of the study, 4/5 patients still required treatment, 2 of them with moderate activity of disease in spite of this. Regarding LS patients, 3 were linear morphea and 1 was plaque morphea. 3/4 presented proximal skin induration and s2/4 had sclerodactyly, one patient showed arthritis and none of them had Raynaud’s phenomenon. Antinuclear antibodies were present in two patients. Two patients presented mild alterations in esophageal micromanometry, without involvement of other systems. All patients had favorable evolution with methotrexate, but in two patients it was changed to mycophenolate and cyclosporine due to poor tolerance. One patient was treated with oral corticosteroid. Conclusion: Clinical and laboratory characteristics of our patients were similar to previous published series. Respiratory system involvement is common in SS and is usually asymptomatic in the early stages. Abnormalities in esophageal manometry were common even in children without self-reported symptoms. It can be difficult to define the pathological significance and we need more experience. Nailfold capillaroscopy is an noninvasive and simple test that can help in the diagnosis and follow up of the disease. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
I. Foeldvari 1 , J. Klotsche 2 , O. Kasapcopur 3 , A. Adrovic 3 , K. Torok 3 , M. T. Terreri 3 , A. P. Sakamoto 3 , B. Feldman 3 , F. Sztajnbok 3 , V. Stanevicha 3 , J. Anton 3 , S. Johnson 3 , R. KhubchandanD. Schonenberg-Meinema 3 , W.-A. Sifuentes-Giraldo 3 , N. Vasquez-Canizares 3 , P. Costa Reis 3 , M. Janarthanan 3 , M. Moll 3 , D. Nemcova 3 , A. PatwardhaR. Cimaz 3 , D. Eleftheriou 3 , L. Harel 3 , G. Horneff 3 , D. Kaiser 3 , T. Kallinich 3 , D. Lazarevic 3 , K. Minden 3 , S. Nielsen 3 , F. Nuruzzaman 3 , S. Opsahl Hetlevik 3 , Y. Uzielthere are significant differences between the clinical presentation of diffuse and limited subtypes. We reviewed clinical differences in presentation of subtypes in patients in the juvenile systemic scleroderma inception cohort up to 2021. Objectives: To study the clinical presentation of jSSc patients with diffuse (djSSc) and limited (ljSSc) subtypes. Methods: We reviewed the clinical baseline characteristics of the patients, who were recruited to the juvenile scleroderma inception cohort (jSScC) till 1 st of December 2021. jSScC is a prospective cohort of jSSc patients, who developed the first non-Raynaud´s symptom before the age of 16 years and are under the age of 18 years at the time of inclusion. Results: 210 patients with jSSc were included in the cohort, 71% (n=162) had diffuse subtype. The median age at onset of Raynaud phenomenon was 10.4 years (7.3–12.9) and the median age at the first non-Raynaud symptom was 10.9 years (7.4–13.2). Median disease duration was 2.5 years (1–4.4) at the time of inclusion. The female/male ratio was significantly lower in the djSSc subtype (3.7:1 vs 5:1, p<0.001). Antibody profile was quite2% vs 2%, p=0.013). Decreased FVC<80% was found in approximately 30% and decreased DLCO<80% was found in around 40% in both subtypes. Pulmonary hypertension assessed by ultrasound was identified in 5% in both groups. Patients with diffuse subtype had significantly higher modified Rodnan Skin Score (mRSS)(16 vs 4.5, p<0.001), sclerodactyly (84% vs 60%, p<0.001), history of digital ulceration (62% vs 31%, p<0.001), decreased Body Mass Index (BMI) ≤-2 z score (20% vs 4%, p=0.003) and decreased joint range of motion (64% vs 46%, p=0.019). Patients with ljSSc had significantly higher rate of cardiac involvement (13% vs 2%, p=0.001). Regarding patient related outcomes djSSc patients had more severe disease, looking at patient reported global disease activity (VAS 0–100)(40 vs 25, p=0.039), patient reported global disease damage (VAS 0–100)(40 vs 25, p=0.021) and patient reported assessment of ulceration activity (10 vs 0, p=0.044). Regarding physician related outcomes the physician reported global disease activity (VAS 0–100)(32 vs 20, p<0.001) and physician reported global disease damage (VAS 0–100)(30 vs 15, p=0.014) was significantly higher in djSSc. Conclusion: In this jSSc cohort, the largest in the world, djSSc patients have a significantly more severe disease than ljSSc patients. Interestingly, we found no differences regarding interstitial lung disease and pulmonary hypertension. This project was supported by an unrestricted grant from “Joachim Herz Stiftung”J. Anton 3 , F. Sztajnbok 3 , V. Stanevicha 3 , S. Appenzeller 3 , T. Avcin 3 , S. Johnson 3 , R. Khubchandani 3 , M. Kostik 3 , E. Marrani 3 , W.-A. Sifuentes-Giraldo 3 , D. Nemcova 3 , M. J. Santos 3 , D. Schonenberg-MeinemaT. Lehman 3 , M. Moll 3 , F. Nuruzzaman 3 , A. Patwardhan 3 , V. Smith 3 , N. Helmus 1 1 Hamburg Centre for Pediatric and Adolescence Rheumatology, Hamburg, 2 German Rheumatism Research Center, Berlin, 3 jSSc collaborative group, Hamburg, Germany Correspondence: I. Foeldvari Introduction: Juvenile systemic scleroderma (jSSc) is an orphan disease with a prevalence of 3 in 1,000,000 children. Currently no medications are licensed for the treatment of jSSc. Due to its rarity, only recently have the first management and treatment guidelines. Objectives: To better understand treatment practices internationally for jSSc, both at baseline and over 24 months observation period and to compare if real world therapies are congruent with the recent SHARE recommendations. Methods: The juvenile systemic sclerosis inceptions cohort (jSScC) is a multinational cohort that prospectively collects clinical data, including medications at baseline and subsequent visits. The jSScC enrollment criteria include age of onset of the first non-Raynaud symptom younger than 16 years and age younger than 18 years at cohort entrance. The frequency of medications was calculated across the cohort at timepoint 0, 12 months and 24 months. Results: We extracted data from the jSScC of patients who were followed for 12 or 24 months. 109 patients were followed at time point 0 and 12 months, and data was available for 77 of them up at 24 months. The mean age of the patients was 13.2 years at the timepoint 0. 75% were female and 75% had diffuse subtype. Disease duration at baseline visit was 3.1 years. The medications the patients were on recorded by the physician were captured at T0, T12 and T24 listed in Table 1. Conclusion: At baseline half of the patients were on corticosteroids. This is more frequent than typical adult SSc practice but coincides with jSSc SHARE treatment recommendations (#1). After 12 months in the cohort over 90% of patients received a DMARD therapy. Methotrexate and mycophenolate mofetil were the most commonly DMARDs, which also reflects the SHARE recommendations (#2, #3). At 12 months the use of glucocorticoid decreased and the use of bDMARDs increased. In general, biological DMARDs are typically considered in severe or refractory (#7), reflecting the lower percentage compared to csDMARDs. Autologous stem cell transplantation was observed in one patient at 12 months, reflecting an option in jSSc with progressive and refractory disease (#8), and its comparison to recently published consensus guidelines.A. Petersen 1 , I. Foeldvari 2 1 Asklepios Campus Hamburg of the Semmelweis-Universität, Budapest, Hungary, 2 Hamburg Centre for Pediatric and Adolescence Rheumatology, Hamburg, Germany Correspondence: I. Foeldvari Introduction: Localized scleroderma in childhood(locSSc) occurs with a prevalence of 3.2 to 3.6 per 10 000 children. There are not many publications assessing in detail the extracutaneous manifestations (EM) of locSSc. It is very important to assess the EM too, because the EM can lead to significant damage and morbidity too. Objectives: To assess the occurrence of extracutaneous manifestations in locSSc in our cohort and the correlation of the occurring EM to the subtype of locSSc and the localisation of the skin involvement. Methods: Retrospective chart review of all consecutive patients, who were followed at our centre from January 2000 to July 2020 with the diagnosis of locSSc. The subtype was classified according Laxer et al. The patients were under the age of 18 years at the time point of the first visit. Demographic and clinical data were extracted. Results: 73 patients could be identified, 71% of them were female. Mean age at disease onset was 8 years (4-14 years). The mean time of follow up was 5 years. The subtype distribution was 42 (57%) linear, 24 (33%) mixed, 6 (8%) circumscribed morphea and 1(1%) pansclerotic morphea. 9 (21%) of the 42 patients with linear subtype had coup de sabre and 4 (10%) of them had Parry Romberg. Fifty-six (76%) patients had EM, 40 (53%) of them had 1 form of EM, 10 (13%) of them 2 forms of EM and 6 (8%) patients 3 forms of EM. 53 (73%) of the 76 patients had arthritis. Twenty (37%) of the 53 arthritis involvement occurred on a localisation without overlaying skin involvement. Most frequent localisation of arthritis without overlaying skin involvement was in the hip joints (18%). Of the 53 patients with articular involvement had 31 (58%) linear, 17 (32%) mixed, 4 (7.5) circumscribed morphea and 1 (2%) pansclerotic subtype. 14(19%) of the 73 had length discrepancy of the extremities and 13 (93%) of them had linear subtype. Neurologic symptoms presenting as headache occurred in 8 (11%) patients, 6 (75%) of them had Parry Romberg subtype and 2 (25%) of them coup de sabre. “White” anterior uveitis was screened according to published recommendations and it occurred in 3 patients, only one of them had coup the sabre the other two linear and mixed subtype without involvement of the face. Conclusion: EM is very common and it occurs in 76% of the patients. Thirty seven percent of the articular involvement occurred in joints without overlaying skin involvement, which suggest the importance of the whole body joint count as in juvenile idiopathic arthritis. Only 1 of 3 patients with uveitis had skin involvement in the face, which emphasize the recommended uveitis screening. Disclosure of Interest : None declared
I. Foeldvari 1 , J. Klotsche 2 , P. Carreira 3 , O. Kasapcopur 4 , A. Adrovic 4 , K. Torok 4 , P. Airò 5 , F. Iannone 6 , Y. Allanore 7 , A. Balbir-Gurman 8 , T. Schmeiser 9 , F. Sztajnbok 4 , M. T. Terreri 4 , V. Stanevicha 4 , J. Anton 4 , B. Feldman 4 , R. Khubchandani 4 , E. Alexeeva 4 , S. Johnson 4 , M. Katsicas 4 , S. Sawhney 4 , V. Smith 4 , S. Appenzeller 4 , T. Avcin 4 , C. Campochiaro 10 , J. de Vries-Bouwstra 11 , M. Kostik 4 , T. Lehman 4 , E. Marrani 4 , D. Schonenberg-Meinema 4 , W.-A. Sifuentes-Giraldo 4 , N. Vasquez-Canizares 4 , M. Janarthanan 4 , H. Malcova 4 , M. Moll 4 , D. Nemcova 4 , A. Patwardhan 4 , M. J. Santos 4 , G. Seskute 12 , M.-E. Truchetet 13 , C. Battagliotti 4 , L. Berntson 4 , B. Bica 4 , J. Brunner 4 , R. Cimaz 4 , P. Costa Reis 4 , D. Eleftheriou 4 , L. Harel 4 , G. Horneff 4 , D. Kaiser 4 , T. Kallinich 4 , D. Lazarevic 4 , K. Minden 4 , S. Nielsen 4 , F. Nuruzzaman 4 , S. Opsahl Hetlevik 4 , Y. Uziel 4 , D. Veale 14 , A.-M. Hoffman-Vold 15 , A. Gabrielli 16 , O. Distler 17Germany, 3 Hospital 12 de Octubre, Madrid, Spain, 4 jSSc collaborative group, Hamburg, Germany, 5 Spedali Civili di Brescia, Brescia, 6 School of Medicine University of Bari, Bari, Italy, 7 Cochin Hospital, Paris, France, 8 Rambam Health Care Campus, Haifa, Israel, 9 Krankenhaus St. Josef, Wuppertal-Elberfeld, Germany, 10 San Raffaele Hospital, Milan, Italy, 11 Leiden University Medical Center, Leiden, Netherlands, 12 State Research Institute for Innovative Medicine, Vilnius, Lithuania, 13 CHU de Bordeaux, Bordeaux, France, 14 St. Vincent’s University Hospital, Dublin, Ireland, 15 Rikshospitalet University Hospital, Oslo, Norway, 16 Università Politecnica delle Marche & Azienda Ospedali Riuniti, Ancona, Italy, 17 University Hospital Zürich, Zurich, Switzerland Correspondence: I. Foeldvari Introduction: Juvenile systemic sclerosis(jSSc) is an orphan autoimmune disease with a prevalence of 3 in 1 000 000 children. Information on long-term development of organ involvement and clinical characteristics of jSSc patients in adulthood are lacking. It was believed that patients in adult cohorts may represent a survival biased population. Objectives: To assess differences in clinical characteristics of jSSc-onset patients from the pediatric age group, with a mean disease duration of 3 years, compared to the adult age jSSc-onset group, with a mean disease duration of 18.5 years. Methods: We extracted clinical data at time of inclusion into the cohorts from the Juvenile Scleroderma Inception Cohort (jSScC) and data from juvenile-onset adult SSc patients from the European Trials and Research Group (EUSTAR) cohort. We compared the clinical characteristics of the patients by descriptive statistics. Results: We extracted data of 187 jSSc patients from the jSScC and 236 patients from EUSTAR. The mean age at time of assessment was 13.4 years old in the jSScC and 32.4 years old in EUSTAR. The mean disease duration since first non-Raynaud was 3.0 years in jSScC and 18.5 years in the EUSTAR. We found significant differences between the cohorts. There were more female patients in EUSTAR (88% vs 80%, p=0.04). More patients had diffuse subtype in jSScC (72% vs 40%, p<0.001). The modified Rodnan skin score (mRSS) was significantly higher in jSScC (14.2 vs 12.1, p=0.02). Active digital ulceration occurred more often in EUSTAR (27% vs 18% p=0.01), but history of active ulceration was more frequent in jSScC (54% vs 43%, p<0.001). Mean DLCO was lower in jSScC (75.4 vs 86.3, p<0.001). Intestinal involvement was significantly more common in jSSc (33% vs 24%, p=0.04). Esophageal involvement was more common in EUSTAR (64% vs 34%, p<0.001). Conclusion: Patients with jSSc-onset who are currently adult age (>18 years of age) are less frequently male and from the diffuse subset, have lower mRSS, less digital ulcers and intestinal involvement. This might represent a combination of both survival bias and/or be explained by the longer observation time with less active disease (i.e. natural progression decreased mRSS over time). Further long-term observational studies with jSSc patients are required to address this issue. Disclosure of Interest : None declared
B. Feldman 3 , J. Anton 3 , M. Katsicas 3 , V. Stanevicha 3 , F. Sztajnbok 3 , S. Appenzeller 3 , T. Avcin 3 , M. Kostik 3 , E. Marrani 3 , W.-A. Sifuentes-Giraldo 3 , S. Johnson 3 , R. Khubchandani 3 , D. Nemcova 3 , M. J. SantosM. Janarthanan 3 , T. Kallinich 3 , K. Minden 3 , M. Moll 3 , S. Nielsen 3 , A. Patwardhan 3 , D. Schonenberg-Meinemathe changes in the clinical characteristics and patient and physician reported outcomes over 12 months observation period from the time of inclusion into the cohort. Methods: The jSScC cohort, who had 12 months follow up from the time of inclusion until 1 st of December 2021. Results: We could extract data of 113 patients. The female/male ratio was 3.5:1. Median age of onset of Raynaud´s was 10.1 years and the median age of onset of non-Raynaud´s was 10.8 years. Eighty-eight percent of the patients were treated with disease modifying anti-rheumatic drugs (DMARDs) at time of inclusion in the cohort (T0) and 93% after 12 months (T12). Median disease duration was 2.5 years at T0. Antibody profile stayed unchanged. Only 3 clinical parameters changed and improved significantly, the median modified Rodnan skin score improved from 13 to 8 (p=0.002), the number of patients with swollen joints decreased from 17% to 8% (p=0.043) and number of patients with joints with pain on motion decreased from 20% to 12% (p=0.048). All other organ involvement did not show any statistically significant change from T0 to T12. All collected patient reported outcomes improved significantly from T0 to T12: the patient reported disease activity (VAS 0 – 100) from 40 to 20 (p=0.011), the patient reported disease damage (VAS 0 – 100) from 40 to 20 (p=0.001), patient reported ulceration activity (VAS 0 – 100) from 10 to 0 (p=0.02) and the CHAQ score from 0.3 to 0.1 (p=0.002). Two of the three physician reported outcomes improved significantly, the physician global disease activity (VAS 0 – 100) from 30 to 20 (p=0.011) and physician reported global disease damage (VAS 0 – 100) from 30 to 25 (p=0.028). Conclusion: Skin and musculoskeletal clinical features improved over 12 months, with almost all patients on DMARDs, supporting likely response of these features to therapy. It was promising that internal organ involvement, like cardiac and lung, although potentially stable, did not significantly worsen or increase. The most striking observation in the positive direction is improvement across several patient and physician reported outcome measures over the 12 month time period in this large international cohort. This project was supported by an unrestricted grant from “Joachim Herz Stiftung”I. Foeldvari 1 , N. Helmus 1 , S. Li 2 1 Hamburg Centre for Pediatric and Adolescence Rheumatology, Hamburg, Germany, 2 Hackensack Meridian School of Medicine, Hackensack, United States Correspondence: I. Foeldvari Introduction: Pediatric scleroderma consists of two diseases, juvenile localized scleroderma (jLS) and juvenile systemic sclerosis (jSSc). While jLS and jSSc share some disease processes, there are major differences in their clinical features and morbidity patterns. Co-existence of LS and SSc has been reported in adults, with a recent adult systematic review reporting a frequency of 2.4-7.4%, and most patients having SSc prior to development of LS 1 To explore this issue in pediatric scleroderma, we conducted a survey over the international pediatric rheumatology email board to ask about the co-existence of jLSc and jSSc. Objectives: To explore the coexistence of jLS and JSSc, and the relative timing of their development. Methods: A 12 question was distributed over the worldwide pediatric rheumatology electronic list-serve. Data was collected in an Excel spread sheet and analyzed using Excel software. Results: From approximately 800 survey recipients worldwide, physicians from 28 centers responded (27 pediatric rheumatologists, 1 pediatric dermatologist, half were practicing for over 20 years). Respondents followed a median of 12 jLS and 2 jSSc patients over the past 12 months, and a total of 916 jLS and 193 jSSc patients over the past 60 months. Only 7 of the patients had concurrent jLS and jSSc (0.8% of total jLS, 3.6% of total jSSc patients). Four of the patients developed jSSc following onset of jLS, with interval for 3 patients reported as 6 months, 1 year, or 6 years. Among the other 3 coexisting patients, one simultaneously presented with jLS and jSSc, and the pattern for the other two patients was not reported. The time between development of jSSc following jLS was 6 months, 1 year, and 6 years, jLS subtype was generalized morphea (2, one as part of mixed morphea), linear of the trunk/limb (2, one as part of mixed morphea), circumscribed superficial morphea, or mixed morphea. Many of the jLS patients who did not develop jSSc were reported to have features associated with jSSc. Raynaud Phenomenon occurred in 397 patients from 7 centers, for a collective overall frequency of 45%(879 total patients), while nailfoldcapillary changes were reported in 359 patients from 4 centers, for a collective overall frequency of 43% (842 patients). Abnormal pulmonary function or imaging tests were found in 145 from 3 centers, collective overall frequency 24%. Gut involvement including gastroesophageal reflux was found in 534 patients from 9 centers, collective overall frequency 61%. Conclusion: In this international survey cohort of 916 jLS and 193 jSSc patients, the overall frequency of co-existence of these 2 diseases was low, only 0.8% of the jLSc patients, 3.6% of the jSSc patients. However, many jLS patients were reported to jSSc-associated symptoms including Raynaud Phenomenon. More work is needed to evaluate the features associated with co-existence of these two forms of scleroderma in children. Disclosure of Interest : None declared
O. Koker 1,2 , A. Adrovic 2 , M. Yildiz 2 , S. Sahin 2 , A. Gunalp 2 , F. Haslak 2 , K. Barut 2 , O. Kasapcopur 2 1 Pediatric Rheumatology, Marmara University-Pendik Training and Research Hospital, 2 PediatricO. Koker Introduction: Pulmonary involvement is the most consequential determinant of morbidity and mortality in juvenile systemic sclerosis (jSSC), which is rare but may have a severe progression. The six-minute walking test (6-MWT) has achieved a noteworthy role in the assessment of cardiopulmonary diseases, and improvement in the walking distance is deemed a favourable predictor for the clinical outcome and treatment response. Data concerning the practicality of the test in the management of jSSC is limited. Objectives: The aim of the study is to assess the six-minute walking distance (6MWD), the modification in the distance with disease progression, and oxygen desaturation in patients with jSSC. It is also intended to evaluate the relationship between disease outcome measures and 6MWD. Methods: The study was conducted with patients with a diagnosis of jSSC, who were followed up at Cerrahpasa Medical Faculty, Department of Pediatric Rheumatology at regular intervals. 6MWT was performed twice at 48-month intervals. The test was conducted according to the guidelines recommended by the American Thoracic Society (ATS). The Borg Scale, a well-validated scoring system, was used to determine the patient self-reported fatigue and dyspnea levels. Results: Twelve female patients with a current age of 19,5 (12-23) and diagnosed with jSSC were included in the study. The median age at diagnosis and the disease duration were 12 (8-17) and 112,5 (59-143), respectively. The disease type (limited/ diffuse) was 5/7 (41,7/58,3). Seven patients had Interstitial lung disease (ILD) according to high resolution computed tomography (HRCT). No patients received new cyclophosphamide therapy during this period, but tocilizumab has been added to the treatment of four patients. There were two patients who continued to require steroid treatment. Mycophenolate mofetil treatment has been commenced Conclusion: Although the walking distances of the patients did not show a direct and notable association with disease activity and progression, a significant improvement has been succeeded in the long-term in the follow-up period of the same patients. Factors related to the treatment process, including the use of biologic agents, may influence the test results. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
D. Alekseev, I. Nikishina Pediatric, V. A. Nasonova Research Institute Of Rheumatology, Moskow, Russian Federation Correspondence: I. Nikishina Introduction: Juvenile localized scleroderma (JLSc) has a special place among other rheumatic diseases and requires special approaches to assessing the status. Objectives: Development of an approach to stratification of patients (pt) with JLSc based on medical history, clinical manifestations and indices to develop an optimal management approach in real clinical practice. Methods: A study of 100 pt (obtained through a sequential sample from the existing database) was conducted, based on complete anamnesis data, clinical, laboratory and instrumental examinations; the LoSSI and LoSDI indices were additionally calculated. As a special task, a global assessment of the status was performed, which we use in daily practice, with the empirical selection of the following 4 gradations: (1) remission, (2) chronic course, (3) chronic slowly progressive course, (4) chronic progressive course. Main purpose: determining the prognosis and, accordingly, the necessary therapy. Ctg1 means no obvious signs of disease activity and progression and allows discontinuation of therapy. Ctg2 implies the possibility of maintaining activity / progression, suggests a gradual decrease in the severity of activity / progression, with a slow transition to an inactive non-progressive status / remission, and is the basis for maintenance therapy. Ctg3 requires continuation of therapy, which can be gradually reduced to maintenance as the pt observes. Ctg4 corresponds to the active status, the progression of the manifestations of the disease is assumed, and is the basis for prescribing or intensifying therapy. Results: The age of the pt was 3.8-17.9 years (у) (m±σ 11.06±3.8), with the disease duration at the time of the study being 0.4-12.8 у (m±σ 4.2±3.2). jLSc forms - morphea 18%, linear with damage to the trunk and extremities 42%, linear with damage in the head area 37%, generalized 1%, mixed 2% (with the presence of forms - morphea and linear with damage to the trunk and extremities) . Localization of lesions: on the face - 37 pt, on the trunk - 40 pt, on the extremities - 52 pt. Antinuclear antibodies were detected in 44% of pt. All of them had no manifestations of Raynaud’s phenomenon, significant capillaroscopic changes, visceral lesions. Therapy at the time of the study: 11 pt were not receiving treatment; 30 - only local drugs; the rest received metipred orally (26% of pt; at a dose of 1-11 mg); methotrexate 53%, mycophenolate mofetil 2%. The status of the course of the disease was assessed. No one was in remission status. Pt were registered to have a Ctg4 (50%), a Ctg3 (31%), a Ctg2 (19%) Based on the data obtained, the following decision was made on further pharmacotherapy: pt in a Ctg4 underwent therapy correction aimed at improving the effectiveness of treatment (pulse therapy with methylprednisolone was performed, methylprednisolone, methotrexate or mycophenolate mofetil or abatacept were prescribed). The rest of the pt either reduced the dose of drugs, or recommended drug withdrawal. When comparing the data of the LoSSI and LoSDI indices with the types of the course of the disease, it was obtained: — pt classified as Ctg4 had indices LoSSI min 4 - max 34 (m±σ 8.9 ± 6.3), LoSDI 2-21 (m±σ 4.4 ± 3.7) — pt categorized as a Ctg3 — LoSSI 2-28 (m±σ 6.7 ± 6.1), LoSDI 2-47 (m±σ 7.4 ± 8.9) — pt classified as Ctg2 — LoSSI 1-14 (m±σ 4.1 ± 3.6) , LoSDI 1-22 (m±σ 5.1 ± 5.2) Conclusion: We present data from our pilot study on the management of children with localized scleroderma, which we consider useful for developing a treatment approach. The specified allocation of 4 subtypes contributes to the formation of a clear verbal judgment about the current status of the patient, which facilitates prediction and makes it possible to make a rational choice of treatment. Requires additional verification and statistical analysis in the future. Disclosure of Interest : None declared
M. Kaleda, I. Nikishina, S. Salugina, E. Fedorov, N. Yudkina, Z. Verizhnikova Moscow, Russian Federation Correspondence: I. Nikishina Introduction: Anti-RNP antibodies are considered specific for mixed connective tissue disease (MCTD). Some authors described that anti-RNP antibodies may be present in defined rheumatic diseases (RD), and associated with particular clinical features, for example, like scleroderma-like features in patients (pts) with systemic lupus erythematosus (SLE). Objectives: According to a retrospective study, to describe clinical and immunological characteristics of children with RD with positive anti-RNP antibodies (antiRNP+), which were observed in our pediatric rheumatology center. Methods: The study included all pts with RD, who had antiRNP+ during past 3 years. We tested pts with antiRNP+ on 4 different sets of criteria for MCTD: Kasukawa’s, Alarcon-Segovia, Kahn and Sharp criteria. Results: A total of 23 pts with RD and anti-RNP+ were selected. All pts were girls. The median age at onset of RD was 11.5 years [IQR 8.6; 13.2]. 9 pts had criteria for defined RD: 2 - SLE, 2 - SLE with Sjögren’s syndrome (SS), 2 - rheumatoid factor-positive (RF+) juvenile idiopathic arthritis (RF+ JIA) with SS, 1 - primary SS, 1 - systemic sclerosis, 1 - juvenile dermatomyositis. 14 pts fulfilled of criteria for MCTD (60.9%): Kahn’s criteria - 12 pts, Sharp’s criteria - 9, Kasukawa’s criteria - 6, Alarcon-Segovia criteria – 5. 3 pts met only one set of criteria, 6 pts – 2 sets of criteria, 3 pts – 3 sets, 2 pts – all 4 sets. The most common combination of criteria – Sharp’s and Kahn’s (8 pts). Sjögren’s syndrome was diagnosed in 18 pts (78.3%). The most common symptoms were arthritis (96.6%), followed by Raynaud’s phenomenon and lymphadenopathy (65.2% each). 60.9% of pts had various skin lesions: 6 - sclerodactyly, 2 - telangiectasias, 3 - malar rash, 2 - heliotrope rash and Gottron’s papules, 2 - erythema nodosum, 1 - livedo reticularis. 52.2% of pts had clinically significant fatigue, 17.4% - myopathy, 8.7% – nephritis, and 4.3% - myocarditis. Interstitial lung disease was observed in 26.1% of pts. Among the pts with SS 16 had isolated involvement of salivary glands, 2 – combined with lacrimal glands. Sicca syndrome was occurred in 12 pts, recurrent parotitis – in 1. All pts had anti-RNP+: 87% of pts - in titre of >200 U/l, others - 72.9, 140, 149 U/l, respectively. All pts had positive antinuclear antibodies (ANA) in high titer: 1/1280 – 65.2%, 1/2560 – 34.8%. Isolated speckled (sp) type of ANA was in 60.9% of pts, isolated homogeneous (h) type - 8.7%, mixed type (h+sp+cytoplasmic) – 30.4%. Other autoantibodies included positive anti-Sm in 10 pts, anti-DNA in 7, anti-Ro in 5. RF+ was in 13 pts. Hypergammaglobulinemia was verified in 11 pts, the median level of IgG was 22.5 g/l [19.6; 26.5], max 43 g/l. Capillaroscopic changes in the nailfold were noted in 15 pts (65.2%): 4 pts had non-specific abnormalities, 5 pts had an early scleroderma pattern, 3 had a late scleroderma pattern with a myopathic component and 3 – a changes characteristic of DM. The most common combination of features of MCTD included fatigue, Raynaud’s phenomenon, arthritis, Sjögren's syndrome, lymphadenopathy and hypergammaglobulinemia (7pts, 50%). Conclusion: In our study, only 60.9% of anti-RNP+ pts had MCTD. Most of the pts with MCTD met the criteria of Kahn and Sharp. The combination of fatigue, Raynaud’s phenomenon, arthritis, Sjögren’s syndrome, lymphadenopathy and hypergammaglobulinemia affected a half of pts with MCTD. Taking into account the high frequency of SS, also Raynaud’s phenomenon in combination with the predominance of scleroderma pattern in capillaroscopy in pts with antiRNP+, we can assume a high probability of the evolution of disorders in these pts along the path of the predominance of the SS and/or systemic sclerosis in the futurscleroderma is a multisystem progressive disease with characteristic changes in the skin, musculoskeletal system, involvement of internal organs in the pathological process, as well as vascular disorders. Objectives: Make a clinical and epidemiological analysis of systemic scleroderma7 patients diagnosed with systemic scleroderma (SS), aged from 6.8 to 17.5 years, of which 73% (n=49) were girls and 27% (n=18) were boys living in Moscow. Results: The median age of patients was 12.5 years, the median age at the disease onset was 5.4 years. The time interval from the onset of the disease to the verification of the diagnosis averaged 10.5 months. Twenty-eight patients (43%) had an initial diagnosis other than SS. When verifying the diagnosis in accordance with clinical criteria, all patients had skin lesions, 11.9% had involvement in the process of the gastrointestinal tract, Raynaud’s syndrome was registered in 10.5%. By the nature of the course, acute was noted in 6 (9%) patients, subacute - in 59 (88%), chronic - in 2 (3%) patients. When assessing the activity of the disease in most patients, the activity was assessed as low - in 39 (58%), moderate - in 23 (34.5%), high - in 4 (6%), remission - in 1 (1.5%) child . Basic anti-inflammatory therapy was received by 97% of patients, 42% of them in a combination of several drugs. Biological therapy (BT) is received by 6% of patients. In the structure of BT, abatacept predominates - 75%. Conclusion: Systemic scleroderma is one of the rarest, but difficult to diagnose diseases in children, for the improvement of the prognosis of which timely diagnosis and early initiation of therapy are importantF. Tirelli 1 , M. Soliani 2 , F. Calabrese 3 , C. Giraudo 4 , G. Martini 1 , A. Meneghel 1 , F. Zulian 1 1 Pediatric Rheumatology Unit, Department of Woman and Child Health, University Hospital, Padova, 2 Department of Pediatrics, ASST di Cremona, Cremona, 3 Department of Cardiac, Thoracic, Vascular Sciences and Public Health, University Hospital of Padova, 4 Department of Medicine, University Hospital, Padova, Italy Correspondence: F. Tirelli Introduction: Connective tissue nevi (CTN) are benign hamartomas of the dermis caused by excessive proliferation of collagen, elastin and proteoglycans that sometimes may mimic juvenile localized scleroderma (JLS). Objectives: To describe a series of patients with CTN misdiagnosed as having JLS and subsequently treated with immunosuppressive drugs. Possible elements of differential diagnosis are discussed. Methods: We included in the study children referred to our Center between 2001 and 2020 for a second opinion in suspected or confirmed JLS with atypical clinical course and/or unresponsive to treatment, who received a final diagnosis of CTN. Clinical, histological and radiological data were retrospectively collected from the patients’ charts. Results: Sixteen children (11 females), with mean age at onset of 4.4 years (range 2 - 9) were included. All patients were referred to our Center for a second opinion with a confirmed diagnosis of JLS (n=12) suspected JLS (n=2) or suspected fasciitis (n=2). Ten patients (62.5%) were already on systemic treatment with methotrexate (MTX) alone (n=3), MTX + corticosteroids (CS) (n=5) or MTX+CS+Tocilizumab (n=2), while 3 (18.8%) were on topical CS; none had shown improvement despite the treatment. Cutaneous lesions were characterized by skin induration involving the lower limbs in 13 (81.2%) patients, with 6 having also involvement of ipsilateral trunk, and 3 of the upper limbs. Erythema or other signs of skin inflammation were not noted. When JLS damage score (LoSCAT) was applied, activity criteria (LoSSI) were grade 1 erythema and 1-2 skin induration, while among damage criteria (LoSDI) only mild pigmentation changes were present (grade 1-2), usually as hyperpigmentation, and no signs of dermal or subcutaneous atrophy were observed; notably, there was a lack of significant changes in the score over time, which is, in contrast, rather typical in JLS. Inflammatory markers and autoantibodies were absent/negative in all patients. Thermography was negative in 15 patients, only one showed mild hyperthermia of the lesion with homogeneous pattern, not typical for JLS. Fifteen patients underwent musculoskeletal MRI that was negative in 10, while showed skin plane thickening without morphological or subcutaneous tissues abnormalities in 5. Each patient underwent a skin biopsy either performed by us (n=3) or at the referring site (n=13) with revision of the specimens at our Center. Pathology examination confirmed the absence of inflammatory infiltrate and allowed a final diagnosis of non-familial collagenoma in 10 (62.5%), mixed CTN in 4 (25%) and familial CTN in 2 (12.5%). Mean diagnostic delay was 5 years (range 1-15). Conclusion: CTN may mimic JLS but some key elements, such as absence of clinical and histological inflammatory features of the skin lesions, negative autoantibody profile, normal thermography and peculiar MRI features are essential to establish a the differential diagnosis between the two conditions and thus avoid unnecessary treatments. Disclosure of Interest : None declared
M. G. Villarreal 1 , M. B. Lucero 2 , A. Uriburu 2 , M. C. Bertinotti 1 , J. Manrique 1 , L. Vasconcellos 1 , M. M. Katsicas 1 1 Service of Immunology & Rheumatology, 2 Service of Pulmonology, Hospital de Pediatría J. P. Garrahan, Buenos Aires, Argentina Correspondence: M. M. Katsicas Introduction: Juvenile systemic sclerosis (jSSc) is a multisystem connective tissue disease characterized by skin fibrosis and internal organ involvement including the lungs. Pulmonary manifestations in jSSc are associated with morbidity and mortality. The low incidence of jSSc and the late diagnosis of its pulmonary features results in progressive lung damage with the associated mortality. Further knowledge of this rare disease may improve patient treatment and lung survival. Objectives: To describe pulmonary features associated jSSc and to evaluate potential predictor variables for progression of interstitial lung disease. Methods: This is an observational, retrospective study. Clinical charts of children with a definite diagnosis of jSSc who were admitted at a tertiary referral center between 2000 and 2021 were reviewed. Only patients who had lung involvement with follow up at least ≥36 months±6 months were included. Demographics variables were recorded. Lung specific data collection includes clinical, pulmonary functional tests (PFT) and imaging (HRCT: high resolution computed tomography). PFT parameters included: forced vital capacity (FVC) and diffusing capacity of the lungs for carbon monoxide (DLCO). Pulmonary outcome measures were recorded at follow up (36± 6 months). Outcome measures definitions were: a) clinically significant change on PFT :10% difference in FVC with respect to basal/previous value and 15% in DLCO2. A cut off value of <80% (predicted for age, weight, height, sex and race) was used to determine and abnomal FCV and DLCO as defined in healthy children; b) Abnormal findings in HRCT; c) Progression of interstitial lung disease was defined as an irreversible change on imaging showing indirect signs of fibrosis such as ground glass opacification and/or interstitial thickening. Descriptive statistics and stepwise logistic regression were used for data analysis. Results: Overall 34 jSSc patients, 15 (14 female) patients met inclusion criteria. Age at first symptom attributable to JSS of 8.09 (1.2-15.0) years. Age at lung involvement was 10.2 (1.2- 15.0) years. Lung data collection showed: lung symptoms (4 patients: cough (2), dyspnea (2)), abnormal PFTS (10 patients: abnormal FCV (10), abnormal DLCO (8)), abnormal HRCT 11patients (micronodules/nodules 8, ground-glass opacification 5, interstitial thickening 5, cystic changes 1). Pulmonary outcome measures at follow-up showed: a) PFTs: Improvement on FCV: 1patient (7%); improvement on DLCO: 3 (20%). Twelve (80%) patients did not show changes; b) Abnormal HRCT: 11 (73%); c) progression of interstitial lung disease in 7 patients (interstitial thickening and/or ground glass opacification). Only DLCO was found as a significant risk factor for lung damage(p=0.004). DLCO (median, range) was 95% (54-108) and 72.5% (49-75) for patients without or with lung damage, respectively. Other clinical features (non-pulmonary) were skin induration 80%, musculoskeletal 73%, gastrintestinal27%, cardiovascular 7%. Autoantibodies profile: ANA 100% and Scl70 20%. Pharmacologic treatment: corticosteroids, cyclophosphamide, mycophenolate, D-penicillamine, methotrexate, and vasodilatadors. Conclusion: Pulmonary disease is the most frequent visceral involvement for jSSc. Abnormal DLCO showed increased risk for progression of interstitial lung disease. Unmet medical need in jSSc-ILD remains difficult to treat, with limited options showing poor effectiveness or progression of lung disease evidenced in our cohort. Prospective, multicentric studies are needed to validate definitions for progression lung disease, as well as functional tests adapted to pediatric population. Disclosure of Interest : None declared
P340. Demographic, time and clinical pattern of Kawasaki disease; the experience in Libya P341. Peripheral tuberculous lymphadenitis presenting as henoch–schönlein purpur: a first child case report in Libya P343. Genetic variants and phenotypic features of childhood large vessel vasculitis: case series and literature review 44. Withdrawn P345. Withdrawn P346. Demographic characteristics of childhood vasculitis other than Kawasaki and Henoch-Schoenlein purpura among arab children: a multicenter study demographic characteristics of childhood vasculitis other than Kawasaki disease and Henoch-Schoenlein P347. Epidemiology of Kawasaki disease in covid times: data from a single center from Eastern India P348. A study on the effectiveness of infliximab following ivig on coronary artery aneurysms in patients with Kawasaki disease P349. A challenging case of fever, recurrent oral ulcers and vertigo P350. Evolution of an immunological disease over a decade P351. The characteristics of Beschet’s disease in Russia: the preliminary data of multicentral retrospective cohort study 52. Takayasu’s arteritis revealed by severe hypertension: about 3 pediatric cases P353. Double coronary aneurisms in Kawasaki disease successfully treated with anakinra: a case report 4. Monogenic mimics of Behçet’s disease 5. The nailfold videocapillaroscopy in pediatric behçet’s disease: a multi-center study P356. Is there an increased future cardiovascular risk in children with vascular behçet’s disease? P357. Longterm kidney prognosis of anca vasculitis in children 8. Association between glutathione s-transferase (gst) m1,t1 and a1 polymorphisms and iga vasculitis: a pilot study P359. Retropharyngeal abscess as an atypical presentation of Kawasaki disease: a case report and literature review 60. The paediatric vasculitis activity score (pvas) and proteinuria in igav nephritis: is there an association with different histologic findings? P361. Clinical profile and medium-term follow up of childhood-onset polyarteritis nodosa in a tertiary care centre in South India P362. GP363. Schoenlein-henoch purpura and enterorrhagia: is this a prelude to IBD? P364. Behçet syndrome in children and adults: discovering similarities and differences by a comparative study 5. Non-typhoid salmonella and Kawasaki disease: randomness or cause / effect? P366. Plasma exchange therapeutic effect in refractory Kawasaki disease P367. Plasmapheresis. therapeutic effect in refractory Kawasaki disease. Case series P368. Infantile takayasu: clinical features and long-term outcome P369. infection and Kawasaki disease: an analytical study from North-India P370. Pediatric Behcet’s disease in india - a single center experience of 14 childrenP371. Clinical presentation and outcome of rare primary systemic vasculitides in 20 children - a single center experience from India P372. Anticoagulation in children with Kawasaki disease and coronary artery aneurysms: our experience at chandigarh, North India P373. Kawasaki disease: incidence figures at Chandigarh, India (2015-2019) 74. Epidemiology and clinical features of pediatric vasculitis: a single-center study 5. Orbital tumor as an initial manifestation of anca-associated vasculitis: a series of three cases P376. Uveitis in Takayasu arteritis - a case report P377. Severe cutaneous manifestations in IGA vasculitis are associated with a more severe clinical course: the experience from the largest international cohort of patients 78. Henoch schonlein purpura (HSP)- case series from newly established pediatric rheumatology cell in North-West India P379. Withdrawn P380. Behcet disease presenting as superior vena cava syndrome P383. Bronchoscopy may be a diagnostic tool in microscopic polyangiitis: case report and review of the literature P384. Pan coronary artery involvement in Kawasaki disease: a unique radiological entity on computed tomography coronary angiography P385. Interval CT coronary angiography in 11 children with Kawasaki disease: our experience at Chandigarh, North India R. Algaryani 1 , H. Alrabti 2 , S. T. Ashur 3 , M. Zletni 4 1 Pediatric Rheumatology, 2 Pediatric Cardiology, Tripoli Children’s Hospital; Faculty of medicine; University of Tripoli, 3 Family and Community Medicine, Faculty of Medicine, University of Tripoli, Libya, 4 Paediatric Rheumatology , Faculty of Medicine,Tripoli university, Tripoli Children’s Hospital , Tripoli , Libya Correspondence: A. A. Abushhaiwia Introduction: Kawasaki disease (KD), an acute, febrile, self-limiting vasculitis of unknown etiology, is a disease that predominantly affects medium- and small-sized arteries of infants and preschool children. Kawasaki disease is the leading cause of acquired coronary artery disease in young children. There is a lack of data on Kawasaki disease and its effect on coronary arteries in Libya and other developing countries. Objectives: This study aimed to describe the demographic, time and clinical patterns of Kawasaki disease (KD) in the Libyan settings. It also examined the association between selected demographic and clinical factors with the presence of abnormal echocardiogram findings at the initial presentation of KD cases Methods: This observational, hospital-based retrospective cohort study was conducted at Rheumatology and cardiac department in Tripoli children’s Hospital in Libya; from January 2012 to December 2020. Those with both complete and incomplete KD were considered. The diagnostic criteria for KD were based on the European and American Heart Association recommendations Results: A total of 71 cases were diagnosed as KD in the period from January 2012 to December 2020, the disease was more prevalent among males (76.1%), and in those aged from 1 to 5 years old (67.6%) than in the younger and older age groups. Most of the identified cases were from Tripoli(67.6%), and the majority were presented first at one of the public health care facilities; primary health care, before being referred to the tertiary hospital. Regarding time distribution, KD cases presented throughout all seasons; however, spring reported the highest percentage (40.8%). Total of them 88.7% were typical KD of patients satisfied the diagnostic criteria for complete KD. Among the cases included,cardiac abnormalities confirmed through echocardiography were documented in 12 (16.9%) ,8 cases out of 12 had changes of the coronary arteries where 2 had ectasia without aneurysm, one case had pericardial effusionall. all of them were detected on the initial evaluation and on the subsequent 2-week evaluation. The presence of abnormal findings dropped to 8 (12.9%) out of 62 cases, and to 5 (12.5%) out of 40 cases in the subsequent echocardiogram examinations done at 6 months and 1 year, respectively Conclusion: Majority of patients fulfilled diagnostic criteria of complete KD, and the presence of coronary artery abnormalities consisted with other international published studies. All patients successfully completely recovered during follow-up, and no mortality was documented Patient Consent: Yes, I received consent Disclosure of Interest : None declared
A. A. Abushhaiwia 1 , N. Abushhiwa 2 , A. Ateeq 3 1 Paediatric Rheumatology , Faculty of Medicine,Tripoli university ,Tripoli Children’s Hospital , 2 Pathology department , Faculty of Medicine ,Tripoli university , 3Tuberculosis is a major public health problem worldwide. It is one of the main causes of infectious disease and mortality, especially in developing countries, Peripheral tuberculous lymphadenitis is the second most common presentation of extrapulmonary tuberculosis in children especially in developing countries, tuberculosis was found to be a triggering factor of Henoch–Schönlein purpura in paediatric patients. Traditionally, cervical lymphadenitis has been the most common site (60-80 %).Given the varied differential diagnoses of lymphadenopathies in paediatrics, PTL is an ongoing challenge for a timely diagnosis and management. Here, we report an unusual case of Henoch–Schönlein purpura associated with peripheral tuberculous lymphadenitis in a young Libyan female child Objectives: we describe a case of peripheral tuberculous lymphadenitis, which was seen to be associated with Henoch-schonlein purpura in a young child. Methods: case report is described Results: A 7 year-old Libyan female child for non-consanguineous parents and had significant past medical history during neonatal period, she had BCG vaccine infected and treated her by anti-tuberculous for 2 months but no family histories neither TB nor autoimmune diseases. Presented to Paediatric rheumatology clinic in April 2022 with progressive bilateral pain over the calves, that rapidly extended to the knees and ankles. Accompanied Cutaneous eruption over the lower extremities followed her since March 2022. The patient also complained of fatigue and painless bilateral cervical lymphadenopathy since December 2021. No fever, no pulmonary, urinary, or abdominal symptoms were noted it. On physical examination, both ankles were erythematous and mild swollen. Palpable purpuric or petechial lesions were covering both lower limbs,there were multiple bilateral cervical lymphadenopathy with bulky (the largest measuring 2 cm in diameter) .Heart and lung auscultation were normal; the abdomen was soft and nontender no organomegaly. Blood tests showed an elevated ESR 103ml,CRP(25) mg/dl),WBC (18X10 3 ) mainly neutrophilia 85%, HGB 10, and PLT 648X10 3. The rest of the analysis was normal and included hepatic and renal chemistry profiles, urinalysis, thyroid-stimulating hormone, complement level, as well as immunoglobulin profile IgA,IgM,IgG,IgE, viral screen(HIV,HBSAg,HCV). A complete auto-immune panel , with anti-neutrophil cytoplasmic antibodies ,cryoglobulin and flowcytomter CD3,CD4,CD8,CD19,CD20,CD56 were sent but still not yet result as we evaluated and considered her immunodeficiency. The tuberculin skin test positive for Tb (PPD: 9 mm) neck, abdominal and pelvis ultrasound showed bilateral cervical lymph node up to 33 mm and multiple bulky and necrotic lymph node enlarged Para aortic &aortocaval. Neck, thoracic and abdominal and pelvis contrast computed topographies demonstrated multiple cervical adenomegaly (up to35x27mm in length), and several retroperitoneal lymph nodes (up to 0.9 cm in length), fine- needle aspiration biopsy in the cervical lymph node was performed,histopathological showed findings compatible with tuberculous lymphadenitis. The patient was initiated of anti-TB treatment (Isoniazid, Rifampicin, Pyrazinamide and ethambutol) it will continue for 6-9 months) and Clarithromycin for 2 weeks. Her vasculitic lesions resolved with anti-tuberculous treatment without the addition of CSs. Conclusion: This is the first paediatric case in Libya that describes an assciocation of Peripheral tuberculous lymphadenitis (TB) infection as a predisposing factor in HSP. This clinical scenario highlights the importance of early suspicion and prompt management. Improved with anti-tuberculosis drugs (ATD) alone. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
S. Alansari, A. AlSaleem, S. Al-Mayouf King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia Correspondence: S. Alansari Introduction: Large vessel vasculitis (LVV) rarely affects children. Clinical characteristics, disease progression and outcomes vary based on the size of blood vessel and organ involved. The precise etiology remains incompletely defined. Recently, genetic variants gained more attention and shed more light on the underlying pathomechanism involved in the various systemic vasculitis. Objectives: To report the phenotypic, genetic findings, outcome of children with LVV. Methods: This work is a retrospective case series of LVV associated with genetic variants from a single tertiary medical center. Also, a systematic literature review was conducted. Results: We identified seven patients with LVV, however two patients were excluded after applying the exclusion criteria. The remaining five patients comprised of 3 males and 2 females. All presented with their primary disease before two years of age. Two patients had recurrent chest infection, skin abscesses and eczema proved to have DOCK8 variants. One patient with FOXP3 variant presented with very early onset inflammatory bowel disease, another patient with DiGeorge syndrome; and one patient had skeletal dysplasia and inflammatory bone disease with homozygous ZNF469 variant and de novo variant in KDM5B. The mean age of the aortitis onset was 12 (±3.6) years and the mean time interval between disease onset and aortitis was 10.6 (±3.7) years. All patients had progressive extensive LVV, affecting mainly aorta and its branches confirmed by radiological study. The most common clinical features were hypertension, abdominal pain, fever and vascular bruit. The most common laboratory findings were high inflammatory markers, leukocytosis, anemia and abnormal hepatic profile. All patients received corticosteroids; three patients received DMARDs. Four patients required surgical intervention, and one underwent hematopoietic stem-cell transplantation. One died and three achieved clinical remission. Furthermore, data of 17 patients extracted from the literature; all included patients had inherited disorders, including hyper-IgE syndrome, Blau syndrome, Wiskott–Aldrich syndrome, Hyper-IgM syndrome, Familial Mediterranean fever, X-linked inhibitor of apoptosis deficiency, Marfan syndrome, Noonan syndrome, Noonan-like syndrome, Juvenile Myelomonocytic Leukemia, and Familial hyper-cholesterolaemia. Eleven patients had genetically proved diagnosis while the diagnosis was based on the expert physician’s opinion and fulfilling the diagnostic criteria. Out of 17 patients, nine patients treated with corticosteroids and eight received DMARDs, one patient completed hematopoietic stem-cell transplantation. Three of them underwent surgical intervention. Most of them showed a reasonable therapeutic response. However, two patients had neurological complications. There were four deaths. Conclusion: LVV associated with genetic variants is a rarely described cluster, which might allow to propose monogenic LVV as a distinct entity. Hopefully, these findings will increase the awareness of the association between LVV with genetic variants, and early recognition can lead to earlier effective intervention in order to improve the outcome. Patient Consent: Not applicable (there are no patient data) Disclosure of Interest : None declared
R. Bakry 1 , S. Alansari 2 , S. Almayouf 2 on behalf of Soad Hashad, Awatif abushhaiwia, Ayah Altawati, Halah Etayari, Magada Altafyl, Abdullatif Alenazi, Khulood Khawaja, Wafaa AlSuwairi, Abdulrahman Alrasheed, Jubran Alqanatish, Sima Abusaoud, Wafa Madan, Raed Alzyoud, Muatasem AlSuwaiti, Djohra Hadef 1 Pediatric Rheumatology, East Jeddah Hospital, Jeddah, 2 Pediatric Rheumatology, king Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia Correspondence: R. Bakry Introduction: Childhood vasculitis is an umbrella term that describes a group of disorders that are characterized by inflammation of blood vessel walls. It could be primary or due to secondary causes. Clinical manifestations are heterogeneous and depend mainly on the size of the vessels involved. Objectives: To report the spectrum and clinical manifestations of childhood vasculitis other than Kawasaki disease (KD) and Henoch-Schoenlein Purpura (HSP) in Arab children, and to highlight the long-term outcome. Methods: We conducted a multicenter retrospective study recruiting patients with childhood vasculitis other than KD and HSP during the period between January 2000 and May 2022, from ten Pediatric Rheumatology clinics in seven Arab countries that are members of the Pediatric Rheumatology Arab Group (PRAG). The collected data comprised demographic and clinical findings, and long-term outcome using the Pediatric Vasculitis Damage Index (PVDI). Results: A total of 150 (78 female) patients diagnosed with childhood vasculitis other than KD and HSP before 14 years old with a median age at onset was 7.2 (IQR 4.3-11) years were enrolled. The initial diagnosis was inaccurate in 59.3% and the interval time to diagnosis was 0.5 (IQR 0-1) years. Consanguinity among parents was 42% and positive family history of vasculitis was (n=18, 12%). The most frequent vasculitis was Bechet’s disease (n=37, 24.6%) followed by ANCA associated Vasculitis (n=24, 16%), Takayasu arteritis (n=16, 10.6%), Polyarteritis nodosa (PAN) (n=15,10%) and cutaneous PAN (n=14, 9.3%). The most common clinical manifestations were fever (n=90, 60%), abdominal pain (n=74, 50%), arthralgia (n=64, 42.6%), headache (n=53, 35%), hypertension (n=43,28%), and palpable purpura (n=31, 20.5%). Most patients had elevated inflammatory markers (73%). ANA was positive in 22%, C-ANCA and P-ANCA were positive in 12% of patients. Angiography showed abnormalities in 22%, while echocardiography was abnormal in 21%. Tissue biopsy was performed in 65 patients. Genetic analysis was done in 36 patients; 19 patients had genetic variants. Most patients (84.7%) received corticosteroids with good response. One hundred-thirty-five patients received immunosuppressive therapy; cyclophosphamide used in 23 patients with good response. Fifty-seven patients used biologic agents, the most frequently used biologic agents was Rituximab (n= 18) followed by Infliximab (n= 12) and Tocilizumab (n= 11) with overall improvement more than 90%. The PVDI showed renal complication (8%) with ESRD (2.5%), cardiomyopathy (7.3%), osteoporosis (6%), and blindness (2.4%). No reported bone marrow depression or malignancies. However, there were four deaths related to the disease or comorbidities. Conclusion: This study presents the first and largest data on childhood vasculitis other than KD and HSP in Arab children. It shows a heterogenous spectrum of vasculitis with high prevalence of genetically confirmed vasculitis. This report intended to increase awareness of these diseases among health care providers. Hopefully, this work will be the first step for a prospective registry for childhood vasculitis in Arab countries. Disclosure of Interest : None declared
J. Bathia 1 , P. Pal 1 , A. Mondal 2 , S. D. Sarkar 2 , H. De 3 1 Department of Pediatric Rheumatology, 2 Department of Pediatric Medicine, 3 Department of Pediatric Endocrinology, Institute of Child Health, Kolkata, Kolkata, India Correspondence: J. Bathia Introduction: Kawasaki disease (KD) is an acute febrile vasculitis in childhood and is the leading cause of acquired heart disease in children. There is a dearth of data on the epidemiology of KD during the COVID times from India. Objectives: The study has been conducted with the objective to estimate the incidence of KD during the two years of the pandemic due to SARS - CoV 2 and to compare it with the pre-pandemic years. Methods: It is an observational, singe center study conducted at the Institute of Child Health, Kolkata. Data of patients admitted with KD during the first and second wave of SARS-CoV2 were compared with the data from the pre-COVID times Results: 1. The first wave (March 2020- December 2020): 33 KD cases, 18 females and 15 males. 2. The second wave (April 2021 - July 2021): 13 KD cases, 4 females and 9 males. 3. In the pre-COVID times, the incidence of KD was: 2018: Total cases 36, females 11 and males 25 2019: Total cases 39, females 14 and males 25 There was a rising trend of KD every year with cases doubling in number from 18 in 2009 to 39 in 2019 4. First wave had 33.33% cases and the second wave had 61.54% cases with coronary artery dilatations but no giant aneurysms (z score> +10) were seen during either waves. In comparison, in 2018, 16.67% cases had coronary involvement with giant aneurysms in 2.78% and in 2019 30.77% cases had coronary involvement with giant aneurysms in 5.13% 5. The first wave had 75 cases of MISC, with 22 (29.33%) KD phenotypes. The second wave had 48 cases of MISC, with 23 (47.9%) KD phenotype. The second wave of MISC had a greater proportion of younger children (median age 6.6 years) with doubling of the KD phenotype Conclusion: Incidence of KD was similar to that of the preceding years, following the same upward trajectory despite the lockdown. If the KD phenotype of MISC is taken into account, there was a two-fold increase in the incidence of KD-like illnesses. Inspite of wide spread lockdown with restriction in transportation facilities hospital admission of patients with Kawasaki Disease remained the same. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
M. Roy 2 , D. Biswas 2 , N. Ahmed 2 , M. Bhelo 2 1 Department of Pediatric Rheumatology, 2 Department of Pediatric Medicine, Institute Of Child Health, Kolkata, India Correspondence: J. N. Bathia Introduction: Kawasaki Disease (KD) is emerging as the commonest childhood vasculitis in India. Cardiac involvement is the major determinant in long term prognosis. Presence of Coronary Artery Abnormalities (CAAs) carries a high risk of complications such as coronary artery aneurysm, ectasia, ischaemic heart disease, arrythmia and sudden death. Both Infliximab (IFX) and corticosteroids have been proposed as add on drugs for patients with CAAs at diagnosis. This study was undertaken to determine effect of IFX on CAAs Objectives: To determine the effect of IFX following IVIG on regression of CAAs. Methods: This single centre, observational s tudy was conducted at the Institute of Child Health Kolkata, India, from January 2016 to December 2019. 33 children aged 6 weeks to 7 years with KD received IFX after 1 or 2 doses of IVIG. IFX was given at 5mg/kg within 24 to 48 hours of completion of IVIG infusion. Of these 33 children, 15 received IFX due to the presence of medium to giant CAAs at diagnosis, increasing CAAs or development of new CAAs following IVIG. Patients were analyzed for change in size of CAAs, as determined by z scores proposed by AHA. Serial echocardiography was done weekly, for the next 4 weeks, then monthly for the next 3 months and then every 3 to 6 months. Results: 15 children with CAAs received IFX, 7 had multiple/ giant CAAs at presentation and 8 with new or enlarging CAAs post IVIG. Seven of them were also IVIG resistant. 7 were infants the youngest being 6 weeks old. 4 infants had giant aneurysms. Diminution in z scores was seen in 80 % (12 out of 15) cases on follow up, giant aneurysms decreased to medium or small sized aneurysms over 6 to 18 months. 50% reduction in the aneurysm size was noted in 60% (n=9) within first 6 months of administration of IFX. Conclusion: 80% of children receiving IFX post IVIG showed progressive decrease in size of CAAs by 18 months. Infliximab therapy has shown to improve treatment response and progression of coronary artery abnormalities Disclosure of Interest : None declared
J. N. Bathia 1 , P. Pal 1 , H. Jagwani 2 , A. GhoshPediatric Behcet’s disease (BD) is a chronic inflammatory vasculitis which can affect any type and size of vessels. It is characterized by recurrent oral ulcerations, recurrent genital ulcerations along with eye and skin manifestations. The global prevalance is reported to be 10/100000 children. Central nervous system manifestations has been reported in 5 to15% of children with BD with variable features such as meningoencephalitis, headache, focal neurological abnormalities, psychosis etc. It is common in adults but rarely seen in adolescent and children. Objectives: To present a six and half yr old female patient with neuro-Behcet’s disease who came with fever, recurrent oral ulcers and vertigo Methods: Six and a half yr old female child presented to us with a history of recurrent oral ulcers along with fever for a duration of 10 days. Initially she was managed as herpetic gingivostomatitis but there was no improvement. Then she developed rashes on face, trunk, palms and soles and limb weakness. Results: On examination we noted the presence of violaceous, nodular rashes, flat topped plaques on knuckles, arthritis of bilateral proximal interphalangeal joints, nodular lesion in bilateral elbows and bilateral hip pain. Initial investigations were haemoglobin 9.6 g/dl, total leukocyte count 7.2 x 10 3 /μL, platelets 193 x 10 3 /μL, serum creatinine kinase 97 units/litre, eye examination was normal and Tzank smear normal. Oral steroids was started but symptoms persisted. HLA B 5 was also negative. With a suspicion of BD oral colchicine was added which decreased the symptoms. After two months the fever reappeared. It was non-responding. Echocardiography was done which was suggestive of left ventricular hypertrophy with myocarditis leading to a diagnostic dilemma. Non remitting fever was worked up. Infection ruled out, ANA profile, ANCA , immunoglobulin profile was normal. Tuberculosis ruled out. Intravenous methyl prednisolone was started. As the fever was persistent the dose was escalated from 2mg/kg/day to 6 and then 30mg/kg/day. In the meantime, she started developing vertigo for which MRI brain with angiography was done. It showed the presence of multiple small white matter signal abnormalities likely ischaemic foci. Itravenous cyclophosphamide for given for 6 cycles during which the fever subsided after the first cycle. After 2 months fever, ulcers and vertigo reappeared and mycophenolate mofetil (MMF) was added. Patient is now on colchicine, tapering dose of steroids and MMF and is in remission Conclusion: Pediatric neuro-Behcet’s disease is a challenging disease and is not well described. Although rare it should be considered even with subtle neurological manifestations in children with recurrent ulcers. Early diagnosis and immunosuppressive therapy is pivotal. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
J. N. Bathia 1 , P. Pal 1 , G. S. H 2 , M. Bhelo 2 , D. Biswas 2 , S. D. Sarkar 2 1 Department of Pediatric Rheumatology, 2 Department of Pediatric Medicine, Institute of Child Health, Kolkata, India Correspondence: J. N. Bathia Introduction: Burkitt’s lymphoma (BL) is a rare and a very aggressive B-cell lymphoma that represents less than 1% of all non-Hodgkin lymphomas (NHL). One of the clinical subtypes of BL is immunodeficiency-related form common among HIV-infected patients, patients with autoimmune diseases and patients with primary immunodeficiency disorders. Autoimmune disorders such as SLE, vasculitis, celiac disease etc have been associated with an increased cancer risk, particularly for certain cancer types. The underlying pathophysiology is yet to be understood. Objectives: We report a case of ANCA- negative small vessel vasculitis who developed Burkitts lymphoma after 10 years of initial diagnosis. Methods: 5 year old girl presented with a history of intermittent painful nodular lesions over her lower limbs for 3 months, two episodes of haemoptysis followed by persistent fever for last 15 days. Investigations showed neutrophilic lymphocytosis, high titres of CRP and ESR and thrombocytosis. Skin Biopsy revealed deep dermis blood vessels with neutrophilic infiltrates and nuclear debris- suggestive of vasculitis. Bronchoscopy showed normal anatomy and congested mucosa. Bronchoalveolar lavage microscopy revealed inflammatory background with many hemosiderin laden macrophages. Antinuclear antibody (ANA), antineutrophil cytoplasmic antibodies (c- ANCA) and perinuclear antineutrophil cytoplasmic antibodies (p-ANCA) were negative. A diagnosis of ANCA negative small vessels systemic vasculitis was made and she was started on steroids and pulse cyclophosphamide. On follow up, she was maintained on mycophenolate but had multiple relapses on attempting to taper steroid. Finally, she was administered Rituximab after two years which had to be repeated after a year for a minor flare. She was in remission but required two hospital admissions at an interval of six months for lobar pneumonia. Investigating for any associated immunodeficiency showed persistent low levels of IgG, which had developed post Rituximab. She was kept on cotrimoxazole prophylaxis and remained asymptomatic. Results: At the age of 15years she presented with pain abdomen with generalized discomfort for 15 days and acute onset right sided ptosis and right medial rectus palsy for 7 days. MRI Brain with contrast revealed irregular enhancing lesion at the confluence of inferior sagittal sinus with the straight sinus- due to venous sinus thrombus/ mass lesion. USG abdomen showed right adnexal mass and biopsy from the mass confirmed the diagnosis of Burkitts lymphoma Conclusion: Several studies have evaluated the association between autoimmunity and cancer. The factors that are speculated to influence the association include medications for autoimmune disease and interaction between treatment and viral exposure. The development of NHL from activated lymphocyte suggests that chronic inflammation might increase the risk of lymphoma in autoimmune diseases Patient Consent: Yes, I received consent Disclosure of Interest : None declared
K. Belozerov 1 , V. Klavdenkova 1 , Z. Shogenova 1 , A. Yakovlev 1 , L. Andaryanova 1 , E. GaidarT. Burtseva 2 , V. Argunova 3 , P. Sleptsova 3 , S. Boeskorova 4 , L. Leonteva 5Saint-Petersburg, 2 Department of Pediatrics and pediatric surgery, North-Eastern Federal University , 3 Cardiorheumatology, State Autonomous Institution of the Republic of Sakha (Yakutia) Republican Hospital No. 1 - National Center of Medicine, 4 Department of Pediatrics and pediatric surgery, North-Eastern Federal University, 5 Yakut science centre of complex medical problems, Rheumatological, Yakutsk, Russian Federation Correspondence: M. Kostik Introduction: Beçet’s disease (BD) is a rare systemic vasculitis, associated with certain nationalities, related to Great Silk Road. BD in Russia is rare and the data about BD in Russia is scarce. Objectives: to describe clinical course of BD in Russia. Methods: in the retrospective cohort study we included data from patient’s case histories. We evaluated demography, family history, clinical and laboratorial features, treatment options and outcomes. The diagnosis was made according criteria of the International Study Group for BD, 1990. Results: from 44 patients with inclusion age 21.5 years (15.7; 38.6) 59.0% (26/44) had pediatric onset (11 females) and 41% (18/44) adult onset (12 females). Asians/Causacians was 9 (20.5%)/ 35 (79.5%) patients. BD positive family history was in 4 patients (9%). The most frequent first symptom of BD was oral ulcers in 31/44 (70.5). Among clinical features patients with BD had the following organ and system involvement: oral ulcers -95%, genital ulcers - 53%, ulcers of both localizations - 52%, eye involvement - 45%, skin - 48.7%, positive pathergy phenomenon - 50%, CNS - 23%, GI - 39%, joints - 59%, thrombotic events/large vessel vasculitis - 7%. Laboratorial features: ESR - 21.0 (12.5; 27.8) mm/h, CRP - 3.9 (0.4; 14.5) mg/l, number of patients with increased ESR - 55%, with increased - CRP 55%, with anemia - 36.4%. The rate of HLAB51 positivity was 50%, HLAB27 positivity - 40%, RF positivity - 18.2%. The main comorbidity included Crohn’s disease 4 (9%) Treatment options included: corticosteroids – 67%, colchicine – 42%, TNF-a inhibitors 37% (etanercept 6.25%, adalimumab 43.75%, golimumab and infliximab 25% each), azathioprine – 26%, cyclophosphamide and methotrexate – 10 % each. Also less frequent medications used: canakinumab (n=1), tocilizumab (n=2), tofacitinib (n=1), cyclosporine A (n=1), MMF (n=1), hydroxychloroquine (n=2), sulfasalazine (n=3). In 5 patients biologics were switched. Conclusion: the data about BD in Russia is limited, prevalence underestimates, big diagnostic delay is typical and further investigations are require, S. Lotfi, A. SakhiCasablanca, Morocco Correspondence: K. Bouayed Introduction: Takayasu’s arteritis (TA) is a chronic panarteritis mainly affecting the aorta and its main branches, which occurs rarely in children. Objectives: We describe the clinical and paraclinical features, the treatment strategies and the evolutive profile of three cases of TA. We draw attention to the association with tuberculosis. Methods: We report three cases of TA, revealed by severe arterial hypertension. Results: Three gilrs with a mean age of 11 years were admitted with severe arterial hypertension, revealed in one case by headaches and tinnitus, and in two cases by a status epilepticus. The clinical examination showed decreased femoral pulses and an audible murmurs at auscultation of abdominal aorta in one case, discrepancy of four limb systolic blood pressure >10 mmHg in another case. Two of our patients had glomerular injury confirmed either by an albuminuria/creatinuria ratio at 539 mg/mmol or by a 24-hour proteinuria at 111mg/kg/day. TA was confirmed by CT angiography which revealed an extensive stenosis of the sub-renal abdominal aorta and of the left renal artery in the first case, a total occlusion of the right renal artery in the second case, and a parietal thickening of the supra- and sub-renal abdominal aorta and the left renal artery in the last case. DMSA renal scintigraphy showed unilateral decline in renal function according to the location of the vascular damage, estimated at 36%, 8% and 5% respectively. All our patients were treated with anti-hypertensives, corticosteroids and azathioprine, with therapeutic failure in both cases leading to nephrectomy, one of them was followed by aorto-aortic bypass surgery. The evolution was favourable for the third patient who presented an associated tuberculosis diagnosed on cervical lymph nodes, an important inflammatory syndrome and a positive Quantiferon and treated with antibacillary drugs with a normalization of the inflammatory markers. Conclusion: Our patients presented a severe clinical features with malignant hypertension very difficult to control. The association with tuberculosis, as described in our case, has already been reported in tuberculosis endemic countries. Disclosure of Interest : None declared
K. Bouayed 1 , S. Lotfi 1 , A. Sakhi 1 , N. Mikou 1 , A. Driguil 2 1 Department of Pediatric Rheumatology and Internal Medicine, Hôpital Mère Enfant Abderrahim Harouchi, 2 Department of Cardiology, CHU Ibn Rochd, Casablanca, Morocco Correspondence: K. Bouayed Introduction: Kawasaki disease is an acute inflammatory vasculitis of the medium and small-caliber arteries, usually occurring in children under 5 years of age. Refractory Kawasaki disease is associated to a major risk of coronary arteries abnormalities and its treatment is not standardized. In this regard, Anakinra, a interleukin-1 receptor antagonist, represents an emerging therapeutic option. Objectives: We report the case of a 9-month-old girl, diagnosed with refractory Kawasaki disease, successfully treated with Anakinra 3 months. Methods: We report the case of a 9-month-old girl, diagnosed with Kawasaki disease, refractory to two doses of intravenous immunoglobulin and acetyl salicylic acid, who developed aneurysms, successfully treated with Anakinra 3 months with 4 years of follow-up Results: A previously healthy 9-month-old girl was admitted with an 8-day persistent fever of 39.7°C, a perineal scaling erythematous rash, bilateral non-purulent conjunctivitis, cheilitis, right cervical lymphadenopathy measuring 2 cm, and asthenia without extremity abnormalities. Blood tests showed normochromic microcytic anemia (Hb:9.9 g/dL, MCV: 69 fl, MCHC: 34 g/dL), hyperleukocytosis at 17.3 G/L with a predominance of neutrophils 11.64 G/L, normal platelets (289 G/L), elevated ESR at 122 mm at the first hour and C-reactive protein at 260 mg/L, hyponatremia (120 mmol/L), hypoalbunemia (28g/L), and transaminases at 69 U/L for AST and 33 U/L for ALT, urine cytobacteriological examination was normal. Kawasaki disease was diagnosed with a Kobayashi score of 4, predicting IVIG resistance. Treatment with IVIG (2 g/kg) and aspirin (100 mg/kg) was initiated on day 9 of fever and an echocardiogram performed on day 11 of fever was normal. The patient received a second dose of IVIG on day 16 because of persistent fever and increased inflammatory markers. Iterative CARDIAC ultrasound revealed an aneurysm exacerbation in the left coronary artery (6 mm with Z-score +11) and in the left anterior descending coronary artery (4 mm with Z-score +11). The blood tests showed a significant increase in platelets (758 G/L) with a decrease in ESR (40 mm/h) and CRP (74 g/L). Regarding the significant coronary lesions, a second-line treatment with Anakinra 5 mg/kg/day was introduced for 3 months associated with a dose of anti-aggregating aspirin 5 mg/kg/day leading to apyrexia on day 6 of Anakinra, complete normalization of inflammatory tests at month 3 and coronary arteries at month 8. Conclusion: Our experience supports the existing data on the efficacy of Anakinra as a second-line treatment in some cases of refractory Kawasaki disease, particularly in cases of severe coronary artery involvurleigh 1,2 , E. Omoyinmi 2 , C. Papadopoulou 3 , E. Al-Abadi 4 , D. Eleftheriou 1,2,3 , P. Brogan 2,3 1 Centre for Adolescent Rheumatology Versus Arthritis at UCL, 2 UCL Great Ormond Street Institute of Child Health, 3 Great Ormond Street Hospital for Children NHS Foundation Trust, London, 4 Childhood Arthritis and Rheumatic Diseases Unit, Birmingham Women’s and Children’s Hospital NHS Foundation Trust, Birmingham, United Kingdom Correspondence: A. Burleigh Introduction: Behçet’s Disease (BD) is a rare, polygenic autoinflammatory vasculitis. Multiple risk alleles across numerous genes have been associated with BD, the strongest association of which is with HLA-B*51. There is, therefore, no single genetic hallmark of BD, making definitive diagnosis challenging. Many rare monogenic diseases can present with symptoms that are indistinguishable from BD 1 , and it is vital that BD is differentiated from these so-called ‘monogenic mimics’, as the treatments and prognoses can differ. Objectives: The aim of this study was to undertake whole exome sequencing in an unselected cohort of patients from the UK with clinically diagnosed BD to investigate the possibility of a monogenetic pathogenesis. This will enable: Informed diagnoses and treatments for the recruited patients. Insight into the genetics of BD-like disease. Potential for novel autoinflammatory disease and variant discovery. Methods: Patients were recruited from five hospitals in London, Birmingham, and Oxford. The only inclusion criterium was a clinically suspected diagnosis of BD. Patients’ DNA was whole exome sequenced, and analysis performed using three approaches: Rare variants in the ~400 genes of our in-house vasculitis and inflammation panel were manually assessed. Potential disease-causing variants were prioritised based on connection to phenotype using Exomiser 2 . HLA genotype was obtained using OptiType 3 . Results: A total of 33 patients were recruited, 13 female, median age 13 (3-51) and median age of onset 5 (0-20). Using our genetic analysis pipeline, eight patients were found to have suspected monogenic BD mimic diseases: Four cases of Haploinsufficiency of A20 with five novel TNFAIP3 mutations (p. p.G316S, p.S548Dfs, p.M112Tfs, p.C657fs, p.E661Nfs). One case of ISG15 deficiency with a novel homozygous ISG15 mutation (p.Q16X). One case of common variable immune deficiency with a pathogenic TNFRSF13B mutation (p.A181E). Two cases of Tumour necrosis factor receptor associated periodic syndrome (TRAPS) with a pathogenic low penetrance TNFRSF1A mutation (p.R121Q). Of the patients for whom a monogenic cause could not be discerned, seven were HLA-B*51 positive, and classified as having typical BD. The remaining 16 patients were classified as having BD without monogenic cause or HLA-B*51 genotype. Two cases remain under analysis. The functional consequences of the novel mutations discovered in this study are under investigation. Conclusion: This work has expanded the genotypic spectrum of autoinflammatory diseases that can mimic BD. The genetic diagnoses made in this study have enabled clinicians to make informed decisions regarding individuals’ treatments and prognoses, highlighting the importance of next generation genetic sequencing in patients with suspected BD. The analysis workflow designed for this study offers a template for which genetic characterisation of BD-like disease could be implemented more routinely, both for identification of monogenic diseases, and for HLA-typing. 1.Papadopoulou C, Omoyinmi E, Standing A, Pain CE, Booth C, D’Arco F, et al. Monogenic mimics of Behçet’s disease in the young. Rheumatology. 2019 2. Smedley D, Jacobsen JOB, Jäger M, Köhler S, Holtgrewe M, Schubach M, et al. Next-generation diagnostics and disease-gene discovery with the Exomiser. Nature Protocols. 2015;10(12):2004-15 3. Szolek A, Schubert B, Mohr C, Sturm M, Feldhahn M, Kohlbacher O. OptiType: precision HLA typing from next-generation sequencing data. Bioinformatics. 2014;30(23):3310-6Çakmak 1 , S. Doğantan 2 , R. İşgüder 3 , M. Kasap Cüceoğlu 4 , S. Çağlayan 5 , H. E. Sönmez 6 , Ö. Akgün 1 , B. Sözeri 5 , A. Paç Kısaarslan 2 , E. Ünsal 3 , S. Özen 4 , N. Aktay Ayaz 1 1 Pediatric Rheumatology, Department of Pediatric Rheumatology, Istanbul University Medical School, Fatih, Istanbul, Turkey, istanbul, 2 Pediatric Rheumatology, Kayseri Erciyes University, Kayseri, 3 Pediatric Rheumatology, Dokuz Eylül University, İzmir, 4 Pediatric Rheumatology, Hacettepe University, Ankara, 5 Pediatric Rheumatology, Umraniye Training and Research Hospital, istanbul, 6 Pediatric Rheumatology, Kocaeli University, Kocaeli, Turkey Correspondence: F. Çakmak Introduction: Behçet’s disease (BD) is a chronic inflammatory disease characterized by recurrent oral aphthous and genital ulcers accompanied by eye, joint, skin, gastrointestinal and central nervous system involvement. The vascular involvement may affect both the arterial and venous systems. Nailfold videocapillaroscopy (NVC) is an easy and non-invasive method used in the evaluation of microcirculation. Objectives: With this study, we aimed to find the characteristics and prevalence of nailfold capillary alterations in patients with juvenile BD and to analyze their possible relationship between clinical characteristics and activity of the disease. Methods: Patients aged 5-21 years with a diagnosis of juvenile BD and followed up for at least six months were included in the study. Demographic and clinical characteristics of the patients were recorded. NVC was performed on 8 fingers of both hands, excluding the thumbs, and four consecutive non overlapping fields for each of fingers were evaluated (32 fields per patient). Capillary density, capillary width (arterial width, venous width, apical loop), capillary morphology and the presence of meandering capillary, microhemorrhage, avascular area, neoangiogenesis, capillary ramification were evaluated from the images. Capillary morphology were evaluated by classifying them into four groups as normal, minor abnormalities, major abnormalities and scleroderma pattern. The presence of abnormilities in at least two fingers were recorded as capillary abnormality. The semiquantitative rating score 1-3 was applied for each capillaroscopic alteration. Results: 37 patients from 6 pediatric rheumatology centers were included in the study. The mean age of patients was 17 years (IOR 13-19) and 20 (54.1%) of them were girls. The patients were evaluated in four clusters according to their clinical presentations. Nineteen patients had mucocutaneous involvement, 9 patients had uveitis, 8 patients had vascular and neurological involvement, and 4 patients had gastrointestinal system involvement. During the follow-up period, genital ulcers developed in 22 patients, erythema nodosum in 9 patients, pseudofolliculitis in 18 patients, uveitis in 10 patients, vascular involvement in 8 patients, and neurological involvement in 5 patients. Anterior uveitis was present in five, posterior uveitis in three, panuveitis in one, and retinal vasculitis in three of the patients with ocular involvement. Four patients had lower extremity venous thrombosis, three patients had central nervous system (CNS) thrombosis, and one patient had both lower extremity and CNS thrombosis. When capillary morphology was evaluated; normal morphology was present in 16 patients, minor abnormality in 13 patients, and major abnormality in 8 patients. Median capillary density was 8, capillary length was 325 μm, arterial width was 12 μm, venous width was 16 μm, apical loop width was 18 μm, capillary width was 39 μm, and intercapillary distance was 107 μm. Neoagiogenesis was seen in 13 patients, enlarged capillaries in 12 patients, capillary meandering in 9 patients, bushy capillaries in 5 patients, bizarre capillaries in 4 patients, and microhemorrhage in 3 patients. Neoangiogenesis was found to be significantly more common in the NVC evaluation of patients with lower hemoglobin values at the time of diagnosis (p=0.014). Conclusion: NVC is an in vivo, non-invasive, and inexpensive imaging technique that allows the direct observation of the capillary network in living tissue throughout the skin and it may be preferred in juvenile BD for evaluating microvascular involvement. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
S. Demir 1 , A. DÜZOVA 2 , O. Cimen 1 , E. Sağ 1 , B. Oguz 3 , T. Karagöz 4 , S. Ozen 1 , Y. Bilginer 1 1 Depatrment of Pediatric Rheumatology, 2 Depatrment of Pediatric Nephrology, 3 Department of Radiology, 4 Department of Pediatric Cardiology, Hacettepe University Medical Faculty, Ankara, Turkey Correspondence: S. Demir Introduction: Behçet’s disease (BD) is a polygenic multisystemic autoinflammatory disorder and vascular involvement is one of the major causes of morbidity and mortality of the disease. The disease is commonly seen in young adults however can occur in childhood, as well. Objectives: We aimed to define whether vascular involvement of pediatric BD is a risk factor for a future cardiovascular disease. Methods: Thirty-one BD patients who followed at pediatric rheumatology outpatient clinic, were enrolled to the study. The pediatric patients (<16 years of age at disease onset and diagnosis) were classified as having BD according to the Pediatric Behçet’s Disease (PEDBD) classification criteria. Demographic data, clinical manifestations, laboratory and radiological findings and outcomes were documented from patient charts. Patients with incomplete BD, and have other known risk factors for cardiovascular disease as obesity and hypertension were excluded. During the same week, carotid intima-media thickness (cIMT) measurement and echocardiography and 24-hour ambulatory blood pressure monitoring (ABPM) were performed. Physical examination and blood tests were also performed on the same day with cIMT. Results: Thirty-one children with pediatric BD (16 female, 51.6%; F/M: 1.06) were enrolled in the study. The patients were classified as having BD according to the Paediatric BehÇet’s Disease (PEDBD) classification criteria. Based on the cumulative disease characteristics; oral ulcer was the most common clinical finding (100%), followed by skin involvement (78%, n=25), arthritis (56%, n=18), genital ulcers (47%, n=15), ocular involvement (28%, n=9) had and vascular involvement (18%, n=6). We grouped patients into two groups as patients with and without vascular involvement. The mean age at disease onset and at the time of BD diagnosis was 8.79 ± 4.23 years, and 11.62 ± 3.22 years, respectively. The median follow-up duration was not statistically different between two groups (59.63 months (IQR: 58.38-63.57) vs 40.28 months (IQR: 11.30-75.47)). There was no significant difference between the two groups in terms of other organ involvement, except for CNS involvement. In patients with vascular involvement, VLDL, Triglyceride and CRP levels were found to be significantly higher and HDL levels were found to be significantly lower (Table). The mean values of right cIMT, left cIMT were higher in patients with vascular involvement, however it did not reach statistical significance. Similarly, the prevalence of abnormal ABPM, non-dipping, and ambulatory hypertension was higher in patients with vascular involvement, they did not reach to statistical significance, as well. In echocardiography measurements, vascular BD patients had significantly higher rates of aortic outflow and velocity integral of the aorta which points out increased stiffness of the aorta. Conclusion: We suggest that, pediatric-onset vascular Behçet’s disease may increase the risk of advanced cardiovascular disease. Long-term follow-up studies in larger series may clarify the value of ABPM. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
M. Drozynska-Duklas, I. Zagozdzon, I. Balasz-Chmielewska, I. Zaluska-Lesniewska, A. Zurowska Paediatrics, Nephrology and Hypertension, ERKNet ERN-Rare Diseases Centre, Medical University of Gdansk, Gdansk, Poland Correspondence: Drozynska-Duklas Introduction: Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a necrotizing vasculitis associated with the presence of antibodies specific for myeloperoxidase or proteinase 3. Objectives: Prognosis in children with AAV has improved but is still associated with morbidity and mortality and the long term kidney outcome in these children is not well described. The aim of this study was to analyze the medical data of patients previously treated in our centre for AAV. Methods: Between 2004-2022 a medical record search identified eight children with AAV at a single pediatric nephrology centre serving a population of 2 million inhabitants. The disease was diagnosed in 6 girls and 2 boys at a mean age of 13.1 (range: 8,6-16,7) years. Microscopic polyangiitis (MPA) was recognized in five and granulomatosis with polyangiitis (GPA) in three subjects. Results: In subjects with MPA the prevailing initial symptoms were those of advanced kidney failure frequently with little extrarenal involvement. Upper and lower respiratory tract involvement predominated in GPA subjects who at onset showed less severe kidney symptoms. Initial eGFR in MPA patients was decreased (mean: 28 ml/min/1,73m 2 ) in contrast to usually normal values in GPA subjects (mean eGFR 125ml/min/1,73 m 2 ). Diurnal proteinuria was more severe in MPA (mean: 3,43 g/day) than in GPA children (mean: 0,83g/day). All patients underwent intensive immunosuppression. After a mean follow-up of 7 years four out of five MPA subjects were on renal replacement therapy but had not experienced further vasculitis relapses. GPA subjects had frequent relapses but after a mean follow-up of 9 years had normal eGFR (mean:105 ml/min/1,73m 2 ). Conclusion: Systemic ANCA-associated vasculitis with renal involvement is an ultra-rare disease in children. MPA carries a poor prognosis with frequent late kidney manifestation of advanced Chronic Kidney Disease. GPA has less severe kidney manifestations at onset with better kidney prognosis in spite of a relapsing courJuras 1 , M. Held 2 , M. Sestan 2 , M. Batnozic Varga 3 , N. Kifer 2 , S. Srsen 4 , A. Gagro 5 , M. Frkovic 2 , S. Huljev Frkovic 2 , M. Jelusic 2 , K. Crkvenac Gornik 1 1 Department of Laboratory Diagnostics, University Hospital Centre Zagreb, 2 Department of Pediatrics, University Hospital Centre Zagreb, University of Zagreb School of Medicine, Zagreb, 3 Department of Pediatrics, University Hospital Centre Osijek, Josip Juraj Strossmayer University of Osijek, Faculty of Medicine, Osijek, 4 Department of Pediatrics, University Hospital Centre Split, University of Split School of Medicine, Split, 5 Children’s Hospital Zagreb, Josip Juraj Strossmayer University of Osijek, Faculty of Medicine, Zagreb, Croatia Correspondence: M. Held Introduction: IgA vasculitis (IgAV) is the most common childhood vasculitis. Considering the clinical heterogeneity, genetic factors might play a role in pathogenesis of IgAV. Glutathione S-transferase (GST) are members of a multigene family of metabolic enzymes divided into four major subfamilies designated as GST α ( GSTA1 ), GSTμ (GSTM1), GSTθ ( GSTT1 ) and GST π ( GSTP1 ), act as cell housekeepers protect cells against oxidative stressors in the environment by detoxifying a wide variety of potentially toxic and carcinogenic electrophiles. Deletions in GSTs lead to reduction in detoxification enzymatic activity. It was identified that detoxification effects modified by GST s polymorphism possibly can aggravate the susceptibility to diseases. Objectives: To investigate the GSTA1, GSTM1 and GSTT1 genes polymorphism and their influence to susceptibility for IgAV. Methods: Clinical data were collected from four Croatian tertiary centers for pediatric rheumatology. GSTA1, GSTM1, and GSTT1 polymorphisms were detected in patients and controls. DNA was isolated from whole blood using the QIAGEN QIAamp kit. GSTA1 (-69C>T) was examined by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method whereas the GSTM1 and GSTT1 were determined by the PCR method. Results: Pilot study included 107 patients diagnosed with IgAV, of whom 56 girls and 51 boys, with median age at the time of diagnosis 6.25 (4.5-8.0) years, as well as 75 sex and age-matched controls. All patients had purpuric rash, 75,7% had arthralgia or arthritis, 36,5% had gastrointestinal involvement, while 31,7% patients developed IgA vasculitis nephritis (IgAVN). The frequencies of GSTM1 (−) null allele and GSTT1 null (−) allele in IgAV patients were 56,1% and 26,2% respectively. There was no statistically significant difference in the null genotype distribution of GSTM1 and GSTT1 between groups (CI 0.49-1.62, OR 0.89, p=0.714; CI 0.35-1.44, OR 0.70, p=0.335). The frequency of GSTA1 C/C, GSTA1 C/T and GSTA1 T/T genotypes in IgAV patients were 36,5%, 44,8% and 18,7% respectively. There was no statistically significant differences in genotype frequencies between patients and controls (CI 0.83-3.00, OR 1.38, p=0.167; CI 0.33-1.09, OR 0.60, p=0.09; CI 0.55-2.65, OR 1.20, p=0.639). Patients with gastrointestinal involvement had statistically significant difference in the null genotype distribution of GSTM1 compared with patients without gastrointestinal involvement (CI 0.15-0.81, OR 0.35, p=0.014). Conclusion: Our pilot study provides evidence that the examinated polymorphisms were not associated with the increase individual susceptibility for IgAV, although GSTM1 genotype proved to have effect on gastrointestinal involvement in IgAV. For precise evaluation of results it is necessary to include larger study populations, however this study offers some essential information for further research. SUPPORT: Croatian Science Foundation IP-2019-04-8822. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
R. Kasem Ali Sliman, M. Hamad Saied Carmel Hospital, haifa, Israel Correspondence: R. Kasem Ali Sliman Introduction: Only 40% of Kawasaki Disease (KD) patients present with adequate clinical criteria for diagnosis, the remainder present with an incomplete or atypical presentation [1][2]. One of the most challenging cases of KD can present with a clinical and radiological manifestation of a retropharyngeal abscess. Objectives: We describe a patient who presented with an atypical retropharyngeal abscess-like lesion, unresponsive to several antibiotics regimens, as well as surgical drainage, but promptly responded to immunoglobulin treatment once a diagnosis of KD was suspected. In addition, we present a literature review of similar cases published over the last 30 years. Methods: A literature search was conducted, the primary database was PubMed (Medline), Embase and Google Scholar. The key words were Kawasaki disease, Pediatrics, Retropharyngeal abscess. Fifteen articles were found. Results: An eight years old girl presented with three days of fever associated with left neck swelling, tenderness and torticollis, CT scan demonstrated left retropharyngeal swelling with a hypodense lesion of 45mm with a suspected abscess. No improvement after five days of different antibiotics regimens and surgical intervention. On the 8 th day of fever, symmetrical arthritis of the PIPs, MCPs and MTPs appeared and a non-purulent conjunctivitis as well as a strawberry tongue with swollen dry lips were noted at her examination. A single dose of IVIG led to a rapid clinical improvement and resolution of fever and other signs and symptoms. Seventeen case reports including our case describing pediatric patients presenting with retropharyngeal abscess, later diagnosed with KD. The patients were predominantly males (82%), age range from 10 months to 9 years with mean age of 5 years, which considered higher than the average of patients with KD. The majority of the cases (94%) initially presented with fever and neck swelling, without additional clinical criteria, leukocytosis was in all patients with average of 18500 WBC/μL. All the cases were initially concerning for possible deep neck bacterial infection, prompting antibiotic therapy and imaging studies. CT imaging were performed in all the cases, with findings suspicious for retropharyngeal disease, but in the majority without enhancement. Intravenous antibiotics were administered in 16 patients (94%), surgical drainage was attempted in 7 patients (41%), with two 2 cases (11%) of purulent fluid, only one of them being culture positive for Staphylococcus aureus. The diagnosis was delayed beyond 9 febrile days in 5 patients (30%). 41% of the patients had a cardiac manifestation compared to less than 20% in the general pediatrics population who are diagnosed with KD. All the patients had a dramatic and fast clinical improvement with resolution of fever within 24-48 hours after IVIG administration. Conclusion: KD requires high index of suspicion and awareness of unusual presentations. In our case and literature review Kawasaki disease mimicked a retropharyngeal abscess that was refractory to antibiotics and surgical intervention. Thus, It should be kept in mind as one of the differential diagnosis of patients with febrile lymphadenitis and/or retropharyngeal abscess who do not respond to antibiotic treatment in the relevant clinical context. This can prevent delay in diagnosis and the detrimental sequelae especially cardiac complications. References [1] Isidori C., Sebastiani L. and Esposito S. A Case of Incomplete and Atypical Kawasaki Disease Presenting with Retropharyngeal Involvement. Int J Environ Res Public Health. 2019 Sep; 16 (18): 3262. [2] Kritsaneepaiboon S., Tanaanantarak P., Roymanee S. and Lee E.Y.Atypical presentation of Kawasaki disease in young infants mimicking a retropharyngeal abscess. Emerg Radiol. 2012 Apr; 19 (2): 159-16Kifer 1 , M. Sestan 1 , M. Held 1 , D. Kifer 2 , S. Srsen 3 , A. Gudelj Gracanin 4 , M. Heshin-Bekenstein 5 , T. Giani 6 , R. Cimaz 6 , M. Frkovic 1 , S. Bulimbasic 1 , A. Gagro 7 , M. Coric 1 , M. Jelusic 1 1 University of Zagreb, School of Medicine, UHC Zagreb, 2 University of Zagreb, Faculty of Pharmacy and Biochemistry, Zagreb, 3 University of Split School of Medicine, UHC Split, Split, 4 CH Holly Spirit, University of Zagreb School of Medicine, Zagreb, Croatia, 5 Dana Dwek Children’s Hospital, Tel Aviv Medical Center, Tel Aviv, Israel, 6 University of Milan, Milan, Italy, 7 Children’s Hospital Zagreb, University of Osijek, Medical Faculty Osijek, Zagreb, Croatia Correspondence: N. Kifer Introduction: IgA vasculitis (IgAV) is usually self-limiting with a favorable prognosis. However, the development of nephritis (IgAVN) can lead to chronic kidney disease and kidney biopsy has been a continued standard in determining the severity of IgAVN. The association between the disease activity, as well as laboratory parameters descibing kidney function, and different histologic classifications used for IgAVN are still left unclear. Objectives: To determine whether there is an association between histologic variables, measures of disease activity (PVAS) and markers of kidney function. Methods: Patients included were diagnosed with IgAV and IgAVN based on EULAR/PRINTO/PRES criteria in the period from 2003 to 2021. Their renal biopsy findings were examined using light microscopy, immunofluorescence, and electron microscopy analyses. Four classifications were used: ISKDC, Haas classification, Oxford classification, and SQC classification. PVAS was determined at the time of diagnosis. Results: The study included 67 patients, with the median (range) age of 10.8 (3.1-28.5) years at the diagnosis. Fifty-eight percent of patients were male, with a male to female ratio of 1.4:1. The median time from IgAV diagnosis to IgAV nephritis was 5 (0-270) days. The median time from the onset of nephritis to kidney biopsy was 30 (2-2555) days. Laboratory parameters were tested for association with all four classifications. Twenty-four hours protein excretion has shown statistically significant correlation with higher grade in Oxford classification (b= 0.58 ± 0.12, p <0.001), SQC classification (b= 0.44 ± 0.11, p <0.001), and ISKDC classification (b= 0.36 ± 0.11, p <0.001), but not with Haas classification. However, it was found that patients with hematuria had a higher grade in Haas classification, in comparison with patients without hematuria (b= 1.93 ± 0.49, p <0.001). The median (range) PVAS of our patients at diagnosis was 15 (2-30). We explored whether there is an association between the activity of the disease and histologic variables. Endocapillary proliferation and tubular atrophy from Oxford classification were found to associate with PVAS. Endocapillary proliferation showed a statistically significant positive correlation (b= 0.70 ± 0.25, p =0.008), while tubular atrophy was negatively associated (b= -1.39 ± 0.55, p =0.015). Conclusion: Our findings suggest that a higher 24h urine protein excretion could be a possible indicator of higher grades in ISKDC, Oxford and SQC classifications. Furthermore, we have found that when the activity index is high, we could expect acute histologic changes such as endocapillary proliferation, while chronic changes, such as tubular atrophy, could occur after the PVAS score decreases. Support: Research is supported by Croatian Science Foundation project IP-2019-04-8822 Patient Consent: Yes, I received consent Disclosure of Interest : None declared
A. Kumar 1 , D. Patro 2 , J. Raghuram 1,3 , A. P. Rao 1,2 1 Pediatric Rheumatology, Indira Gandhi Institute of Child Health, 2 Pediatric Rheumatology, Manipal Hospital, 3 Pediatric Rheumatology, Aster Whitefield, Bengaluru, India Correspondence: A. Kumar Introduction: Polyarteritis nodosa (PAN) is a systemic necrotizing vasculitis predominantly affecting medium-sized vessels. Though it is a multisystem disease, it predominantly involves the skin, gastrointestinal tract (GIT), musculoskeletal system (MSK), and kidneys. Cutaneous PAN is a limited form of the disease wherein the disease remains limited to the skin without any visceral organ involvement. Objectives: To study the clinical profile, laboratory findings, and outcome of childhood-onset PAN. Methods: A retrospective study of 20 children fulfilling the European league against rheumatism (EULAR)/ Paediatric rheumatic European Society (PRES)/ Paediatric Rheumatology International Trials Organization (PRINTO) classification criteria of PAN who were seen over 9 years. Results: The findings are summarized and compared in the table below. The median age of onset of symptoms was 9.4 years (Range: 4- 15 years) with M: F ratio being 1:1. The most common presentation is skin involvement (100%) followed by fever (95%) and MSK manifestations (80%). Deficiency of Adenosine Deaminase 2 (DADA2) was detected in two cases. Labs revealed anemia in 50%, leucocytosis in 65%, and thrombocytosis in 30%. In follow-up, three patients were in complete remission whereas two were lost to follow-up. 9 patients had relapsed on medication whereas 6 patients had relapsed off medication. The mean duration of treatment was 3.5 years. One child succumbed due to a complicated varicella infection and another patient died due to an unrelenting PAN. Conclusion: Childhood PAN is chronic pediatric vasculitis characterized by dermatologic and musculoskeletal manifestations. Early diagnosis and treatment may minimize sequelae in patients with PAN. Relapses occurred more frequently in those with systemic involvement and poor compliance with medicationR. Kumrah, R. Rikhi, A. Rawat, S. Singh, Ankur Kumar Jindal Advanced Pediatrics Centre, PGIMER, Chandigarh, India Correspondence: R. Kumrah Introduction: Kawasaki disease (KD) is an acute medium vessel vasculitis, predominantly affecting children <5 years of age. It is the most common childhood vasculitic disorder causing inflammation of the medium sized coronary arteries. The initial inflammatory insult to the endothelium during the acute phase of KD leads to endothelial injury, while the persistent chronic & smouldering inflammation during the chronic phase leads to endothelial dysfunction. Objectives: To do gene expression analysis of inflammation-induced endothelium dysfunction markers in Kawasaki disease patients from North India Methods: KD patients were enrolled at different time intervals in 3 groups (20 each) as per AHA guidelines and 20 age matched healthy controls. Group 1 : KD diagnosed > 6months-1.5 years; Group 2 : >1.5 - 3 years ; Group 3 : > 3 - 4.5 years prior to enrollment. Complementary DNA (cDNA) converted from extracted whole blood RNA was used to perform real-time PCR analysisReal-time PCR analysis for intra group 1 revealed elevated CXCL8, pecam-1, osteopontin in KD patients with coronary artery aneurysms (CAA) as compared to KD patients without CAA (p=0.038 ), (p= 0.05) , (non-significant) respectively. Increased levels of endoglin, resistin, VEGF-A and leptin was found in patients without CAA as compared to patients with CAA (non-significant). Intragroup analysis for group 2 showed increased Pecam-1, CXCL8, resistin, osteopontin and decreased levels of leptin, pentraxin-3 in KD with aneurysms as compared to patients without CAA (non-significant). Levels of endoglin, VEGF-A were comparable. Intragroup analysis for group 3 patients revealed elevated VEGF-A, CXCL8, Leptin, Pentraxin-3, resistin in patients with CAA as compared to KD without aneurysms while pecam-1, endoglin and osteopontin were comparable in both. Significant difference was also found for Leptin gene in intergroup analysis (1&2) in patients without CAA. Conclusion: Real time analysis revealed altered gene expression in Kawasaki disease patients with aneurysmsM. C. Maggio, S. AccomandoSevere gastrointestinal bleeding is rare but potentially life-threatening in patients with Henoch-Schönlein purpura (HSP). The management is not universally codified. Intra venous steroids are a first-line therapy. Nonsteroidal immunomodulatory treatment has been choosed to treat steroid-resistant patients. Objectives: We report the case of a 4-year-old boy who presented acutely with life-threatening gastrointestinal haemorrhage to describe the difficult therapeutic choice to control the clinical presentation. Methods: : a 4-year-old boy with fever, round worsening vasculitic lesions in the legs, gluteal region, auricle, associated with left gonarthritis. Skin lesions were typical of Seydlmayer purpura. The day after, he showed testicular pain, abdominal pain, positive occult blood, proteinuria. The diagnosis of HSP with renal and intestinal involvement was done. Infectious diseases were excluded. A low title p-ANCA antibodies was detected. Results: He was treated with methylprednisolone (2 mg/Kg/day i.v.), parenteral nutrition and high dose of methylprednisolone (30 mg/Kg/day, for 3 days, followed by 4 mg/Kg/day), IVIG (2 gr/Kg) for a massive intestinal bleeding. After a partial improvement, he presented a further intestinal bleeding some days later. Mycophenolate mofetil (600 mg/m2) was started for the persistence of intestinal bleeding. The clinical manifestations resolved, and a polymeric diet was introduced. 4 months later, he stopped treatment and he still is in remission. Conclusion: the control of the clinical manifestations followed the start of Mycophenolate mofetil and polymeric diet. The winning therapeutic choice was focused on Mycophenolate mofetil and polymeric diet. A multidisciplinary follow-up will support the patient, to exclude a bowel inflammatory disease. Disclosure of Interest : None declared
V. Mastrolia 1 , A. Bettiol 2 , E. Marrani 1 , I. Maccora 1 , E. Taddei 2 , I. Pagnini 1 , M. Canfora 2 , G. Emmi 2 , E. Silvestri 2 , D. Prisco 2 , G. Simonini 1 1 Rheumatology Unit, Meyer Children’s University Hospital, Neurofarba Department, University of Florence, 2 Unit of Internal Interdisciplinary Medicine, Careggi University Hospital, Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy Correspondence: M. V. Mastrolia Introduction: Behçet’s syndrome (BS) is a rare disorder with a relapsing-remitting course. Clinical variance across geographical regions and different age groups has been observed. Objectives: To match demographic, clinical, treatment-related and outcome features of two cohorts of paediatric and adult BS patients. Methods: Two clinical databases of BS patients were retrospectively compared. The paediatric BS database was collected at the Meyer Children’s Hospital, Florence, while the adult one at the Careggi University Hospital, Florence. Medical charts of BS patients were reviewed for age, gender, familiar and genetic predisposition (HLA-B51), clinical symptoms and treatments at onset and over the disease course. The fulfillment of the International Criteria for Behçet’s Disease (ICBD) criteria and/or of the International Study Group Criteria (ISG) for BS was assessed in both adult and paediatric groups, while the paediatric BD (PEDBD) classification criteria were applied only to the paediatric population. All patients met the ICBD and/or ISG and/or PEDBD (where applicable) at BS diagnosis or subsequently, during the whole medical history. In both cohort, disease activity at BS onset and at last available follow-up was assessed by the Behçet’s Disease Current Activity Form (BDCAF). Results: Thirty-three paediatric patients diagnosed with BS, over a median time of 23 months (IQR 11-70 months) were included in the study. They were compared with a cohort of 165 adult BS patients, considered representative of the total BS adult population (n=340), followed over the same median time. In the paediatric cohort, BS onset had occurred at a median age of 12 (IQR 7.7-14.3) years, while in the adult cohort the mean age at symptoms beginning was of 36.6 (IQR 26.5-43.5) years. The female sex was significantly more represented in the adult cohort [105/165 (63.6%) vs 14/33 (42.4%), p=0.032]. A familiar predisposition was significantly more frequent in the paediatric cohort (3/33 vs 1/165, p=0.015). No difference emerged in terms of prevalence of HLA-B51 positivity. The proportion of patients meeting the revised ICBD and/or the ISG criteria at BS diagnosis was comparable. No significant difference emerged in terms of muco-cutaneous, ocular and neurological involvement, and gastrointestinal symptoms. Articular manifestations resulted more common in the paediatric cohort [14/33 (42.4%) vs 35/165 (21.2%), p=0.015], whereas venous vascular events were more frequent in the adult cohort [37/165 (22.4%) vs 2/33 (6.1%), p=0.031]. Regarding treatment strategy, paediatric patients more frequently received no treatment [14/33 (42.4%) vs 61/165 (37%) p<0.001] or corticosteroid monotherapy [17/33 (51.5%) vs 54/165 (32.7%) p<0.001]. Conversely, the use of DMARDs, both traditional and biologics, was significantly higher in the adult cohort [traditional DMARDs: 23/165 (13.9%) vs 1/33 (3%), biologic DMARDs: 86/165 (52.1%) vs 8/33 (24.2%) p<0.001]. Conclusion: Remarkable differences between juvenile-onset and adult-onset BS, both in terms of gender, familiar predisposition, and clinical manifestations have been observed and a different therapeutic approach in the real clinical practice of the two settings emerged. Prospective, comparison studies with a longer follow-up are encouraged to provide further data about the disease course for juvenile and adult-onset BSPalamone 1 , G. Macchini 2 , R. Caiazzo 2 , E. Alterio 1 , V. Tipo 2 , A. Mauro 3 1 Pediatrics, Villa Malta Hospital, Sarno, 2 Pediatrics, Santobono-Pausilipon Children’s Hospital, Naples, 3 Rheumatology Unit, Fatbenefratelli Hospital, Milan, Italy Correspondence: A. Mauro Introduction: Kawasaki disease (MK) is an acute systemic vasculitis of small and medium caliber vessels. The etiology is unknown, probably multifactorial, the major complication of which, if not treated, is represented by aneurysms of the coronary arteries. Non-Typhoid Salmonellae are gram-negative bacteria that cause mild gastroenteritis and, more rarely, invasive forms almost exclusively in defected subjects Objectives: We report the case of S. (age 6 months) hospitalized for persistent fever. Methods: Seven days before entry, S. had presented with mucoematic diarrhea and fever lasting 4 days. Family members also had the same symptoms. After about 48 hours of apyrexia the little S. again manifested high fever for which he was hospitalized, showing a course of continuous fever (T Max 39.6 ° C) which was associated with oropharynx inflammation, tonsillar exudate, and then, to follow, laterocervical lymphadenitis, polymorphic skin rash on the trunk and limbs and finally bulbar conjunctivitis. In Kawasaki’s suspicion, he was practicing therapy with IvIg 2g / kg just over 72 hours after the onset of the fever. After 24 hours of therapy, he presented apyrexia and remission of conjunctivitis and polymorphic rash. In the meantime, the results of the I and II co-cultures that had identified an ESBL + strain of Salmonella Enteritidis, which were no longer detectable at subsequent culture controls, were received. Echocardiographic controls, both acute and follow-up, were all negative for coronary artery disease. Results: Recent evidence in the literature correlates cases of NTS and Kawasaki disease, both as a “complicating”(1) coinfection, and as a potential risk factor or trigger of MK (2). Therefore, the temporal relationship between the episode of salmonellosis from probable domestic infection and the subsequent Kawasaki disease appears to be of considerable interest. It has also been suggested that there is a relationship between intestinal microbiome and MK (3): an alteration of the balance in the intestinal flora in conjunction with external infectious factors, could induce MK in genetically predisposed children. Conclusion: In our patient, enteritis preceded the onset of the clinical picture which in a few days configured the diagnostic criteria of MK, and NTS therefore presented itself as a potential “trigger” agent of MK. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
H. F. Menchaca Aguayo, T. Tamayo Espinosa, E. Faugier Fuentes Centro Médico ABC, Ciudad de Mexico, Mexico Correspondence: H. F. Menchaca Aguayo Introduction: - Objectives: To describe the therapeutic effect of plasmapheresis in refractory Kawasaki disease. Methods: Description of a clinical case and review of the literature. Results: A 4-year-old female patient who presented with a 5-day history of persistent fever of 40 oC, abdominal pain, vomiting, diarrhea, maculopapular exanthema on the trunk and non-suppurative conjunctivitis of 5 days of evolution, was taken to the emergency room with signs of shock, vital signs: blood pression 84/40, heart rate: 160 T: 39°C Respiratory: 45. On physical examination, pale, decreased pulses and prolonged capillary filling, cervical lymphadenopathy greater than 2 cm is palpated, crystalloid solutions are administered, aminergic support and supplemental oxygen. Ceftriaxone antibiotic is started. Laboratory tests: normal blood count, renal and hepatic function preserved, hyponatremia, elevated D-dimer, CRP, troponins and proBNP; Ig G serology for SARS CoV-2 positive. Admitted to intensive care unit (ICU). Echocardiogram myocarditis and valvulitis. PIMS Kawasaki phenotype was diagnosed and gamma globulin was administered, methylprednisolone pulses 30 mgkgdo 5 doses, and acetylsalicylic acid 5 mgkg/day, he presented favorable clinical evolution and was discharged from ICU. Control echocardiogram at 7 days with coronary ectasia and increased troponins and proBNP, so a second dose of gamma globulin is administered and full dose continues, persists with elevation of acute phase reactants, new echocardiogram with appearance of aneurysms so infliximab 5 mgkgdo is indicated. Angio tomography at 12 days showed giant aneurysms in anterior descending coronary artery an right coronary artery, not extra coronary. Due to refractoriness, azathioprine 2 mg/kg per day and bolus cyclophosphamide bolus 750 mgm2sc, prophylactic enoxaparin, acetylsalicylic acid and statins were started. Echocardiogram on day 27, there is evidence of increased size of aneurysms so plasma exchange begins 5 sessions with subsequent echocardiogram where there is evidence of decreased size of the same. Conclusion: Patient with a diagnosis of refractory Kawasaki disease, with presence of inflammation and increased size of aneurysms for which therapeutic alternatives were used infliximab, azathioprine, and cyclophosphamide, the latter being a medium vessel vasculitis. Given the refractoriness, plasma exchange was started, a therapeutic alternative used in Kawasaki disease and other autoimmune pathologies to stop inflammatory activity immediately, with which good results were obtained by stopping inflammation. The use of plasma exchange showed efficacy by decreasing the size of aneurysms. Disclosure of Interest : None declared
E. R. Mercedes-Pérez, P. P. Ramos-Tiñini, H. F. Menchaca-Aguayo, M. A. Balba-Aguilar, A. Guzmán-Revilla, K. Primero-Nieto, H. Bermudez-Canales, N. De la Rosa-Encarnación, M. I. De La Cera-Rodriguez, S. Rodriguez-Aguayo, E. Faugier-Fuentes Hospital Infantil de Mexico Federico Gomez, Cuidad de Mexico, Mexico Correspondence: E. R. Mercedes-Pérez Introduction: Kawasaki disease (KD) is a vasculitis of unknown etiology and refractory in 10 to 20%. In these cases of refractory, management is controversial. Several therapeutic alternatives that have proven effective in other vasculitides have been used: Glucocorticoids, tumor necrosis factor (TNF) inhibitors, other immunosuppressive agents and plasmapheresis. We present two cases of KD, refractory to two doses of IVIg, steroids, infliximab and cyclophosphamide, with aneurysm progression and successfully treated with plasmapheresis. Objectives: To describe the therapeutic effect of plasmapheresis in refractory kawasaki disease. Methods: Descrition of clinical case and review of the literature. Case 1. Infant younger than 7 months, previously healthy, with a diagnosis of refractory Kawasaki. Harada 6 points. He did not respond to two doses of intravenous immunoglobulin, three boluses of methylprednisolone, and infliximab. Refractory to cyclophosphamide, azathioprine; in addition to a second regimen of 3 methylprednisolone boluses. Due to persistent inflammatory activity, elevated C-Reactive Protein, thrombocytosis, progressive increase in aneurysm size, treatment was escalated to 3 plasmapheresis sessions. The subsequent evolution was favourable. Case 2 .A 2-year-old male patient, previously healthy, with a diagnosis of refractory Kawasaki, did not respond to two doses of intravenous immunoglobulin and three boluses of methylprednisolone. Refractory to cyclophosphamide, azathioprine, and infliximab. However, due to persistence of inflammatory activity, elevated C-reactive protein, thrombocytosis and progressive increase in aneurysm size, treatment was escalated to 5 sessions of plasmapheresis with controls after the last session where negative reactants were reported, as well as a decrease in the Z score size of reported aneurysms. Results:The therapeutic management in both cases was attached to the AHA 2017, SHARE 2019 and Japanese 2020 guidelines for refractory Kawasaki. Given the evidence of persistent inflammatory activity documented by a progressive increase in acute phase reactants and aneurysm size; It was decided to use azathioprine, cyclophosphamide because it is a medium caliber vasculitis and gives long-term therapeutic effect. Affection in other blood vessels was ruled out by CT angiography. To immediately stop the inflammatory effect, the use of plasmapheresis was decided. Obtaining satisfactory result. Individualized therapeutic decision made with the experience of the medical staff and accessibility of hospital resources. Situation referred to in the literature. Conclusion: Plasmapheresis is one of the most effective therapies for the treatment of patients refractory to IVIG, when other additional therapies are not effective. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
A. Miller-Barmak 1,2 , F. Sztajnbok 3 , Z. Balik 4 , A. Borzutzky 5 , L. A. Fogel 6 , O. Goldzweig 7 , S. Ozen 4 , Y. Butbul Aviel 1,2,8 1 Department of Pediatrics B, 2 Pediatric Rheumatology Service, Ruth Rappaport Children’s Hospital, Rambam Health Care Campus, Haifa, Israel, 3 Pediatric Rheumatology Division, Pedro Ernesto University Hospital, State of Rio de Janeiro University, Rio de Janeiro, Brazil, 4 Department of Pediatric Rheumatology, Hacettepe University Faculty of Medicine, Ankara, Turkey, 5 Department of Pediatric Infectious Diseases and Immunology, Pontificia Universidad Catolica de Chile, Santigao, Chile, 6 Department of Pediatrics, Washington University in St. Louis, St. Louis, United States, 7 Pediatric Rheumatology Service, Kaplan Medical Center, Rehovot, 8 The Ruth and Bruce Rappaport Faculty of Medicine, Technion - Israel Institute of Technology, Haifa, Israel Correspondence: A. Miller-Barmak Introduction: Takayasu arteritis (TA) is a large vessel vasculitis rarely reported in children, with an extremely low incidence in infants. Thus, most articles on pediatric TA have not focused on infants. We present the largest case series of infantile TA. Objectives: Identify demographic and clinical characteristics of infantile TA and compare them with existing data on older children. Methods: We conducted an international multicenter retrospective cohort study. Patients with the diagnosis of TA at age <5 years were included in the study. Epidemiological and clinical data were collected from patients’ charts from six rheumatology centers. All patients met both the EULAR/PReS endorsed Ankara 2008 classification criteria and the 1990 ACR/EULAR criteria for TA. Results: Twelve patients were included (50% female) meeting Median age of symptom onset was 11 months, with a diagnostic delay of 4 months, and median follow-up of 7.5 years. The most common symptoms at presentation were hypertension, Blood pressure differences between upper and lower limbs, and fever. The most commonly involved arteries at diagnosis were the abdominal aorta, renal artery, and superior mesenteric artery. Medications used included steroids, conventional and biological disease-modifying antirheumatic drugs, and other immunosuppressive therapies. Half of the patients received biologic agents of which infliximab had the highest complete remission rate (40%). Other medications resulting in complete remission were cyclophosphamide (40%) and methotrexate (38%). Invasive procedures were required for 58% of patients. The most common complications were cardiac (50%), stroke (42%), and serious infections (33%). No patients died. Conclusion: This study presents the largest series of infantile TA. Compared to other reported series on older children, infants with TA have more severe disease where they were more likely to receive biologic agents, develop complications, and require invasive interventions. Disclosure of Interest : None declared
S. Mondal, R. K. Pilania, A. K. Jindal, D. Suri, S. SinghS. Mondal Introduction: Kawasaki disease (KD) is the most common vasculitis in children. The etiology of KD remains an enigma despite more than 50 years of extensive research. However, multiple lines of evidence have supported the role of infections as possible triggers for KD. It is believed that KD develops in a genetically susceptible host on exposure to an environmental agent that is most likely an infectious agent. Objectives: Objective of this study is to report various infections identified in a cohort of patients with KD at the. Methods: We carried out a review of case records with KD during 1994-2019. Of 950 cases of KD during this period, 102 children had some evidence of infection during the course of their illness. This subgroup of patients was subsequently analyzed in detail. Results: Overall 10.73% children had evident active infection during the course of KD. Of 102 children (67 boys), majority (68/102; 66.6%) was diagnosed as incomplete KD. Mean delay in diagnosis was 11 days (range 4-35 days). Only 1 sibling developed KD at the same time. Fever was present in all and periungual peeling was seen in 98 (96%%) patients. Microorganisms were isolated in 72 children (70.5%) that were bacteria (n=52); virus (n=15); fungus (n=4); protozoa (n=1). Superficial and deep-seated abscess was most common infective features (28/102; 27.45%). Other clinical features were pneumonia (n=27), gastrointestinal manifestations (n=26), shock (n=9), blood stream infection (n=5), urinary tract infection (n=4), splenomegaly (n=8) and arthritis (n=9, septic arthritis 2). Amongst the bacterial etiology, while staphylococcus and streptococcus were frequently encountered, dengue virus was commonest amongst viruses. Neutrophilic leukocytosis (86/102; 86.27%) was most commonly noted amongst supportive laboratory criteria followed by thrombocytosis (76.2%), anemia (62.5%), hypoalbuminemia (59.8%), hyponatremia (50%) and transaminitis (26.4%). Sterile pyuria was noted in 7 (5.83%) children. Ninety-six children were treated with first dose of IVIg (2gm/kg) and 5 required adjunctive therapy (2 received second dose of IVIg and 3 received both infliximab and steroids) along with low dose aspirin. Coronary artery abnormalities (CAAs) were seen in 12 (11.7%) patients during acute phase, which became normalized with in first 6 weeks in all patients. One child had myocarditis and succumbed to his illness. Conclusion: Based on these results it may be concluded that approximately one tenth of all children with KD may be associated with an infection in our set up and mostly it presents as incomplete KD. This infection may be a possible trigger for the disease. One should not exclude the diagnosis of KD even if there is evidence ofBehçet’s disease is a rare variable vessel vasculitis characterised by recurrent oral ulcers , genital ulcers , arthritis , fever with systemic manifestations including ocular disease, skin lesions, gastrointestinal disease, neurologic disease and vascular disease. The disease is usually seen in young adulthood with a peak age of onset of 25–30 years, but it is also occasionally seen in children before the age of 16 years in 4–26% of cases. 1 There is limited published data about the profile of Behcet’s disease in children seen at centers across India . Objectives: We aim to look at the spectrum of behcet’s disease in Indian children at A tertiary level pediatric rheumatology center. Methods: This is a retrospective analysis of fourteen children who were diagnosed with Behcet’s disease at our pediatric rheumatology center (PRC) , New Delhi , India between January 2013 to August 2021.Our collected data included demographic details , clinical presentation , management and follow up details. Results: This table is showing characteristics of fourteen Indian children with Behcet’s Disease Conclusion: Young age boys found to be affected more commonly. Recurrent oral ulceration remains the most common manifestation at onset . Systemic involvement can be depicted as MSK ~ Skin > Eye ~ GI > CNS > CVS ~ RS. Colchicine and methotrexate alone was not able to control disease in most of the patients. Azathioprine remains the drug of choice in our cohort . Thalidomide was found to be effective in some of our patients as an add on therapy. Adalimumab was effective especially in eye involvement. Most of the patients in our cohort are in remission as per their last follow up data. Trial registration identifying number: 1. Karincaoglu Y, Borlu M, Toker SC et al. Demographic and clinical properties of juvenile-onset Behçet’s disease: A controlled multicenter study. J Am Acad Dermatol 2008 Disclosure of Interest : None declared
, M. Aggarwal, S. Sawhney Pediatric & Adolescent Rheumatology Department, Sir Gangaram Hospital , New Delhi , India Correspondence: D. B. Pandya Introduction: Acute primary vasculitides like IgA Vasculitis & Kawasaki Disease are common in pediatrics while the others described below are rare and their exact frequency is unknown. It includes mainly Polyarteritis nodosa , Takayasu Aortoarteritis , ANCA associated vasculitis and primary CNS angiitis. 1 Objectives: To study clinical presentation and outcome of rare primary systemic vasculitides in 20 children at our center. Methods: This is a retropsective analysis of 20 children with rare primary systemic vasculitides who were diagnosed and managed at our center between Aug 2013 to March 2022. Our collected data includes demographics , clinical presentation , management and follow up details. Results: Table is showing clinical presentation & outcome of 20 children with rare primary systemic vasculitides at our center Conclusion: Females found to be affected more with rare primary vasculitides. Fever , constitutional symptoms , hypertension & elevated inflammatory markers remains the most common initial manifestations. Systemic involvement can be depicted as Gastrointestinal system > Renal > Skin > CNS > MSK > CVS > RS > Eye. Most children with severe systemic vasculitides responded to IV Cyclophosphamide as Induction agent & Oral Azathioprine as Maintenance agent. One patient with ANCA associated vasculitides responded to IV Rituximab as Induction & Maintenance agent. Three patients with Takayasu Aortoarteritis responded to IV Tocilizumab. Four patients with DADA-2 sydrome have been treated with TNF inhibitors but there was no adequate response. In these patients, there was a significant delay between initial presentation and final diagnosis of DADA-2 syndrome. Most patients are in remission off steroids. There are two deaths reported in our cohort and both these cases were diagnosed very late as DADA-2 syndrome with gastrointestinal perforation. Trial registration identifying number: 1. Rheumatology (Oxford). 2019 04 01; 58(4):656-671. European consensus-based recommendations for the diagnosis and treatment of rare paediatric vasculitides - the SHARE initiativR. K. Pilania 1 , M. Singhal 2 , S. Siniah 1 , S. Basu 1 , A. Thangraj 1 , J. Ahluwalia 3 , S. Singh 1 1 Pediatric Allergy immunology Unit , 2 Department of Radiodiagnosis and Imaging , 3 Department of Hematology, PGIMER, Chandigarh, India Correspondence: R. K. Pilania Introduction: Kawasaki disease is a common childhood vasculitis with special predilection for coronary arteries. Patients with large coronary aneurysms need long-term antiplatelet and anticoagulation therapy. Objectives: To describe safety and efficacy of antiplatelet and anticoagulation therapy (aspirin and low molecular weight heparin (LMWH)/warfarin) in a cohort of KD patients with large coronary artery aneurysms. Methods: Records of all children diagnosed to have KD during 1994-2021 were analyzed. Of the 1076 patients with KD, clinical details of children who had received aspirin and either LMWH/warfarin were retrieved. Results: Thirty-eight (3.53%) children (28 boys; 10 girls) with KD, were put on aspirin and LMWH/warfarin. Median age of diagnosis was 18 months (range 1.5 months-12 years). Ten children (26.3%) below 1 year at diagnosis. Twenty-seven patients (71%) have received LMWH, while 18 (47.4%) received warfarin. Five patients initially received LMWH for period ranging from 12-31 months, followed by oral warfarin. Giant aneurysms were present in 35 patients while 3 patients had medium sized complex aneurysms. Thromboses developed in acute phase of disease in 4/38 (11.4%) and most common coronary artery affected was left anterior descending (LAD) coronary artery. All patients were continued on oral aspirin (3-5 mg/kg/day) along with anticoagulation therapy and 5 patients also received a second antiplatelet agent (clopidogrel). Median duration of LMWH was 14 months (range: 3-32 months), and median warfarin duration was 42 months (range: 2-126 months). In 18 patients we were able to monitor factor Xa activity and median activity was 0.46 IU/mL (0.32-0.81). Median INR in patients receiving warfarin was 1.55 (0.99-2.73). There were no significant complications related to anticoagulation in any of the patients, although parents frequently complained of local bruising. Serial 2D-echocardiogram during follow-up showed remodeling of coronary arteries. None of the patients developed thrombosis or symptomatic stenosis during follow-up. Duration of follow-up was 1414 patient-months. Conclusion: Although the recommended INR in patients with KD and large aneurysm who are receiving anticoagulation therapy is 2-3, we maintained our patients on lower INR. Our results show that even on a much lower INR, these children have had no significant complication. Trial registration identifying number: NOT APPLICABLE, R. Kumrah, S. Loganathan, M. Arora, D. Suri, S. Singh tric Allergy Immunology Unit , PGIMER, Chandigarh, India Correspondence: Introduction: There is paucity of literature on epidemiological data of Kawasaki disease (KD) from developing world. Objectives: The aim of present study was to establish the current incidence of KD, seasonal variations and to assess incidence of coronary artery abnormalities (CAAs) in patients with KD from Chandigarh, India. Methods: All children diagnosed with KD from Chandigarh UT (below the age of 15 years) during the period January 2015 to December 2019 were analyzed. Annual incidence rates were calculated on the basis of National Census data, 2011. The methodology was similar to previously published studies from our centre pertaining to the period 1994 - 2008 and 2009 - 2014. Results: During the period 2015-2019, 79 patients (63 boys, 16 girls) were identified from Chandigarh, UT. Incidence rates varied during the 5 years period were 5.64, 9.25, 9.11, 9.87 and 9.72 /100,000 children below 5 in year 2015 - 2019 respectively. Maximum number of cases were observed in the year 2018 followed by 2019 while less cases were noted in 2015 below 5. While annual incidence varied between 2.65 in 2015 and 5.07/100,000 children in 2018 below 15. Comparison of yearly data revealed increasing incidence of disease from 11 cases (2009–2014) to 16 cases per 100,000 children below 15 cases/years from 2015-2019. There were clustering of cases during the months of April and September (10 each) with highest peak in these two months followed by August (9 cases) while nadir was seen in January and February. Mean age at diagnosis was 59 ± 43 months (median=48 months) ranging from 12 days - 15 years. There is increase in overall number of KD cases, as 2015-2019 Chandigarh incidence is higher as compared to our previous figures. 50% increase in annual incidence of KD in children below 5 and a 46.2% increase in children below 15 (from 2015-2019) as compared to our previous data from 2009-2014. CAAs during acute phase of illness was as high as 17.7%, while at 6 weeks of illness was reported in 7.6% of patients with KD despite treatment. Conclusion: This study highlights that incidence of KD in Chandigarh has increased over period 2015-2019 compared to previous years. This may reflect true increase in the incidence of KD or may be due to increased ascertainment of disease as a result of increased awareness among pediatricians and physicians. Despite treatment CAAs have been reported in as high as 17.7% of patients with KD during the acute phase of diseasG. Morais 1 , T. Trindade 2 , A. Maia 1 , M. Rodrigues 3 , I. Brito 3 1 Department of Pediatrics, Centro Hospitalar e Universitário São João, 2 Faculty of Medicine of the University of Porto, 3 Unidade de Reumatologia Pediátrica e Jovem Adulto, Centro Hospitalar e Universitário São João, Porto, Portugal Correspondence: M. Rodrigues 3 Introduction: Vasculitides are rare systemic conditions which may occur in childhood; pediatric studies done so far are few and have small sample sizes which limits the interpretation of results. Objectives: This population-based study aims to develop knowledge regarding the clinical presentation of vasculitides in children. Methods: In this retrospective study we gathered data regarding epidemiology and clinical presentation of all children diagnosed with vasculitides in a tertiary Portuguese center. All patients were assessed for demographics and clinical presenting symptoms. Results: In our study, we included 138 patients. Kawasaki’s disease (KD) had the youngest average patients with a median age of 2.26 years old. In Behçet’s syndrome (BS) group patients were older (13.41). The gender ratio was only higher in females in BS’s patients (1:1.86), as opposed to other vasculitides. Cutaneous involvement was >90% in both IgA Vasculitis (IgAV) and KD. Gastrointestinal symptoms were common in all groups (15-50%); rarer in BS (17%). Arthritis and arthralgia were highly prevalent in IgAV (65%); ophthalmic involvement was frequent in KD (70%). Renal manifestations in IgAV (11%) and ophthalmic involvement in BS (22%) were lower than expected. There was a notable number of children reporting joint involvement in KD (27%). We also noticed a slightly higher prevalence of vascular findings in BS (30%). Conclusion: Clinical symptoms observed in our patients were similar to previous reports in published studies, with some exceptions. The authors suggest that pediatric multicentric population-based studies are needed; such networks will be invaluable to facilitate research and clinical triaodríguez Aguayo, H. Bermudez Canales, M. A. Barba Aguilar, K. Primero Nieto, A. Guzmán Revilla, E. Faugier Fuentes Hospital Infantil de México Federico Gómez, Ciudad de México, Mexico Correspondence: S. Rodríguez Aguayo Introduction: Orbital tumor is a rare manifestation of ANCA-associated vasculitis, occurring in approximately 15% of patients with polyangiitis and 1% of patients with eosinophilic polyangiitis. Objectives: To present 3 clinical cases of pediatric patients diagnosed with granulomatosis with polyangiitis with orbital tumor as initial presentation on the Hospital Infantil de México Federico Gómez. Methods: Description of a clinical cases and review of the literature. Results: Case 1: an 8-year-old female who was treated for a 2-month-old right orbital tumor. She underwent a biopsy, reporting histopathologically nonspecific chronic inflammation and fibrosis. Within the approach, a renal biopsy was performed with diffuse endocapillary and extracapillary proliferative glomerulonephritis with cellular crescents, high resolution computed tomography of the lung with ground glass areas and pseudonodules, and positive p-ANCA and MPO antibodies. Case 2: a 7-year-old female with a history of recurrent epistaxis. She was attended to for a 6-month-old right orbital tumor. The results of the lacrimal gland biopsy were consistent with small-vessel vasculitis with inflammatory infiltrate. High resolution computed tomography was reported with a pulmonary infiltrate, and positive p-ANCA and MPO antibodies. Case 3: a 7-year-old female with a history of recurrent epistaxis. She was attended to for an orbital tumor, left ptosis, pain, and bilateral conjunctival injections for a month of evolution. The orbital computed tomography was reported with the presence of occupied lesions in both orbits. She underwent a biopsy, reporting histopathologically chronic inflammation and granulomatous reaction. In pulmonary high-resolution computed tomography was seen ground glass areas. IgG4 was negative, but p-ANCA and MPO were positive. Conclusion: Orbital tumor is a rare ophthalmologic manifestation of ANCA-associated vasculitis. Its diagnosis and treatment continue to be a challenge. Clinicians should have a high index of suspicion in patients who present these clinical characteristics at the onset of the disease to carry out a systematized and exhaustive approach that allows us to make an earlyM. C. Santos, F. D. Kuchiki, R. Tupinamba Takayasu’s arteritis ( TA) is an arteritis granulomatous that affects the large arteries such as the aorta and its main branches and less frequently the pulmonary arteries; can lead to stenosis, occlusion, dilation and aneurysm formation. It mainly affects women in their 40s, but does not exclude other age groups. In the pediatric age group, 30% of cases were described in adolescents and less than 2% in children under 10 years of age. Its etiopathogenesis is still not well understood, but it is an autoimmune process and an association with infectious processes is observed. There are few reports of ocular involvement in Takayasu’s arteritis, which may be associated with events associated with the disease itself, due to hypoperfusion or to hypertension, may result from its treatment. Uveitis is no longer a frequent manifestation, but it can occur, affecting both the anterior and posterior segments of the eye, either acutely or chronically, transiently or permanently . Objectives: To describe a case of a pediatric patient with Takayasu’s arteritis who presented ocular involvement as the initial manifestation. Methods: Case report with obtaining clinical, laboratory, imaging and treatment data from the review of the patient’s chart followed up at the pediatric rheumatology center. Results: A seven years old boy complaining of hyperemia in the left conjunctiva, was diagnosed with anterior uveiti , associated with the diagnosis of viral myocarditis and hypertension. In addition, due to abdominal pain, a USG of the abdomen was performed, showing lymph nodes in the hepatic hilum and retroperitoneum, without renal changes. Angiotomography of the abdomen initially showed a lymph node mass that determined a reduction in the caliber of the aorta, at the level of the kidneys, which remained patent. Cervical lymph node excision was also performed, with reactive lymphoid hyperplasia . He evolved with worsening of the ocular hyperemia in the left eye, in addition to arthritis of the wrists and ankles and subcutaneous nodules in the lower limbs and upper limbs, and he was referred to our service. On admission, he had a galloping rhythm on cardiac auscultation with ocular hyperemia, mainly on the left; arthritis in wrists, left elbow and ankles, subcutaneous nodules in the extensor face of the arms and in the tibial extensor region, present and symmetrical pulses. Laboratory tests were performed which showed elevated acute phase tests. It was evaluated by ophthalmology, and biomicroscopy showed uveitis in the left eye (3+/4+ cells), flare (2+/4+), PK’s granulomatous . Low visual acuity , and topical treatment with corticosteroids and mydriatics was started , with improvement, allowing for an evaluation of the eye fundus examination, which showed no changes. A new CT angiography showed significant adenomegaly and a hyperdense halo periaortic artery that suggested aortitis , in addition to aortic stenosis at the level of the renal arteries, not justified by lymph node compression , which associated with evidence of high inflammatory activity, arterial hypertension and difference in blood pressure between the limbs, allowed the diagnosis of Takayasu’s arteritis, treatment being started. Conclusion: Takayasu arteritis can present ocular involvement, with different manifestations, resulting from the disease itself ( hypoperfusive , hypertensive or inflammatory condition) or from the treatment, which can be early or late. Uveitis is not the most frequent ocular manifestation in TA, but it can occur and its early diagnosis allows for early treatment, avoiding serious complications. Thus, patients with AT should undergo routine ophthalmologic evaluation at the onset of the condition and during the courseSestan 1 , N. Kifer 1 , B. Sozeri 2 , F. Demir 2 , K. Ulu 2 , C. A. Silva 3 , R. T. Campos 3 , E. D. Batu 4 , O. Koker 5 , M. Sapina 6 , S. Srsen 7 , A. Gagro 8 , A. Rodrigues Fonseca 9 , M. Rodrigues 9 , D. Rigante 10 , G. Filocamo 11 , F. Baldo 11 , M. Heshin-Bekenstein 12 , J. Kataja 13 , M. Held 1 , M. Frkovic 1 , S. Ozen 4 , M. Jelusic 1 on behalf of PReS Vasculitis Working Party 1 University of Zagreb School of Medicine, UHC Zagreb, Zagreb, Croatia, 2 University of Health Sciences, Umraniye Training and Research Hospital, Istanbul, Turkey, 3 ICr-HC-FMUSP, Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil, 4 Hacettepe University Faculty of Medicine, Ankara, 5 Marmara University-Pendik Training and Research Hospital, Istanbul, Turkey, 6 University of Osijek, Medical Faculty Osijek, UHC Osijek, Osijek, 7 University of Split School of Medicine, UHC Split, Split, 8 University of Osijek, Medical Faculty Osijek; Children’s Hospital Zagreb, Zagreb, Croatia, 9 Universidade Federal do Rio de Janeiro, Instituto de Puericultura e Pediatria Martagão Gesteira, Rio de Janeiro, Brazil, 10; Università Cattolica Sacro Cuore, Rome, 11 UOC Pediatria a Media Intensità di Cure, Clinica de Marchi, Milan, Italy, 12 Dana Dwek Children’s Hospital, Tel Aviv Medical Center, Tel Aviv, Israel, 13 Turku University Hospital, Turku, Finland Correspondence: M. Sestan Introduction: Although skin changes in IgA vasculitis (IgAV) are most commonly typical, in about 2% of children, the most severe changes may develop, including hemorrhagic vesicles, bullae, ulcerations or necroses. Objectives: We investigated whether such changes were also associated with a more severe clinical course of the disease and the need for more intensive therapy. Methods: The retrospective multinational study was conducted in 11 tertiary university medical centers. Patients were diagnosed according to the EULAR/PRES/PRINTO criteria. Data were analyzed descriptively and using the Fisher’s exact and χ2 test. Results: A total of 64 patients with the most severe cutaneous manifestations in IgAV were included in the study, of which 41 (64.1%) were male, with a mean (standard deviation, SD) age of 8.49 (4.06) years at the disease onset. They were older than control group of 596 IgAV patients who did not develop bullae or necroses, with a mean (SD) age of 7.17 (3.57) years (p=0.006). The median (25-75p) time from the onset of the first symptom to the first bullous/necrotic change was 5 (2-10.5) days. The total duration of bullous/necrotic changes was 10 (7-16) days. The most important triggers of IgAV were infections, which were present in 68.8% of patients. The distribution of bullae and necroses followed the distribution of the purpuric changes. Scars and pigmentation changes persisted in 48.5% of children. The patients with severe cutaneous manifestations developed nephritis more frequently (40.6% vs. 20.6%, p=0.001), particularly with a combination of hematuria and proteinuria. The renal disease outcome was worse than the control group (p=0.001). They were more likely to have an affected gastrointestinal system (64.1% vs. 45.5%, p=0.007) and to develop the most severe gastrointestinal manifestations (p<0.001). The majority of these patients (90.6%) were treated: 84.4% of them received systemic glucocorticoids and methylprednisolone was the most frequently used with a median (25-75p) cumulative dose of 12 (6-88.7) mg/kg for a median (25-75p) of 12.5 (4-30) days, while 57.8% were treated with nonsteroidal anti-inflammatory drugs for 7 (3.5-18.5) days. Other drugs were administered sporadically. They were significantly more often treated with systemic glucocorticoids (84.4% vs. 37.2%, p < 0.001). Conclusion: This is the largest international cohort study showing that IgAV patients with severe cutaneous manifestations developed nephritis with worse renal outcome more frequently compared to the controls, requiring systemic glucocorticoids. These patients also presented severe gastrointestinal involvement. Support: Croatian Science Foundation,A. Sharma Paediatrics, Dr RPGMC Tanda, Dharamshala, India Correspondence: A. Sharma Introduction: Immunoglobulin A (IgA) vasculitis, previously known as Henoch Schönlein Purpura (HSP) is a common vasculitic disorder of childhood. Once considered the commonest vasculitis of childhood, may no longer be so. Nephritis is a potentially organ damaging complication that needs regular monitoring for early diagnosis and appropriate management. Relapses are known in IgA vasculitis. Objectives: To study the epidemiological and clinical characteristics of patients diagnosed with IgA vasculitis at Pediatric Rheumatology Clinic (PRC) in Dr. Rajendra Prasad Government Medical College (Dr.RPGMC) Tanda, India, which is a state-funded, tertiary-care centre in the state of Himachal-Pradesh, India. Methods: We analysed records of all patients with IgA vasculitis below 18 years enrolled in PRC at Dr.RPGMC Tanda from Jan 2018 to July 2021. We also compared the number of cases of Kawasaki Disease (KD) seen in PRC during this period. Results: During the above mentioned period, 14 children were diagnosed with IgA vasculitis, out of which 9 were male (M:F= 1.8:1). Majority of cases (6/14, 43%) were aged between 11-15 years, 4 were between 6-10 years. Noe of the patients was below 2 years of age. Skin rash occurred in all 14 patients. Pain abdomen (57%) was the most common initial clinical presentation. Other symptoms being arthritis (3/14), blood in stools (1/14) and scrotal swelling (1/14). Among joint involvement, knee joint (83%) was commonly affected followed by ankle joint (50%), elbow joint (33%), wrist joint(33%) and small joints of hand (1%). Renal involvement occurred in 6 patients (43%). Manifestations of renal involvement were microscopic hematuria and sub-nephrotic albuminuria in all and hypertension (3/6). Mean age of those who had nephritis was 12.2 years as compared to 8.37 years of those without nephritis. Renal biopsy was performed in 2 of them. One, that of a 16 years girl, revealed crescentic glomerulonephritis and the other, that of a 14 years boy revealed mesangioproliferation. Pulse methylprednisolone was given to 4 of these and all were treated with oral gluco-corticoids. Angiotensin converting enzyme inhibitor (enalapril) was used in 3 and other treatment modalities used were immunosuppression with azathioprine and mycophenolate mofetil. Interestingly during the same time period, 21 cases of Kawasaki Disease were seen in the PRC. Therefore the number of cases of Kawasaki Disease outnumber those with IgA vasculitis. Conclusion: Mild disease resolves spontaneously while systemic steroids are recommended for moderate to severe disease including those with nephritis in IgA vasculitis. Prognosis depend on the extent of renal involvement. Close follow-up is required for early recognition of multiorgan involvement. Recognition of Kawasaki Disease is increasing in many parts of the world. Epidemiological studies are needed to prove that true incidence of Kawasaki Disease is increasing, especially in South-east Asian countries. Disclosure of Interest : None declared
J. Sheekha 1 , A. Sharma 2 , D. Dhiman 1 1 Pediatrics, 2 Immunology and Pediatric Rheumatology, Dr Rajendra Prasad Governement Medical college, Tanda at Kangra, HP, Kangra, India Correspondence: J. Sheekha Introduction: Behçet’s disease (BD) is a multisystemic inflammatory disease. It is a variable vessel vasculitis, characterized by the involvement of any size and type (arteries, veins) of vessel. Objectives: To report a case of BD who presented without characteristic features and had an unusual manifestation. Methods: Case report: Eleven-year-old male child presented with swelling over the neck and face for 5 days which was insidious in onset and gradually progressive. The child also had 2 episodes of hemoptysis, 2 days back. In past history, 3 months back he had fever and cough with hemoptysis for one month and rash for 5 days. On evaluation at that time, he had severe thinness, hilar lymphadenopathy. Investigations revealed erythrocyte sedimentation rate (ESR) 120 mm in 1st hour, mantoux test and sputum cartridge based nucleic acid amplification test (CBNAAT) for Mycobacteria was negative. He was empirically initiated on anti-tubercular therapy (ATT) but according to parents, there was no improvement. On presentation this time, he had tachypnea with tachycardia, swelling with prominent veins over the face, neck and chest. Fullness in supraclavicular and infraclavicular fossae. A clinical diagnosis of superior vena-cava (SVC) syndrome was made. On respiratory system examination, air entry was decreased in right superior axillary and mammary area. Ophthalmologic evaluation showed panuveitis. Results: Investigations revealed, anemia (haemoglobin 7.6 gm/dl), thrombocytosis (platelets 4,97,000,cmm), elevated ESR (50 mm in 1st hour) and C-Reactive Protein (CRP) (48 mg/L). His coagulation profile and urine examination were normal. Chest X-ray showed homogenous opacity in left upper lobe. Computed tomography thorax showed aneurysm in main pulmonary artery with changes of fibrosing mediastinitis with mediastinal lymphadenopathy with venous thrombosis involving superior vena cava (SVC) and bilateral branchiocephalic trunks on either sides with no involvement of cervical vasculature. His antinuclear antibodies (ANA), anti-phospholipid antibodies (APLA) were negative and angiotensin converting enzyme (ACE) level was normal. With these clinical features, aneurysm in pulmonary artery with thrombosis with SVC syndrome, panuveitis, with elevated inflammatory parameters, possibility of Behcet disease was considered. His pathergy test was negative (on prednisolone). HLA B51 assay was planned. He was given topical betamethasone, pulse methylprednisolone (30 mg/kg/day) for 3 days followed by oral prednisolone (2 mg/kg/day). He was planned for injection infliximab but he had rupture of aneurysm and succumbed to the illness. Conclusion: Classic features of oral and genital ulcers may not always be seen in children with BD. Pulmonary aneurysms are the most severe feature of pediatric BD. SVC syndrome is also a rare manifestation of BD. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
A. T. Suchi 1,2 , Y. Uziel 1,2 , A. Ziv 1,2 , T. Schoenfeld 3 , R. Haviv 1,2 1 Sackler School of Medicine, tel aviv university, tel aviv, 2 Pediatric rheumatology, 3 Pediatric Pulmonology Unit, Meir Medical Center, Kfar Saba, Israel Correspondence: A. T. Suchi Introduction: Microscopic polyangiitis (MPA) is a vasculitis that affects both adult and pediatric populations, mostly involving the lower respiratory and renal systems. MPA is defined as a necrotizing vasculitis, with few or no immune deposits, predominantly affecting small vessels. Epidemiological data on MPA are limited, because of its origins as a subset of polyarteritis nodosa, and because it is now frequently described collectively with the other ANCA-associated vasculitis. MPA is very rare, in general and especially so in pediatric populations. The incidence in the UK was estimated at 3.6 cases per million and the median age of onset in children is 9 to 12 years. There are no data regarding typical bronchoscopic findings in pediatric patients diagnosed with MPA. Objectives: To report the unique bronchoscopy findings in conjunction with the clinical signs and symptoms, and laboratory test results of a female patient diagnosed with MPA, and to review the literature. Methods: We collected data from the patient’s medical record and pictures taken during a bronchoscopic evaluation prior to the diagnosis. In addition, we searched databases and reviewed relevant articles. Results: A 16-year-old girl was admitted to the Department of Pediatrics after 2 weeks of paroxysmal hemoptysis. She also reported weakness, 5 kg weight loss, arthralgia, night sweats and shivering. Physical examination upon admission was generally unremarkable, with normal vital signs, and without any significant findings on chest auscultation. Blood work-up at admission revealed mild leukocytosis and neutrophilia, mild normocytic anemia and mildly elevated inflammatory markers. Serum creatinine was mildly elevated. Chest radiographs showed bilateral opacities and computed tomography showed ground-glass like opacities in the lower left lobe. Bronchoscopy was performed and diffuse bronchial mucosal hemorrhage was reported (images will be presented on the poster). The abnormal appearance of the bronchial tree during the procedure prompted the pulmonologist to form an immediate working diagnosis of small-vessel vasculitis, strongly suggestive of MPA. The morning after the bronchoscopy, leukocyturia, erythrocyturia and proteinuria were reported and 24-hour urinary collection further revealed nephrotic range proteinuria. A renal biopsy was performed, and along with results of serologic tests (anti-myeloperoxidase and pANCA positivity), MPA was finally diagnosed. The patient was started on a course of corticosteroids and rituximab, and is now under remission. Searching medical databases did not reveal any description of diffuse bronchial mucosal hemorrhage in MPA patients. However, diffuse alveolar hemorrhage is reported in patients 16–86 years of age. Limited data are available regarding bronchoscopic findings in MPA and bronchoscopy is not considered a diagnostic tool in this disease. Conclusion: In this case, the diagnosis was based on the unique bronchoscopy findings obtained several days before receiving the renal biopsy and serology results. As there are no relevant data in the literature, we propose that bronchoscopy might have diagnostic value for vasculitis, in patients presenting with hemoptysis and constitutional symptoms. We recommend collaborating with other pediatric medical centers, that are willing to perform bronchoscopies and to the gross findings that may lead to another diagnostic tool for this very rare disease. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
hangaraj 1 , H. Chaudary 1 , R. K. Pilania 1 , M. Singhal 2 , A. SharmaKawasaki disease (KD) is one of the most common childhood vasculitides. KD may result in coronary artery abnormalities (CAAs) in ~25% of untreated patients. Transthoracic 2-dimensional echocardiography (2DE) has been used as a standard of care for the diagnosis of CAAs patients with KD. Computed Tomography Coronary Angiography(CTCA) has shown to be superior in picking up a host of coronary artery aneurysms (CAAs) in KD patients that are missed on 2DE. CAAs involving all 3 coronary arteries along with their branches (pan coronary artery involvement-pan-CAAs) represent a severe spectrum of KD and have never been reported. In our observation, we have seen that a minority of patients with KD can have diffuse coronary artery involvement involving all segments of the right and left coronary circulation on CTCA. Objectives: To study the prevalence of pan coronary artery aneurysms in our cohort and to analyze the difference in CTCA and 2DE in identifying pan coronary artery aneurysms Methods: We have reviewed records of children who were diagnosed with KD and underwent CTCA for assessment of coronary artery dimensions between November 2013 to April 2021.CTCA was planned in children who had CAAs on 2DE. Patients with KD who had CAAs in all 3 coronary arteries (left coronary artery, left circumflex coronary artery and right coronary artery), but where the branches of these coronary arteries were spared were not included in this study. Results: Two hundred fifteen patients with KD underwent CTCA between November 2013 to April[2021. Of these, 13 (6.04%) patients were noted to have pan-CAAs on CTCA. The mean age at diagnosis of KD in the patients was 7.4 years (range: 4 months-13 years). Eleven patients satisfied the complete criteria for KD, 2 patients were labelled as incomplete KD. Out of these 13 patients, 3 patients satisfied the clinical criteria for Multisystem inflammatory syndrome associated with coronavirus disease-2019 (MIS-C). All CTCAs were performed in the acute state. CAAs were in the form of aneurysms (saccular in 7, fusiform in 32). Non opacified segments suggesting thrombosis were noted in 4 coronary arteries. 2DE (performed within 48 hours of CTCA) demonstrated left main coronary artery (LMCA) abnormalities in 12(92.3%), proximal segments of left anterior descending (LAD) abnormality in 13(100%), right coronary artery (RCA) abnormalities in 11(84.6%) and left circumflex artery (LCX) abnormalities in 5(38.4%). Distal coronary artery aneurysms (5) and luminal thrombi (2) diagnosed on CTCA were entirely missed by 2DE. All CTCA were performed in the acute stage of KD. All children received an intensified therapy; twelve children received infliximab (5mg/kg) in addition to IVIg(2g/kg), high dose corticosteroids (10mg/kg for 3 doses) were given to intensify therapy in a child with MIS-C along with IVIg(2g/kg). All children received tapering doses of corticosteroids Aspirin (3-5mg/kg/day) is continued in all patients. Anticoagulants were added to patients with giant aneurysms and/or thrombosis. Conclusion: Pan-CAAs in KD are a unique radiological entity and its better diagnosed by CTCA. Although 2DE is excellent in the diagnosis of CAAs in proximal coronary arteries, it is limited in identifying the distal extent of CAAs or distal CAAs and complications like thrombosis. We recommend that CTCA should be considered whenever significant CAAs are detected on 2DE as it provides information on the entire course of coronary arteries along with complications. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
R. K. Pilania 1 , M. Singhal 2 , S. Mondal 1 , H. Chaudary 1 , A. Sharma 1 , A. Jindal 1 , S. Singh 1 1 Pediatrics, 2 Radiology, Post Graduate Institute of Medical Education and Research, Chandigarh-160012, India Correspondence: A. Thangaraj Introduction: Coronary artery abnormalities (CCAs) can occur in ≈25% of children with KD. Transthoracic 2D-Echocardiography (2DE), hitherto the imaging modality of choice, has several limitations as an imaging modality. Computed Tomography Coronary angiography (CTCA) has enabled the comprehensive evaluation of coronary arteries in children with KD. This study pertains to interval CT coronary angiography in 11 children with KD at a tertiary care centre in Chandigarh, India Objectives: To analyse the long term consequences and prognosis of patient with coronary artery aneurysms Methods: CTCA was carried out on 128-slice dual-source CT scanner (Siemens, Erlangen, Germany). There were 11 children in whom interval CTCA was performed. Results: Median age at diagnosis of KD was 37 months [range 4-96 months]. Median interval between the first and second CTCA examination was 34.5 months [range 6-61 months]). Findings of CTCA at presentation revealed 21 aneurysms: left main coronary artery (LCA) – 5; left anterior descending artery (LAD) – 8; right coronary artery (RCA) – 4 and left circumflex artery (LCx) -4. Giant aneurysms were present in 6 patients (LAD – 5; RCA-3). Thrombosis was noted in 1 patient with giant aneurysm in LAD. Interval CTCA was completely normalized in 5/11 (45.4%) patients. Remaining 6 patients showed persistent (albeit regressed/remodelled) coronary artery aneurysms: LCA-5; LAD-4; RCA-3; LCx-2. Two patients had shown mural calcifications. Interval CTCA in 1 patient showed focal stenosis just beyond remodelled aneurysm in LAD. Two patients in whom CTCA was performed at intervals of 14 months and 72 months after diagnosis of KD, revealed long segment stenosis in LAD and ostioproximal segment of LCx. Conclusion: Children with KD and CAAs require prospective long-term follow-up as they may develop complications like thrombosis, stenosis and calcifications. CTCA provides more detailed and comprehensive evaluation in comparison to 2DE. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
P386. Comparison of clinical characteristics and risk factors of recurrence in Kikuchi-Fujimoto disease between children and adult 87. The role of the paediatric rheumatologist in the management of children with Non-Specific Orbital Inflammation (NSOI): a single centre experience 8. Successful treatment of Satayoshi syndrome with dantrolene: a case report P389. Neonatal HLH : rare is not so rare P390. Empowering primary care providers to manage Benign Joint Hypermobility Syndrome (BJHS) with an educational packet P391. A girl with painful deformities, immune dysregulation and persistent inflammation P392. Adolescent with idiopathic myositis ossification in deltoid: a case repor393. With juvenile Sjogren’s syndrome followed in the pediatric rheumatology clinic clinical characteristics and follow-up of patient P394. Importance of nursing consultation to adolescents with chronic rheumatological disease: pilot study P395. Juvenile Sjogren’s syndrome: the need for validated paediatric diagnostic criteria. a retrospective review from a tertiary rheumatology centre 96. A 9-year-old girl with persistent back pain and erythema nodosum P397. A 3-year-old child with persistent fever P398. Retrospective evaluation of pediatric sarcoidosis patients P399. Case report of Behcet’s disease in pediatric population 400. Blood transcriptomics to facilitate diagnosis and stratification in paediatric rheumatic diseases P401. Interleukin-1 inhibitors - a new hope for fibrodysplasia ossificans progressiva? P402. In pediatric rheumatologic disease, methotrexate leads to mildly changed bloodwork on the second day after administration 403. The musculoskeletal manifestations of paediatric Inflammatory Bowel Disease (IBD): the gosh experience 4. Withdrawn P405. Ultra-high frequency ultrasonography of labial glands in pediatric Sjögren’s syndrome: a preliminary study P406. Maternal and neonatal outcomes in pregnant women with rheumatic diseases - a retrospective cohort study P407. Primary sjogren’s syndrome in children: a experience from a single center P408. Foreign body synovitis (case report) P409. Paediatric musculoskeletal education in the Republic of Ireland P410. Prednisone effectiveness in children with Sydenham’s chorea based on a 27-year-experience of a Tertiary Center P411. Case series of three patients with pachydermotactily P412. Neuromyelitis optica spectrum disorder in pediatric sjogren’s syndrome 13. A case of synovial chondromatosis of the shoulder in a young girl P414. Role of the paediatric rheumatologist in the management of tubulointerstitial nephritis and uveitis, a single-centre experience P415. CACP syndrome (camptodactyly, arthropathy, coxa vara, pericarditis). clinical case –16 years follow-up E. Vrtikova, E. Košková J. G. Ahn 1 , J. Y. Baek 2 1 Department of Pediatrics, Severance Children’s Hospital, Yonsei University College of Medicine, 2 Department of Pediatrics, Severance Children’s Hospital, Yonsei University College of Medicine, Seoul, Korea, Republic Of Correspondence: J. G. Ahn Introduction: Kikuchi-Fujimoto disease (KFD) is a rare, benign and self-limited disease that is characterized by cervical lymphadenopathy and fever. Objectives: We analyzed the differences in clinical manifestations and risk factors of recurrence between children and adults. Methods: We retrospectively reviewed the medical records of patients who were diagnosed with KFD at a tertiary referral hospital between 2005 and 2019. We divided the patient groups into children (<19 years of age) and adults (≥19 years of age). Results: During the 14-year study period, a total of 127 patients were diagnosed with KFD. Among them, 34 (26.8%) were children and 93 (73.2%) were adults. There were no clinically significant differences between children and adults except for a longer fever duration before diagnosis and higher frequency of myalgia in adults. For lymph node evaluation, ultrasound was mainly used in children (61.8%), whereas CT was most often used in adults (78.5%). Antibiotics were used more in children than in adults (76.5% vs. 54.8%, P =0.027). In adults, multivariable logistic regression analysis showed that ANA positivity (OR: 7.813; 95% CI=1.818–33.333; P= 0.006) was a risk factor of recurrence. Conclusion: KFD in children and adults showed similar clinical features, but showed differences in evaluation method and frequency of use of antibiotics. In adults, ANA positivity was associated with recurrence of KFH. Alkhdher 1 , O. Kul Cinar 1 , S. Gore 2 , S. Compeyrot-Lacassagne 1,3 1 Rheumatology Department, 2 Ophthalmology Department , 3 Biomedical Research Centre, Great Ormond Street Hospital, London, United Kingdom Correspondence: O. Kul Cinar Introduction: NSOI is a term used to describe a heterogenous group of non-infectious, non-neoplastic disorders characterized by orbital inflammation with no identifiable local or systemic causes. Although rare in children, orbital inflammation may be associated with systemic inflammatory conditions; thus, careful evaluation and follow up is essential. Objectives: We aimed to describe the clinical assessment and work-up of patients presenting with non-specific orbital inflammation, and to identify the role of the Paediatric Rheumatologist in the monitoring of extra-ocular involvement and the management of immunosuppressive treatment. Methods: A retrospective descriptive case series of 15 children who had swelling of the lacrimal gland and a diagnosis of NSOI and were followed in the Paediatric Rheumatology and ophthalmology department at Great Ormond Street Hospital between September 2000 and March 2022. Results: 15 patients, median age at referral of 12 years and 9 months (range 4 years and 9 months – 15 years and 11 months) were identified. Orbital imaging was performed in all 15 patients: CT orbit (1/15), MRI Orbit (11/15), both MRI and CT orbit (3/15). The most common manifestation on orbital imaging was lacrimal gland involvement in 87% (13/15), of which 46% (6/13) had bilateral involvement. All 15 patients had an orbital biopsy with the following histopathological diagnoses: NSOI 87% (13/15), no significant pathology: 13% (2/15). Ophthalmic symptoms were present in all 15 patients, with the most common being (eyelid swelling and/or erythema, pain, blurred vision). Around half of the patients 53% (8/15) reported extra-orbital symptoms/findings: 20% (3/15) abdominal pain, 13% (2/15) arthralgia, 7% (1/15) headaches, 7% (1/15) arthralgia with weight loss and malaise, 7% (1/15) right facial nerve palsy. An associated underlying diagnosis was found during follow up in 13% (2/15) patients: 1/15 Inflammatory bowel disease (IBD), 1/15 SRP positive necrotizing myositis with thyroiditis. Work-up of all patients by the Paediatric Rheumatology team included a comprehensive history with a detailed system review and laboratory investigations including baseline blood tests with inflammatory markers (FBC, liver and renal functions, CRP, ESR) and an extensive autoimmune screen. All patients had a negative ANCA and 20% (3/15) were found to have a positive Antinuclear Antibody (ANA). ACE was raised in 27% (4/15). Faecal calprotectin was tested in 3/15 and was raised in 2. The mainstay of treatment for NSOI is corticosteroids (CS) used in 80% (12/15). 3/15 did not receive CS, 2 underwent ophthalmic surgery and 1 was lost to follow up. 17% (2/12) achieved and maintained remission with a short course of steroids only, 8% (1/12) is awaiting intra-orbital CS injections and 75% 9/12 required disease-modifying antirheumatic drugs (DMARDS) with methotrexate being the most used in 67% (6/9), followed by mycophenolate mofetil in 33% (3/9). Whilst a short course of CS’s was effective in 75% (9/12) of patient, around 78% (7/9) relapsed when stopped. Anti-TNFα agents (infliximab and adalimumab) were used in 2 patients. In our study, 13% (2/15) lost to follow-up, 60% (9/15) achieved remission and 27% (4/15) had recurrent flares. 50% (2/4) patients with on-going dacryoadenitis are awaiting intra-orbital CS injections. Conclusion: Although NSOI is a rare entity in the paediatric population, careful clinical monitoring by a Paediatric Rheumatologist/Paediatrician is warranted to rule out underlying systemic inflammatory conditions. Treatment with short course of steroids is usually effective but recurrence is frequent requiring DMARDS or Anti-TNF agB. Arabshahi, C. Saldarriaga, A. Hoang Pediatrics, Inova LJ Murphy Children’s Hospital , Fairfax, United States B. Arabshahi Introduction: Satoyoshi syndrome is a rare multisystemic disorder of unknown etiology, which presents with diarrhea, alopecia and musculoskeletal symptoms. This disease is often incapacitating, difficult to diagnose, and may lead to severe malnutrition and death if left untreated. There are very limited reports of Satayoshi Syndrome in literature and approaches to treatment have been variable and often unsuccessful. Objectives: We present the case of a 15-year-old male with alopecia and severe progressive bilateral calf spasms, and assess response to treatment with dantrolene and systemic corticosteroids. Methods: Our patient underwent extensive workup for neuromuscular diseases including MRI of the lumbar spine and lower extremities, EMG of bilateral calves, serum paraneoplastic autoantibody panel, inflammatory markers, muscle enzymes, and genetics evaluation -including metabolic myopathy panel and organic acids. Once all other causes were excluded, patient was diagnosed with Satayoshi Syndrome and started treatment with corticosteroids and dantrolene. Prednisone was initiated at 40mg daily and tapered off over the course of 6 weeks, and dantrolene was started at a dose of 25mg daily and titrated up to three times a day. Results: Patient had full resolution of myalgia with no recurrence in muscle spasms on the above treatment regimen. One year post steroid discontinuation, patient remains asymptomatic and independent with his activities of daily living, and has had no recurrence in muscle spasms despite weaning dantrolene dose down to 25mg daily. Conclusion: We present this case report of Satayoshi Syndrome to highlight the importance of prompt diagnosis and treatment of this debilitating and potentially fatal disease, and propose dantrolene as an effective steroid-sparing agent for management of this condition. Written informed consent for publication of patient’s clinical details was obtained from the patient/guardian. A copy of the consent form is available for review. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
K. U. Ghosh 2 , P. Khemka 2 , R. Hassan 2 , S. Kabir 2 1 Department of Pediatric Rheumatology, 2 Department of Neonatology, Institute of Child Health, Kolkata, India Correspondence: J. N. Bathia Introduction: Hemophagocytic lymphohistocytosis (HLH), a rare clinical entity in neonatal period with an incidence of around 1 in 50,000-1,50,000 live births. Objectives: We present a case series of 5 neonates who were diagnosed as neonatal HLH Methods: We noted the data of five neonates admitted in the neonatal intensive care unit of Institute of Child Health, Kolkata during the last one year and were diagnosed as HLH. Results: The five neonates presented to NICU at 7,11,15,20 and 27 days respectively with a mean age of 16 days. All five had fever, two of them had rashes and two had purpuric spots. Hepatosplenomegaly, bicytopenia, deranged liver function tests was seen in all. At diagnosis the mean hemoglobin was 7.54gm/dL, total leucocyte count 9 x10 3/ μL, platelet 26.4 x10 3/ μL , mean ferritin 10350 ng/ml, mean triglyceride 279mg/ dl. They were all initiated on dexamethasone and cyclosporine as per HLH 2004 protocol. in unresponsive cases methyl prednisolone was also used. Due to lack of response to dexamethasone and cyclosporine, one patient was given low dose etoposide on two occasions. Whole exome sequencing could be done in 3 of 5 patients. 2 showed PRF1 gene mutation, one homozygous and the other compound heterozygous, thus confirming the diagnosis of primary HLH however both of them succumbed. Two others in whom whole exome sequencing could not be done also succumbed to the illness. The fifth patient had no pathogenic variant on whole exome sequencing. This patient was diagnosed as secondary infection associated HLH and could be successfully discharged after appropriate treatment of the infection. Conclusion: Neonatal HLH is a definite entity. Babies presenting with fever, multisystem involvement, cytopenias, rashes, hepatopathy should have an estimation of ferritin. and one must look beyond sepsis. Majority of the cases are primary. Genetic analysis, though expensive, should be offered. Secondary HLH has a good prognosis with timely management. Stem cell transplantation is the definitive treatment for primary HLH and is presently a viable option in many Indian centres. HLH is a definite entity in neonates and not so rare if looked for. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
S. Brooks 1 , R. Carrasco 1 , K. Lewis 2 1 Pediatric Rheumatology, Pediatric Rheumatology Consultants of Austin, 2 St. David’s Medical Center Nursing Administratio, Austin, United States Correspondence: S. Brooks Introduction: Benign joint hypermobility syndrome (BJHS) is an under-diagnosed connective tissue disorder found in 7-36% of the pediatric population which can cause frequent joint injury, fatigue, and arthritis-like pains. Many BJHS patients present to pediatric rheumatology offices to rule out inflammatory arthropathy. There is no single sub-specialty that owns the diagnosis of BJHS in the United States, and therefore treatment and coordination of care (i.e., physical therapy) is primarily provided by primary care providers (PCPs). Diagnosis, treatment, and guidance for self-management of BJHS can be overwhelming for PCPs with lack of knowledge and confidence regarding the syndrome. Objectives: The purpose of developing this educational packet is to provide succinct and necessary information regarding diagnosis and treatment of BJHS to empower PCPs to manage BJHS with less reliance upon subspecialists. Methods: We utilized pediatric rheumatology, physiotherapy/physical therapy, occupational therapy, and pain management journals, textbooks, and resources to develop an evidenced-based educational packet for PCPs which addresses general overview of BJHS, diagnostic criteria, treatment plans, genetic testing information, and a physiotherapy home treatment guide developed by Ashford and St. Peter’s Hospitals. In the future, a feedback survey will be created and disseminated to PCPs who share a patient with a new diagnosis of BJHS. The survey will be completed prior to receiving the packet and measure the PCPs clinical confidence in managing BJHS. A secondary survey will be sent 3 days after receiving the packet and will measure the PCPs clinical confidence in managing BJHS after receiving the packet. Results: The data regarding effectiveness of the developed packet has not yet been gained as this is a preliminary abstract. We anticipate that by distributing the BJHS educational packets, they will be used in the following ways. First, PCPs will utilize user friendly treatment plans which are outlined in the packet. Second, PCPs will coordinate with subspecialists, including physical therapy and occupational therapy, to provide care. Third, PCPs will practice utilizing diagnostic criteria for BJHS when presented with a patient who has joint pain without noted swelling on exam. We plan to seek if PCP confidence levels in managing BJHS significantly change prior to and post receiving and utilizing the BJHS packet. Conclusion: A succinct yet comprehensive educational packet was developed to provide research and resources for primary care providers to manage the care of BJHS patients with less reliance on subspecialists. Improvement measures in clinical confidence of PCPs in managing BJHS after receiving the packet have not yet been conducted. Disclosure of Interest : None declared
G. Capata 1 , S. Pastore 2 , A. Taddio 1,2 , A. Tesser 2 , V. Boz 1Italy Correspondence: A. Taddio Introduction: Histiocytosis-Lymphadenopathy (HL) plus syndrome is caused by biallelic mutation of SLC29A3 gene encoding ENT3, a nucleoside transporter largely expressed on the outer mitochondrial membrane and lysosomal membrane of different cells, including monocytes/macrophages, glial cells, ocular and inner ear cells and epithelial cells. ENT3 plays a crucial role in maintaining intracellular homeostasis by transporting nucleotides to the cytoplasm. SLC29A3 mutation leads to the accumulation of nucleosides within the lysosomes and mitochondria resulting in stimulation of TLR7 especially in immune and glial cells. The spectrum of clinical features is characterized by multisystemic manifestations with variable expression and unclear pathogenesis. Features such as histiocytosis, recurrent fever, type 1 diabetes mellitus, thyroiditis and arthritis might be caused by both immune dysregulatory and autoinflammatory patterns. Pathogenesis of other manifestations such as exocrine pancreatic insufficiency, sensorineural hearing loss, hypertrichosis and skin hyperpigmentation is yet to be defined. Objectives: To describe clinical and laboratory features and response to treatment in a girl with HL plus syndrome. Methods: Clinical assessment was performed in a 21-year-old girl presenting to our Pediatric Rheumatology and Immunology Service with infancy-onset painful deformative contractions of hands and feet leading to progressive limitation in daily activities, bilateral sensorineural hearing loss, insulin-dependent type 1 diabetes, pancreatic insufficiency and hypothyroidism. At the age of 15 she underwent a genetic analysis that was diagnostic of HL plus syndrome due to the finding of compound heterozygous mutation in SLC29A3. Laboratory investigation included IFN score. Results: At our evaluation, she complained severe walking difficulties due to painful deformities in her feet. Blood tests showed high inflammation indexes (ESR 47 mm/h, CRP 15.7 mg/L, IFN score 23.7). Hematological, cardiological and pneumological involvement were unremarkable. The association of deforming arthritis with predominant interferonic inflammation recalled Jaccoud arthropathy, a known clinical manifestation of adult SLE. On the other hand, recent studies have shown that IL-6 inhibition could be involved in controlling inflammatory phenomena in HL plus syndrome. Therefore, we started a treatment with baricitinib, a non-selective JAK inhibitor which can simultaneously counteract the effects of interferon and IL-6. At 6 month-follow-up visit, a minimal clinical improvement was reported with persistently active inflammatory indexes. Therefore, a treatment with hydroxychloroquine (HCQ) was added in order to obtain a synergic effect with baricitinib as HCQ acts as a lysosomal stabilizer by reducing the stimulation of TLRs. After 3 months, our patient reported such a significant clinical improvement that she was able to start a physiotherapeutic program and, for the first time in recent years, inflammation indexes were normalized. Conclusion: To the best of our knowledge, this is the first case of HL plus syndrome treated with JAK inhibitors together with HCQ. We hypothesized that this treatment may have a great potential in this rare syndrome by targeting simultaneously IL-6 and IFN inflammation. Moreover, since the disease arises from the mutation of a lysosomal transporter, it may be interesting to disclose the potential benefit of HCQ as a lysosomal active medication. Longer follow-up and further studies are needed in order to confirm our hypothesis. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
Focal myositis belongs to rare type V idiopathic inflammatory myopathy. Though it is rare, prognosis is generally good. Objectives: Herein, I presented a case of left deltoid focal myositis, of which responded well to nonsteroidal anti-inflammatory drugs. Methods: Case Report Results: A 17 years old girl presented with two months’ history of left arm myalgia without proximal muscle weakness. No history of trauma. No constitutional symptoms. No dysphagia. No symptoms suggestive of connective tissue disease. Physical examination revealed localized redness and tenderness over left deltoid but did not show any cutaneous signs of dermatomyositis. Inflammatory marker of erythrocyte sedimentation rate was elevated (17 mm/hr). Creatinine kinase level was normal. Antinuclear antibody, myositis panel, extractable nuclear antigen as well as culture for tuberculosis and fungal were negative. MRI showed diffuse short tau inversion recovery hyperintense signal over the left deltoid muscle. No evidence of enhancing mass and no collection. Muscle biopsy demonstrated inflammatory infiltrate in the perimysium and endomysium with presence of CD4+ T cells. No granuloma. Sarcoma was ruled out. A diagnosis of focal deltoid myositis was made. She was started on nonsteroidal anti-inflammatory drugs and so far no relapse was seen. Conclusion: The current case has shown the importance of excluding differential diagnosis before reaching conclusion on such a rare entity. Second line agent may not be required in this case as the pain did not recur and there were no extra-skeletal manifestationsT. Coşkuner 1 , B. Topatan 2 , B. Sözeri 1 1 Pediatric Rheumatology, 2 Pediatric Health and Disease, University of Health Sciences, Umraniye Training and Research Hospital, Istanbul, Turkey Correspondence: B. Sözeri Introduction: Juvenile Sjogren’s syndrome (JSS) is a chronic inflammation of exocrine glands such as salivary and lacrimal glands. This disease is very rare in childhood, and the onset clinic differs from that in adults. It is more common in girls than in boys (5:1-7:1), with an average onset of 10 years of age. There are no confirmed diagnostic criteria for the pediatric group yet, so diagnostic criteria published for adult patients in 2016 are used. Objectives: The aim of this study is to evaluate the clinical findings, treatment options and treatment results of pediatric patients followed up with the diagnosis of JSS. Methods: Seventeen pediatric patients (16 female patients) who were followed up in Ümraniye pediatric rheumatology clinic after 2016 and diagnosed according to 2016 ACR/EULAR criteria were included in the study. Patients with sicca symptoms that did not meet the diagnostic criteria were excluded from the study. Results: The median age of diagnosis of the patients included in the study was 14 years and the mean age of symptom onset was 13 years. The most common symptoms were found to be dry mouth, musculoskeletal system complaints and dry eyes. Fever was seen in two patients, and Raynaud’s phenomenon in four patients. 18% of patients had another autoimmune disease with overlapping clinical features. There was one patient with a history of recurrent parotitis. While ANA positivity was detected in 88% of the patients, the titer was >1/160 in all of them, and a granular pattern was detected. Anti SSA /RO antibody positivity was observed in 76% of the patients. Only 2 patients had rheumatoid factor positivity. In 3 of 5 patients who underwent parotid ultrasonography, involvement compatible with parotitis was observed. Minor salivary gland biopsy was performed in all patients; while no feature was found in biopsy in 3 patients (17.6%), it was grade 2 in four patients, grade 3 in five patients, and grade 4 and above in five patients. Complaint and age at diagnosis were not found to be significant in terms of clinical and laboratory findings (p>0.05). While 23% of the patients required systemic steroid treatment, methotrexate and/or mycophenolatmofetil treatment was used in 52% of the patients. Rituximab treatment was used in 2 patients because of the persistence of clinical findings in one patient and the development of renal involvement in 1 patient. Conclusion: Sjogren’s syndrome progresses with various systemic findings in addition to dry mouth and eyes in pediatric patients as in adults. There may be a need for immunosuppressive therapy. Disclosure of Interest : None declared
J. C. O. A. Ferreira 1 , A. A. Costa 2 , A. C. S. Monteiro 2 , H. S. Robba 2 , R. C. D. Vaz 1 1 Clinical Research Center, 2 Nursing Division, Instituto Da CRIANÇA DO HCFMUSP, São Paulo, Brazil Correspondence: J. C. O. A. Ferreira Introduction: The care of adolescents with chronic rheumatologic disease must include all the care demands of this phase marked by the need to consolidate identity, develop body image, establish social relationships and achieve independence. The nursing consultation contributes to the identification of difficulties faced by adolescents, reveals points of improvement in care and impacts on adherence to treatment. Objectives: To characterize the profile of adolescents with chronic rheumatologic disease; To highlight the importance of nursing consultations for adolescents with chronic rheumatologic disease and To identify points of improvement in patient care. Methods: Cross-sectional pilot study with adolescents with rheumatologic disease. Instruments were used to characterize the population studied, including sociodemographic and anthropometric data, vital signs, pain, drug addiction and adherence. Approval by the local Ethics Committee number 3.163.835. The parents and adolescents provided written informed consent. Results: Partial results: initial data from thirteen adolescents revealed considerable dependence on parents/guardians, especially regarding medication. Attending the adolescent alone in part of the consultation was essential to identify problems such as bullying and depression, medication adherence and resistance to the use of sunscreen. Conclusion: This work, although hampered due COVID-19 pandemic that caused the interruption of study, has already shown the need for nursing consultations for adolescents with specific instruments that allow diagnosing possible problems, understanding concerns and getting to know this clientele. The continuation of the project will use the results to develop a practical guide for the care of adolescents with rheumatologic disease. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
C. Foley 1 , D. McKenna 1 , K. Gallagher 1 , C. Ciurtin 2 , M. Al Obaidi 1,3 1 Great Ormond Street Hospital, 2 Centre for Adolescent Aheumatology Versus Arthritis Department of Medicine, University College London (UCL), 3 NIHR Biomedical Research Centre GOS Institute of Child Health, GOSH NHS Foundation Trust, London, United Kingdom Correspondence: C. Foley Introduction: Sjögren’s syndrome (SS) is a chronic multisystem autoimmune disease, characterised by inflammation of the exocrine glands, predominantly salivary and lacrimal glands resulting in xerostomia and xerophthalmia. Juvenile-onset SS (jSS) is rare; however, it is probably underrecognised and underdiagnosed. There are no studies reporting accurate incidence or prevalence of jSS. Diagnosing jSS can be challenging. The cardinal signs and symptoms are non-specific. There is no gold standard biomarker of disease. Between 1965–2002 11 diagnostic criteria were developed, none of which have gained universal acceptance or been validated in a paediatric population. Until recently, the most widely used criteria were those developed by the American-European Consensus Group (AECG). It remains well recognised that international consensus on classification is important for standardisation, particularly in relation to research and monitoring treatment outcomes. However, there remains a paucity of validated criteria for diagnosis of jSS. Paediatric focussed criteria are required as features of SS in children differ from those in adults. Children experience less dryness and more frequently experience systemic symptoms and parotid enlargement. Applying adult criteria to a paediatric population may lead to mis- and/or under-diagnosis. Bartunkova et al. (1999) proposed paediatric criteria for diagnosis of jSS. These have not been validated. Objectives: Describe clinical, laboratory and imaging characteristics of children diagnosed with jSS at Great Ormond Street Hospital (GOSH) over a 10-year period Apply the AECG and proposed paediatric criteria to our cohort and report percentage fulfilling each criteria Methods: Retrospective chart review of children diagnosed with jSS, January 2012-January 2022 at GOSH. Demographic, clinical, laboratory and imaging findings were documented. Features at presentation were compared to the AECG and proposed paediatric diagnostic criteria. Percentage of the cohort fulfilling each criteria was calculated. Results: 28 cases of jSS were diagnosed from January 2012-January 2022, 22 female (79%). Referring clinicians to Paediatric Rheumatology included Paediatricians (n=21,75%), Ophthalmologists (n=1,3.6%), Rheumatologists (n=1,3.6%), Dermatologists (n=1, 3.6%), Nephrologists (n=2,7.1%) and Oral surgeons (n=2,7.1%). Main reasons for referral included facial swelling, lymphadenopathy, rash, constitutional symptoms, and positive investigation findings suggestive of jSS i.e. autoantibody profile, US salivary glands. Median age at diagnosis was 11 years (3.4-14.8). The most common presenting feature was facial swelling (n-18,64%), followed by lethargy (n=12,43%) and rash (n=11,39%). Reports of dry eyes (n=7,25%) and mouth (n=9,32%) were reported less frequently. Laboratory features identified included anaemia, elevated amylase and ESR, positive ANA and Ro, and hyper IgG. All children who had an USS of their salivary glands had features consistent with a diagnosis of jSS (n=24,86%). 11 children had salivary gland biopsy, 10 (91%) of which provided tissue confirmation of the jSS diagnosis. A total of 8/28 (28.6%) cases satisfied the AECG criteria; 22/28 (78.6%) satisfied the proposed paediatric diagnostic criteria. Conclusion: A low percentage of our cohort fulfilled the AECG diagnostic criteria, therefore we would suggest application of adult criteria for diagnosis of jSS maybe unhelpful. Inclusion of recurrent parotitis and additional laboratory features in the proposed paediatric criteria may increase sensitivity. Our cohort highlights the need for validated jSS diagnostic criteria as paediatric and adult presentations of SS can differF. Gicchino, S. Colosimo, M. Bartiromo, A. Amodio, A. N. Olivieri Department of Woman, Child and General and Specialistic Surgery, University of the Study of Campania Luigi Vanvitelli, Naples, Italy Correspondence: M. F. Gicchino Introduction: Back pain is a relatively common presenting symptom in children and adolescents. Typical causes include spinal deformities (such as scoliosis), spondylolysis, bulging or herniated intervertebral disks, functional pain (such as fibromyalgia), spondylodiscitis, post-traumatic injuries, benign or malignant tumors, osteomyelitis, juvenile idiopathic arthritis. Erythema nodosum (EN) is the most common panniculitis in childhood. EN lesions are localized at lower limbs in particular on the pretibial region, upper limbs and trunk are rarely involved. EN can be associated with general symptoms such as fever, weakness, sever pain, but skin lesions resolve without skin damage. EN can be associated with infectious diseases (beta-hemolytic Streptococcus, Chlamydia and Mycoplasma Pneumoniae, Epstein-Barr virus, Mycobacterium Tuberculosis), inflammatory bowel diseases, coeliac disease, rheumatologic diseases, malignant tumors. Objectives: To report the case of a 9-year-old patient with back pain and erythema nodosum. To report the case of a 9-year-old patient with back pain. Methods: A 9-year-old girl comes to our Department because of back pain and asthenia. Two weeks before the back pain onset the patient got a trauma due to a fall from a swing. Then the patient presented fever (with a temperature up to 38 ° Celsius) for 2-3 days, associated with epistaxis, asthenia and lumbar pain. At first these symptoms were treated with non steroidal antinflammatory drugs (NSAIDs). Since symptoms got worse she underwent to an orthopedic evaluation that found scoliosis in dorsal and lumbar column and bilateral valgus foot. For the persistence of both back pain and asthenia the patient came to our Department. Our clinical examination revealed: pain in lumbosacral column and on the pressure of the right sacroiliac joint; erythema nodosum on the pretibial region of the left lower limb. Results: Complete blood count, liver and kidney function, iron levels waswas negative. Thyroid profile was in the norm and coeliac disease screening was negative. Inflammatory parameters were slight increased: erythrocyte sedimentation rate (ERS) was 39mm/h (normal value<15mm/h); C-reactive protein (CRP) was 3,7mg/dL (normal value<1mg/dL). Fecal calprotectin was in the normal range. Throat swab was negative. Chest x-ray, magnetic resonance imaging (MRI) of the lumbosacral column and sacroiliac joints, mantoux intradermal reaction and quantiFERON test were also performed. Chest x - ray was in the norm. MRI revealed: reduction of physiological lordosis in supine position; increased representation of intracanal epidural tissue from L4 region to the sacrum; fluid effusion in correspondence of the right sacro-iliac joint. Mantoux intradermal reaction was positive at 48 hours, with a diameter of 25 millimeters. QuantiFERON test was positive. Conclusion: According to clinical features, laboratory and radiological findings Tubercular sacroileitis was diagnosed. Patient was entrusted to the care of our colleagues expert in infectious disease and treatment with ethambutol, pyrazinamide, rifampicin and isoniazid was started. A follow up spine MRI performed two months later was in the norm. In a child presenting with back pain also uncommon causes should be considered such as tubercular infection, so it is very important an accurate and complete medical examinations, in fact in the case described the detection of erythema nodusum on the left lower limb made us think about a possible tubercular infectio, A. Amodio, F. G. AbbateFever is a common symptom of many clinical conditions, infection is the most common cause especially in children. Fever of unknown origin (FUO) is defined as a well-documented fever of at least three weeks’ duration without an apparent source after 1 week of investigation. FUO is a challenging problem in clinical practice, and patients presenting with FUO often undergo to unnecessary laboratory tests and antimicrobial therapies. Among children the most common causes of FUO are: infectious diseases, malignancy, autoimmune diseases, immunodeficit, Kawasaki disease, autoinflammatory syndromes. Objectives: To describe a case of FUO in a 3 years old child. Methods: A 3-year-old child came to our department for episodes of recurrent nocturnal fever for about 8 months (Temperature was up to 39 °Celsius). Fever was treated with antibiotics and non steroidal anti-inflammatory drugs as prescribed by her pediatrician. Psoriasis was diagnosed at the age of two years. On physical examination we found: erythematous and desquamated plaques on the scalp and on the left retro auricular region, and lymphadenitis in left latero-cervical region. We did not find any sign of arthritis and/or enthesitis. Laboratory tests revealed: microcytic anemia (Hemoglobin 9,5 mg/dL; Mean Corpuscular Volume 68) elevation of platelets (689/mm 3 , normal value <450/mm 3 ), erythrocyte sedimentation rate (ESR) (25mm / h, normal value <20 mm/h), and C-reactive protein (CRP) (4mg / dL , normal value <1mg/dL) and serum amyloid A (SAA) (80 mg/L, normal value <6.4mg/L). Liver and kidney function, iron levels, C3/C4 levels, LDH , blood coagulation profile, lymphocyte subpopulations were in the norm. Both Antinuclear antibodies and ENA were negative. Virological screening (including Sars-Cov2) and QuantiFERON test were negative. Thyroid profile was in the norm and coeliac disease screening was negative. Both blood and urine cultures were negative, fecal calprotectin was in the normal range. Both heart and abdomen ultrasound were in the norm. The dosage of vanillylmandelic acid in the 24-hour urine was negative, so a neuroblastoma was ruled out. Bone marrow aspirate did not reveal any blastic cells. The patient finally underwent skin biopsy of the erythematous-desquamative lesions. Results: Skin biopsy revealed : a widespread infiltrate in papillary dermis region that was composed of medium-large cells with round-oval nuclei and some incisions. Some eosinophilic granulocytes were found among cell population. A huge area of microulceration was found on epidermis. Immunoassayed sections with CD1 A, S100 and CD68 were positive. So, the morphological and phenotypic findings were compatible with Langerhans cell histiocytosis (LCH). Conclusion: According to clinical condition and histopathological finding a Langerhans cell histiocytosis was diagnosed. LCH is a rare condition in childhood characterized by the proliferation and accumulation of a particular kind of immune system cells called Langerhans cells or histiocytes . This disease could affect any organ or system and the most affected sites in children are bones and skin. When disease involves the skin LCH most commonly presents as a seborrheic dermatitis or eczematous eruption on the scalp or trunk. The clinical presentation is quite heterogeneous, ranging from an isolated and asymptomatic skin or bone lesion to a life threating multisystem disease, characterized by fever, skin rash , anemia, thrombocytopenia, lymphadenopathy and hepatosplenomegaly. Prognosis depends on involvement of risk organs (liver, spleen, bone marrow) at diagnosis and particularly on presence of organ dysfunction and response to initial therapy. Treatment is systemic, incorporating steroids and cytostatic drugV. Guliyeva 1 , F. G. demirkan 2 , R. E. Yiğit 3 , E. Esen 4 , Y. Bayındır 5 , R. Torun 6 , D. Gezgin Yıldırım 7 , G. Kılbas 8 , G. Otar Yener 9 , M. Cakan 10 , F. Demir 11 , K. Özturk 12 , E. Baglan 13 , B. Bora Makay 14 , S. A. Bakkaloglu Ezgü 15 , A. Pac Kısaaslan 16 , Y. Bilginer 17 , S. Yuksel 8 , S. Ozen 18 , B. Sozeri 19 , N. Aktay Ayaz 1 1 Pediatric Rheumatology, Faculty of Medicine, Istanbul University, Istanbul, Turkey., 2 İstanbul School of Medicine, 3 Department of Pediatric Rheumatology, Ümraniye Research and Training Hospital, University of Health Science, Istanbul, Turkey., İstanbul, 4 Department of Pediatric Rheumatology, Erciyes University School of Medicine, Kayseri, 5 Division of Pediatric Rheumatology, Department of Pediatrics, Hacettepe University, Ankara, Turkey, ankara, 6 Divisions of Pediatric Rheumatology, Dokuz Eylül University Faculty of Medicine, İzmir, Turkey, izmir, 7 Department of Pediatric Rheumatology, Gazi University Faculty of Medicine, Ankara, Turkey., ankara, 8 Pediatric Rheumatology, Faculty of Medicine, Pamukkale University, Denizli, Turkey., Denizli, 9 Department of Pediatric Rheumatology, Sanliurfa Training and Research Hospital, Sanliurfa, Turkey, Sanliurfa, 10 Department of Pediatric Rheumatology, University of Health Science, Umraniye Training and Research Hospital, Istanbul, Turkey., 11 acibadem hospital pediatric rheumatology department, 12Istanbul, Turkey., İstanbul, 13 Pediatric Rheumatology, University of Health Sciences,., ankara, 14 Department of Pediatrics, Division of Rheumatology, Dokuz Eylül University Faculty of Medicine, İzmir, Turkey., izmir, 15 Department of Paediatric Rheumatology, Faculty of Medicine, Gazi University, Ankara, Turkey., ankara, 16 Department of Pediatric Rheumatology, Erciyes University School of Medicine, Kayseri, Turkey., Kayseri, 17 Department of Pediatrics, division of Pediatric Rheumatology, Faculty of Medicine, Hacettepe University, Ankara, Turkey., 18 Pediatric Rheumatology, Faculty of Medicine, Hacettepe University, Ankara, Turkey., ankara, 19 Pediatric Rheumatology, University of Health Sciences, Ümraniye Research and Training Hospital, Istanbul, Turkey., İstanbul, Turkey Correspondence: V. Guliyeva Introduction: Sarcoidosis is a very rare granulomatous condition among childhood rheumatic diseases. It may affect various organ systems resulting in different clinical presentations in pediatric patients. Objectives: we aimed to evaluate the demographic and clinical characteristics of pediatric sarcoidosis patients and treatments used. Methods: Pediatric patients diagnosed with sarcoidosis in 12 centers between 2011-2021 were included in the study. Patient records were retrieved from the Academy of Pediatric Rheumatology (PeRA)-research group (RG) database. Demographic features and clinical spectrum were evaluated retrospectively. Results: The mean age of 12.3±4.6 years and 33 (63.5%) of them were females. Demographics are outlined in Table 1. The clinical findings were ocular symptoms (40.4%), arthritis (25%), dermatologic symptoms (13.5%),multi-organ involvement (11.5%),and features of other systems (9.6%).In 9 (17.3%) patients uveitis developed under treatment. The development of uveitis in BS/EOS group was significantly more frequent (p=0.01). Arthritis, lymphadenopathy, and anemia was significantly more common in the EOS/ BS group (p=0.014). Lung involvement was present in 16 (30.8%) patients. 8 (15.4%) patients had stage I, 5 (9.6%) stage II, 2 (3.8%) stage III and 1 (1.9%) stage IV lung involvement had. Six (11.5%) patients showed a low functional vital capacity (FVC). Bronchoscopy was peculiar in two cases The elevated ACE levels with lung involvement was not significant (p=0.2). Fourteen (26.9%) patients received pulse methylprednisolone. Methotrexate was preferred in 47 (90.3%) patients. The median duration of methotrexate was 24 months. Methotrexate was given to 20 (42.5%)of the patients as monotherapy. Fourteen patients received biologic agents in addition to methotrexate. Remission was in 15 patients (30.7%),partial response was in 28 (53.8%) cases and refractory disease was present in 4 (7.7%) patients. Flare occurred in 13 (25%) patients. Conclusion: This is the largest series of pediatric sarcoidosis in the literature according to our knowledge. Sarcoidosis is very rare in children and its presentation and prognosis are different from adults;can develop at any age and requires long-term follow-up Patient Consent: Yes, I received consent Disclosure of Interest : None declared
A. Guzman, E. Faugier, H. W. Bermudez, S. Rodríguez Aguayo, N. D. L. R. Linne, M. A. Barba Aguilar, M. I. De la Cera Rodríguez, E. . Mercedes Pérez, H. F. Menchaca Aguayo, P. Ramos Tiñini, A. K. Primero Nieto Reumatologia Pediatrica, Hospital Infantil De Mexico Federico Gómez, CDMX, Mexico zman Introduction: Behcet’s disease (BD) is a systemic vasculitis that can affect any type and size of blood vessel and involve almost any type of organ including gastrointestinal, nervous, musculoskeletal and cardiovascular. (1) although most of the patients develops the disease between the second and fourth decade of life, up to 15-20% develops in the pediatric age, for this reason is important to be aware of this pathology, therefore we present three cases of BD that we have documented in our institution. (2) Objectives: Describe three cases of BD on pediatric population and review of the literature Methods: Description of three cases of BD Results: Case 1: Female starts at 10 years old with painful oral ulcers 4 episodes per year, is managed symptomatically and receiving multiple antibiotic and antiviral schemes, at 11 years old presents genital ulcer biopsy with inflammatory and necrotic infiltrate, starts therapy with Prednisone for 1mgkgdosis for 14 days with remission of the lesions. After 2 years she presented new oral and genital lesions, she was sent to the Rheumatology service, PCR for herpes virus was negative and HLB-51 negative, acneiform lesions were documented, Patergia positive and EB diagnosis was integrated, other organ involvement was ruled out, she started treatment with steroid 2mgkgdosis and Thalidomide. Case 2: 13-year-old female with a history of oral and genital ulcers with a 1-year evolution, accompanied by arthralgias, headache and acneiform lesions. History of cervical lymphadenopathy and solitary thyroid nodule of 1 year of evolution, assessed by Oncology and Endocrinology, cervical lymph node biopsy with nonspecific inflammatory result, malignancy data is ruled out, thyroid function tests without alterations. When episodes of genital and oral ulcers persist, infectious causes were ruled out and she was sent to our service. Brain magnetic resonance angiography was performed with no alterations, genital ulcer biopsy with inflammatory and necrotic tissue. Ophthalmologic disease is ruled out. HLA-B51 negative and Patergia positive. Diagnosis of EB is integrated, therapy with short course of steroid and colchicine is initiated with adequate response. Case 3: 15-year-old male with pain on ejaculation of 3 months of evolution, is evaluated by urologist who during urethrocystoscopy evidences penile ulcers. Oral ulcers, conjunctival hyperemia, photophobia and headache were documented, he was sent to Rheumatology. Cerebral magnetic resonance angiography showed stenosis of the left internal carotid artery associated with vasculitis. HLA-B51 negative. Diagnosis of EB was documented. Therapy was started with methylprednisolone and Cyclophosphamide doses, followed by Prednisone and Azathioprine, 6 doses of Cyclophosphamide were completed with adequate evolution. Conclusion: As can be seen in the previously reported cases, EB is a pathology that can affect different organs, and it can take a long period of time for the expression of symptoms to conclude its diagnosis, being important to make known the heterogeneity of the disease to give the appropriate follow-up to make the diagnosis and timely treatment. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
K. Ha 1 , E. Bartholomeus 1 , L. Van Os 2 , J. Dandelooy 2 , J. Leysen 2 , O. Aerts 1,2 , V. Siozopoulou 2 , E. DeSmet 2 , J. Gielen 1,2 , K. Guerti 2 , M. De Maeseneer 3 , N. Herregods 4 , B. Lechkar 2 , R. Wittoek 4,5 , E. Geens 5 , L. Claes 2 , M. Zaqout 2 , W. Dewals 2 , A. Lemay 6 , D. Tuerlinckx 7 , D. Weynants 7 , K. Vanlede 8 , G. Van Berlaer 3 , M. Raes 9 , H. Verhelst 4 , T. Boiy 2 , P. Van Damme 10 , A. Jansen 2 , M. Meuwissen 1,2 , V. Sabato 1,2 , G. Van Camp 10 , A. Suls 10 , J. Van der Werff ten Bosch 3 , J. Dehoorne 4 , R. Joos 2,4,5 , K. Laukens 11 , P. Meysman 11 , B. Ogunjimi 1,2,3,5 1 Faculty of Medicine and Health Sciences, University of Antwerp, 2 University Hospital Antwerp, Antwerp, 3 University Hospital Brussels, Brussels, 4 University Hospital Ghent, Ghent, 5 Antwerp Hospital Network, Antwerp, 6 Turnhout General Hospital, Turnhout, 7 University Hospital Namur, Namur, 8 Nikolaas General Hospital, Sint-Niklaas, 9 Jessa Hospital, Hasselt, 10 University of Antwerp, Antwerp, Belgium, 11 Department of Computer Science, University of Antwerp, Antwerp, Belgium Correspondence: M. K. Ha Introduction:lack of optimal diagnostic criteria caused by the heterogeneity of many rheumatic diseases, variable clinical presentations, and complex pathophysiology. Objectives: The objective of this study is to improve the early diagnosis of paediatric rheumatic diseases via whole blood transcriptomics combined with machine learning. We aim to investigate the gene expression of whole blood from children with rheumatic diseases in comparison with viral infection cases during active infection and convalescence, then apply machine learning on the transcriptomic data to develop classification models for identifying different disease groups. Methods: Whole blood was collected from a cohort of 46 children during active viral infection (35 of whom also had blood collected at a second time point during convalescence) and 48 paediatric rheumatic patients including children with (i) auto-inflammatory diseases, (ii) juvenile idiopathic arthritis (JIA), (iii) chronic recurrent multifocal osteomyelitis (CRMO), (iii) an interferonopathy (IFN) such as juvenile dermatomyositis or systemic lupus erythematosus, (iv) vasculitis or (v) diseases related to the human leukocyte antigen B51 serotype. RNA sequencing was performed on the participants’ whole blood and the resultant transcriptomic data were used to develop classification models with the Random Forest algorithm. The performance of these classifiers was evaluated by leave-one-out cross-validation. Analyses of differentially expressed genes (DEG), gene ontology (GO), and interferon-stimulated gene (ISG) score were also conducted based on the transcriptomic data. Results: Our first classifier could differentiate paediatric rheumatic patients from viral infection and convalescence cases with high area-under-the-curve (AUC) values (AUC = 0.8 ± 0.1 and 0.7 ± 0.1, respectively). Three other classifiers could distinguish CRMO, JIA, and IFN from viral infection and convalescence cases with AUC ≥ 0.8. DEG and GO analyses indicate that the pathophysiology of CRMO, IFN, and JIA involves innate immune responses including myeloid leukocyte and granulocyte activation, neutrophil activation and degranulation. IFN patients in particular had the highest mean ISG score among all disease groupss to : Yes, I received consent Disclosure of Interest : M. Ha: None declared, E. Bartholomeus: None declared, L. Van Os: None declared, J. Dandelooy: None declared, J. Leysen: None declared, O. Aerts: None declared, V. Siozopoulou: None declared, E. DeSmet: None declared, J. Gielen: None declared, K. Guerti: None declared, M. De Maeseneer: None declared, N. Herregods: None declared, B. Lechkar: None declared, R. Wittoek: None declared, E. Geens: None declared, L. Claes: None declared, M. Zaqout: None declared, W. Dewals: None declared, A. Lemay: None declared, D. Tuerlinckx: None declared, D. Weynants: None declared, K. Vanlede: None declared, G. Van Berlaer: None declared, M. Raes: None declared, H. Verhelst: None declared, T. Boiy: None declared, P. Van Damme: None declared, A. Jansen: None declared, M. Meuwissen: None declared, V. Sabato: None declared, G. Van Camp: None declared, A. Suls: None declared, J. Van der Werff ten Bosch: None declared, J. Dehoorne: None declared, R. Joos: None declared, K. Laukens: None declared, P. Meysman: None declared, B. Ogunjimi Grant / Research Support with: Abbvie, Pfizer, and Roche
R. Haviv 1,2 , E. C. Hsiao 3 , L. Zeitlin 4 , N. Rabinowicz 1 , F. De Benedetti 5 , G. Prencipe 5 , Y. Uziel 1,2 1 Pediatric Rheumatology Unit, Meir Medical Center, Kfar Saba, 2 Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel, 3 Division of Endocrinology and Metabolism, University of California at San Francisco, San Francisco, United States, 4 Pediatric Orthopedic Department, Dana-Dwek Children’s Hospital, Sourasky Medical Center, Tel Aviv, Israel, 5 Division of Rheumatology, Bambino Gesù Children’s Hospital IRCCS, Rome, Italy Correspondence: R. Haviv Introduction: Fibrodysplasia ossificans progressiva (FOP) is the most catastrophic form of heterotopic ossification (HO), due to ongoing intracellular signaling through the BMP pathway. It results in rapid, irreversible bone accumulation and loss of mobility. Unfortunately, there is no effective therapy to cure the disease or dramatically change its course [1-16]. The recurrent, paroxysmal appearance of inflammatory “lumps” and the fact that studies of patients with FOP and other forms of HO, indicate that several cytokines, including interleukin-1 (IL-1), are increased, indicate a similarity between FOP and other auto-inflammatory diseases [17-26]. We hypothesized that anti-IL1 agents would ameliorate disease progression. Objectives: To report our long-term experience using canakinumab (CKB, anti-IL-1β) to prevent FOP flares. Methods: Patient data were analyzed, to characterize the efficacy of canakinumab in ameliorating FOP progression. Results: Four FOP patients are currently being treated with 4mg/kg/month CKB at Meir Medical Center, Kfar Saba, Israel and the University of California, San Francisco, USA, with a total experience of over 9 patient years. Two patients were also treated with 100mg anakinra (ANA), daily, before switching to CKB. Markedly lower rates of HO flares were documented, with a decrease from 0.7–3.1 to approximately 0.27 flares per month (see Table 1). In general, no new HO sites were documented, but some existing sites kept growing under treatment, although at a much slower rate. Response to corticosteroids was improved and some lumps diminished under CKB alone. Conclusion: Our data suggest that FOP may be an auto-inflammatory disease and flares are mediated by IL1β. Anti-IL1 agents may have a role in ameliorating FOP progression, which offers new hope to FOPB. Hügle, N. Fischer, J.-P. Haas erman Center for Pediatric and Adolescent Rheumatology, Garmisch-Partenkirchen, Germany B. Hügle Introduction: Methotrexate (MTX) is the most commonly used DMARD in the treatment of juvenile idiopathic arthritis (JIA). Recommendations regarding MTX monitoring recommend measurement of aspartate aminotransferase (GOT), alanine aminotransferase (GPT) and differential blood count. MTX is given as a single weekly dose creating a serum drug level during the following approximate 24 hours. There have been concerns that blood work taken during that time would show a transient increase in blood parameters, especially transaminases [5]. Objectives: The aim of this retrospective study was to determine levels of transaminases and blood counts comparing them by the number of days following MTX administrationpediatric rheumatologic diseases admitted between 2018 and 2021 and treated with MTX. Weekday of blood sampling and last methotrexate dose was determined to calculate the time difference in days. Laboratory values for GOT, GPT, lymphocyte and neutrophil count were determined and normalized. Statistical analysis using ANOVA was performed, as well as Student’s test for levels beyond normal range. Results: A one-way ANOVA revealed that there was a statistically significant difference for GOT (F(6, 966)=8.535, p<0.0001), GPT (F(6,966)=3.657, p=0.001) and neutrophil count (F(6,966)=4.841, p<0.0001) in days of difference, with the highest/lowest values on day 2 after adminstratio However, abnormal values were not found significantly more frequently on any day for all parameters. Conclusion: In a large cohort of children with pediatric rheumatic disease significant change in bloodwork is found on the second day, rather than the first. his effect is too small to merit any clinical note of caution. Bloodwork can be done on any day of the week irrespective of MTX administr, S. Compeyrot-Lacassagne Jashmi Introduction: Children with Inflammatory bowel disease (IBD) have extra-intestinal manifestations. Musculoskeletal (MSK) manifestations are known to be the most common extra-intestinal manifestations of IBD. Though it is well described that IBD can have systemic manifestations, the exact mechanism of joint disease and its relation to intestinal disease is still unclear. Patient and disease characteristics, treatments, and outcomes of p-IBD-associated MSK disease are not well established. Objectives: This study aims to describe the musculoskeletal manifestations seen in children with inflammatory bowel disease. Methods: A retrospective cohort study was carried out between February 2011 and April 2022 looking for all children with inflammatory bowel disease who presented at any time of their disease with inflammatory musculoskeletal manifestations (chronic arthritis or chronic non-infectious osteomyelitis (CRMO) at Great Ormond Street Hospital for children. Results: Out of the 770 patients followed at GOSH for IBD, twenty-four patients (3.1%) were found to have inflammatory musculoskeletal manifestations of IBD. Median age at diagnosis of musculoskeletal manifestations was ten years. Most of the patients were male (66.7%) and had Crohn’s disease (19/24). Twenty-one patients had chronic arthritis, nine patients (42.9%) had axial arthritis and twelve patients (57.1%) had peripheral arthritis. Six patients had chronic non-infectious osteomyelitis (CNO). Three patients had both. Twenty-two patients had confirmed chronic arthritis or CNO by imaging. Two patients had florid peripheral arthritis on examination and imaging was not requested. Eight patients had ultrasound findings: arthritis and tenosynovitis. Twenty patients had MRI changes (on focal or WB-MRI): Bone marrow oedema, enthesitis, synovitis and bone erosions. There was a positive family history in five patients (20.8%): mostly IBD and psoriasis. Seven patients (29.2%) were found to have skin manifestations mostly described as psoriasis (3/7) and Keratosis Pilaris (2/7). Twenty-three patients (95.8%) had constitutional symptoms. Twenty-one patients (87.5%) had raised inflammatory markers. Seven patients were ANA positive, 2 patients Rheumatoid factor positive and 3 patients HLA-B27 positive. Six patients received enteral feeds, 19 azathioprine and 5 sulfasalazine. 17/24 patients were treated with biologics. Anti-TNF was the most used biologics (infliximab was used in 13 patients and adalimumab was used 9 patients). Ustekinumab was used in four patients only. There was a statistically significant positive correlation between age of diagnosis of IBD and age of diagnosis of arthropathy (r= 0.593, P= 0.002), suggesting that they occur at the same age. There was no statistically significant association between IBD remission and arthropathy remission (Fisher’s exact, P= 0.082). Conclusion: This small study described the musculoskeletal manifestations (chronic arthritis and CNO) of patients with IBD. There is a significant correlation between age of diagnosis of IBD and arthropathy. Larger studies are needed to identify predictors of outcome in children with musculoskeletal manifestations of IBDE. Marrani 1 , G. Fulvio 2 , C. Virgili 1 , R. Izzetti 3 , V. Dini 4 , T. Oranges 5 , C. Baldini 2 , G. Simonini 1,6 1 Pediatric Rheumatology, AOU Meyer, Firenze, 2 Unit of Rheumatology, 3 Dentistry and Oral surgery, 4 Dermatology Unit, Azienda Ospedaliero-Universitaria Pisana, Pisa, 5 Dermatology Unit, AOU Meyer, 6 NEUROFARBA, University of Firenze, firenze, Italy Correspondence: E. Marrani Introduction: Sjögren’s Syndrome (SS) is a chronic autoimmune disease, primarily affecting lacrimal and salivary glands. The 2016 American-European Consensus Group criteria are not validated in pediatric population. Therefore, the diagnosis of pediatric SS mostly relies on clinical suspect, resulting in a significant diagnostic delay. Recently, Ultra-High Frequency Ultrasound (UHFUS) of labial glands has been proposed as a diagnostic method in adults with suspected SS. Objectives: The aim of the study is to evaluate the potential role of Ultra-High Frequency Ultrasound (UHFUS) of minor salivary glands for the diagnosis of Sjogren’s Syndrome in a cohort of pediatric population. Methods: Consecutive paediatric patients with pediatric-onset SS seen at AOU Meyer between 1/4/2021 and 30/04/2022 were evaluated with UHFUS. To be eligible patients should be received a clinical diagnosis of Sjogren’s Syndrome before the age of 16 years, according to a combined set of clinical, serological, and instrumental findings. Clinical, radiological, and histopathological findings were retrospectively collected using a dedicated CRF. Intraoral UHFUS scan of the lip mucosa was performed with Vevo MD equipment, using a 70 MHz probe with a standardised protocol and the images were independently reviewed by two operators. Lips salivary glands were assessed by using a four-grade semiquantitative scoring system (0-3), similar to the OMERACT scoring system used for major salivary glands. Results: Twelve patients with paediatric SS were included in the study (n = 12, 11 females; 10 Caucasian, 2 Asian), with a median age at the diagnosis of 14.1 years (range 7.75-17) and a median disease duration of 13.5 months (range 1-94) . Eleven patients were ANA positive; Ro/SSA were positive in 6/12 while none tested positive for LLA/SSB. Minor salivary gland biopsy was performed in 9/12, showing inflammatory chronic sialadenitis in 8/12. Treatment with hydroxychloroquine was ongoing in 11/12. When applying UHFUS to this cohort of patients, all patients showed a UHFUS grade of ≥1 with 8/12 showing a mild glandular alteration (i.e. grade 1), 2/12 a moderate glandular alteration (i.e. grade 2) and finally 2/12 a severe glandular alteration (i.e. grade 3). A moderate intraglandular vascularization was seen in 9/12, with only 3/12 showing a mild intraglandular vascularization. Due to limited size of the sample, the relationship between histological findings, autoantibodies status and UHFUS grade could not be performed Conclusion: Pediatric SS is a rare condition, and its prevalence is under-estimated due to the lack of standardized diagnostic criteria and the subtle early clinical presentation. New approaches to a faster diagnosis are urgently needed in clinical practice. This preliminary pilot study seems to report UHFUS as feasibility technique to identify salivary gland alterations in children with a clinical suspect of SS. This technique might contribute to drive guided lip biopsy, thus reducing the rate of false negative. Further studies are currently in progress in our clinics to identify the exact role of UHFUS and its potential predictive role of the various patterns observed in pediatric SS. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
L. Martínez, E. Moreno, M. López, A. Pujol Unit - Rheumatology Department, Vall d’Hebron University Hospital, Barcelona, Spain Correspondence: L. Martínez Introduction: Inflammatory and autoimmune rheumatic diseases especially affect women of childbearing age. These diseases are associated with a greater risk of pregnancy complications including preeclampsia, preterm delivery and low birthweight infants. Frequency, variety, and severity of these complications are dependent on maternal diagnosis, serologic profile and disease activity at the moment of pregnancy. In general, it is recommended to achieve remission or low disease activity before pregnancy. In our center, female patients with rheumatic diseases who are planning pregnancy or are pregnant are followed by a multidisciplinary team which includes obstetricians, pediatric rheumatologists and neonatologists. In 2019, a follow-up and treatment protocol was established, including assessment of babies in the pediatric rheumatology unit in the first year of life as well as cardiologic evaluation in cases where it was indicated. Objectives: We herein present data of the center experience in daily practice following protocol implementation. Methods: We conducted a retrospective observational study based on data obtained from clinical practice. Maternal diagnosis and serological profile, treatments administered during pregnancy and complications of previous and recent pregnancies affecting both mother and/or children were collected. Results: A total of 23 women and 26 children (3 twin pregnancies) were included in the present study. 4 out of 23 mothers (17.3%) had previous pregnancy complications including spontaneous abortions (2/23) and stillbirths after intrauterine growth retardation (2/23). Regarding current pregnancies, 7 of the 23 pregnancies presented complications (table 1). Out of the 26 babies evaluated in the pediatric rheumatology unit, 3 presented neonatal lupus with skin involvement, all of them being born to mothers with Anti Ro+. In all cases, lesions were self-limited before 3 months of age. Of these cases, only one mother had received preventive treatment with hydroxychloroquine during pregnancy. The 12 babies born to mothers with Anti Ro, La, U1 RNP antibodies underwent evaluation by pediatric cardiology at birth and at one year of life and none presented atrioventricular block. Eight of these mothers received preventive treatment with hydroxychloroquique. None of the babies presented infections that could be associated with the immunosuppressive maternal treatment during pregnancy. A 7-month-old girl born to a mother with rheumatoid arthritis, presented with pyelonephritis due to Escherichia coli : the mother had not received immunosuppressive treatment during pregnancy. Conclusion: In our series, 25% of the children (3/12) who were born to mothers with anti-Ro/La or RNP antibodies developed neonatal lupus with cutaneous involvement. In all cases lesions were resolved in the first three months of life without secondary scarring. There were no cases of atrioventricular block in those children and 66.7% (8/12) of their mothers were receiving preventive treatment with hydroxicloroquine during pregnancy. As atrioventricular block is a low-prevalence complication, larger series studies are needed to establish the efficacy of this treatment for the prevention of atrioventricular blockD. Patro 1 , L. B 2 , I. Rajarathinam 2 , J. Raghuram 2,3 , A. P. Rao 1,2 1 Pediatric Rheumatology, Manipal Hospital, Bengaluru, 2 Pediatric Rheumatology, Indira Gandhi Institute of Child Health, 3 Pediatric Rheumatology, Aster Whitefield, Bengaluru, India Correspondence: D. Patro Introduction: Primary Sjogren’s Syndrome(pSS) is a rare disorder often known as autoimmune exocrinopathy or epithelitis. Unlike in adults, it does not present with sicca-like manifestations in children which leads to a delay in diagnosis. Objectives: 1. To describe the varied clinical manifestation of pediatric Sjogren in our cohort. 2. To assess the EULAR Sjogren’s Syndrome Disease Activity Index (ESSDAI) at the onset. Methods: We reviewed 9 cases of pSS which fulfilled the 2016 ACR- EULAR Classification criteria and Proposed Juvenile pSS by Bartunkova et al. Results: In our series, the majority were females (89%). The mean age of presentation was 11.5 years (±2.45). The findings are summarized in the table below. Extra-glandular manifestations were more common at presentation. 66.7% of patients had joint manifestations followed by renal tubular acidosis (RTA) in 44.4%. 2 patients each initially presented with Immune-mediated thrombocytopenia (ITP) and Ig A vasculitis (IAV). The mean ESSDAI score at onset was 6.88 ± 1.98. The most frequently scored domain in ESSDAI is constitutional followed by the articular and glandular domain. Activity level was more in the articular and hematological domain. The drawback of our study was objective involvement of the salivary gland could not do due to invasiveness and financial constraints. Conclusion: Sjogren’s should be kept in the differential of U-CTD with positive surrogate markers in a resource-poor setting which helps in bridging the gap between diagnosis and timely interventional. ESSDAI score at the onset with involvement of multiple domains guides us for aggressive immunosuppression. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
F. Phoya 1 , W. Slamang 2 , C. Scott 1 , A. Horn 3 , S. Singh 4 1 Rheamatology, University of Cape town, 2 Rheamatology, Red Cross Hospital , 3 orthopaedic, University of Cape town, 4 Histopathology , Red Cross Hospital, Cape town, South Africa Correspondence: F. Phoya Introduction: Joint pain or swelling with limitation of movement is one of the most common presenting complaints in paediatric A&E departments. The wide range of differential diagnoses make it challenging to narrow down a particular cause, which can lead to delays in the initiation of appropriate management. Arthritis is characterized by joint pain, swelling, limitation of movement and sometimes periarticular erythema. These symptoms may be the initial presentation of a number of diseases, the differential diagnosis in children including septic arthritis, Lyme arthritis, foreign body synovitis, pigmented villonodular synovitis, hemarthrosis, avascular necrosis, malignancy, juvenile idiopathic arthritis (JIA) and rarely crystalline arthritis among others. An accurate history, physical examination and initial investigations are invaluable in differentiating a chronic inflammatory arthritis like JIA, from the other causes. A previous history of trauma may be helpful in acute cases but may also detract from the underlying diagnosis. Skill and care is therefore required in identifying the most pertinent information in each case. Objectives: Here we focud on synovitis in a young child caused by a missed foreign body. Methods: We present a case to highlight the importance of a thorough history, including that of previous trauma, and the utility of ultrasound as a go to investigation when there are inconclusive findings on traditional tests like plain radiograph Results: n/a Conclusion: With a clear history of trauma or penetrating injury, every effort should be made to rule out a foreign body, particularly when there is no improvement after treatment. While plain radiographs are not very sensitive at picking up radiolucentforeign bodies, ultrasound may be a helpful tool to narrow down the diagnosis and also to localize the exact position of a foreign body. Although MRI has been shown to be sensitive and the investigation of choice, it might not always be easily available or accessible in many settings Patient Consent: Yes, I received consent Disclosure of Interest : None declared
B. Power 1,2 , S. McGlacken-Byrne 2 , E. MacDermott 3 , O. Killeen 4 1 Dept. of Rheumatology, Children’s Health Ireland, Crumlin, Dublin, 2 Clinical Education, National University of Ireland, Galway, 3 Dept.of Rheumatology, 4 Dept of Rheumatology, Children’s Health Ireland, Crumlin, Dublin, Ireland Correspondence: B. Power Introduction: In July 2020, The Paediatric Task Force for Global Musculoskeletal Health expressed a ‘call to action’ amongst the musculoskeletal community. Internationally, suspected deficits in paediatric musculoskeletal education and training have been cited as potential factors contributing to delayed referral of patients to specialist rheumatological services. Resultant delays may lead to delayed diagnosis and treatment leading to a reduction in physical function, persistent pain, and potentially irreversible joint damage. Objectives: Our study aimed to investigate the provision of paediatric musculoskeletal education in the Republic of Ireland. In addition, we wished to evaluate the relationship between paediatric musculoskeletal education and doctors’ confidence in clinical examination skills and knowledge of common rheumatological disorders of childhood. Methods: A quantitative, cross-sectional, questionnaire-based study was performed. This was based on previously validated instruments. All Consultant General Paediatricians and paediatric doctors currently enrolled in formal paediatric training in the Republic of Ireland (July 2020 to July 2021 training year) were included. The questionnaire was distributed to the participants using the online survey tool, SurveyMonkey. The data was analysed using standard statistical software (SPSS). Kruskal-Wallis and Chi-Square tests were used to investigate potential associations between educational experience, confidence in skills and knowledge-base of physicians. A p value of <0.05 was considered statistically significant. Ethical approval was granted by the Research and Ethics Committee of the Royal College of Physicians of Ireland. Results: In total, 471 doctors were included (82 Basic Specialist Trainees, 150 Higher Specialist Trainees and 239 Consultant Paediatricians). There was an overall response rate of 41% (n=192). Forty-nine percent (n=94) and 48% (n=92) recalled receiving teaching on paediatric musculoskeletal examination skills during undergraduate and postgraduate training respectively. Seven percent (n=14) were ‘not at all confident’ in musculoskeletal assessment, and 61% (n=117) were ‘confident in some aspects only’. Doctors were less confident in musculoskeletal examination than all other bodily systems, except for ophthalmology assessment. There was a statistically significant association between receiving postgraduate musculoskeletal skeletal education and higher confidence in clinical skills, x2 (3, N=191) = 25.655, p = 0.000, but no significant association between receiving undergraduate musculoskeletal skeletal education and confidence, x2 (3, N=192) = 1.844, p = 0.606. There was no significant association between confidence in clinical skills and knowledge of rheumatic disorders, H(2) = .792, p=0.673. Conclusion: Our study has been the first study to explore paediatric musculoskeletal education in the Republic of Ireland, the self-reported confidence of paediatric doctors in paediatric musculoskeletal examination skills, and their knowledge of rheumatic diseases of childhood. Using a learner-centred educational approach, our research has offered valuable feedback from learners within the speciality of paediatrics. Our research has identified deficiencies in the paediatric training curriculum which will help to inform future curriculum planning for undergraduate and postgraduate clinical education in paediatric rheumatology. This study will encourage further reflection and deliberation on paediatric musculoskeletal education, which will ultimately improve patient safety and quality of care for paediatric patients in the Republic of Ireland. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
G. Rogani 1 , A. Petaccia 1 , G. B. Beretta 1 , S. Testa 1 , E. A. Conti 1 , F. Chironi 1 , F. Lucioni 1 , F. Baldo 1 , A. M. Cappellari 2 1 Pediatric Rheumatology, 2 Neuroscience, Fondazione IRCCS Ca’Granda Ospedale Maggiore Policlinico, Milan, Italy Correspondence: G. Rogani Introduction: Sydenham’s Chorea (SC) is a neuropsychiatric autoimmune disorder occurring in up to 40% of patients with acute rheumatic fever and there are no guidelines for its treatment. Objectives: The aim of the study is to evaluate the efficacy of prednisone therapy in children with SC in term of time of remission and recurrence rate, compared to other treatments. Furthermore, correlation has been investigated between duration of symptoms and the following clinical and biochemical features at chorea onset: age, throat culture positivity for streptococcus, presence of hemichorea, psychiatric symptoms, carditis and/or arthritis, reactive C protein (RCP), erythrocyte sedimentation rate (ESR) and anti-streptolysin-O (ASO) titer. Methods: This is an observational, retrospective, single-center study involving all patients diagnosed with SC, according to 2015 revised Jones criteria, admitted to Pediatric Rheumatology Unit of Policlinic Hospital of Milan (Italy), between January 1995 and March 2022. Clinical data of patients were collected from medical records. For statistical analysis Pearson chi squared, Pearson correlation coefficient and the non-parametric Mann Whitney test were used, when appropriate. P-value lower than 0.05 were considered significant. Results: Of 59 enrolled patients (74% female; median age 9.3, range 7.4-10.6 years), 49 were considered eligible (10 were excluded due to incomplete data) and, among these, 79% received steroid therapy. The remaining patients were treated with symptomatic drugs (neuroleptics or valproate). The analysis shows that time of remission is significantly shorter in patients treated with prednisone therapy (median time 31 vs 41 days, p=0.023). Moreover, the presence of arthritis at SC onset was associated to longer symptoms duration (median time 90.5 versus 39 days, p=0,02). Recurrence occurred in 12% among the eligible patients and seemed to be related only to a younger age at onset (p=0,01). Conclusion: Our study demonstrates the superiority of prednisone compared to symptomatic treatments for SC remission. Major strengths of this study are the large number of investigated patients and the long follow-up time (27 years). Disclosure of Interest : None declared
I. Rukavina 1 , M. Frković 1 , I. Brnadić 2 , M. Jelusic 1 1 Department of Pediatrics, 2 Department of Pathology, Clinical Hospital Center Zagreb, Zagreb, Croatia Correspondence: I. Rukavina Introduction: Pachydermodactily (PDD) is a rare digital fibromatosis of unknown origine mostly found in adolescent boys. It is manifested by simetrical, painless swelling of the periarticular soft tissues of the fingers without joint involvement. It is thought to be related to repetitive mechanical trauma where patients also can have different cutaneous responses to the attrition of the skin (for example neurodermitis). Treatment is often not indicated given its benign prognosis. Objectives: These reports aim to define characteristics and increase awareness of the disease that would differentiate it from other disorders that may produce PIP joint swelling, particularly juvenile idiopathic arthritis. Methods: We present clinical, laboratorial and radiological characteristics of 3 male adolescents (aged 17 to 18 years) with simetrical periarticular soft tissue thickening of the fingers. Results: First patient is a 17-year-old boy who noticed bilateral second to fifth PIP joint and thumb swelling that have developed through several years together with hyperkeratose nodulations. No pain was noted neither morning stiffness. He does hard physical work at his family farm. Hand MRI showed periarticular thickening of the PIP joints bilaterally with discrete subcutaneous edema and no signs of synovitis. Histological analysis of the skin showed hyperkeratosis with discrete perivascular inflammation in papilar dermis. Results of the other diagnostic tests specific for rheumatic diseases were normal. Another patient is a 18- year-old boy who developed painless swelling of the PIP joints as well as second and third MTP joints bilaterally. He was examined by dermatologist and the diagnose of contact allergic dermatitis was made. Results of the diagnostic tests for rheumatic diseases were normal. Histological analysis of the skin showed hyperkeratosis with proliferation of fibroblasts and increase in the bands of collagen in the dermis. The third patient is a 17-year-old boy who have developed bilateral second to fifth PIP joint and second to third MTP joint skin thickening with hyperkeratose nodulations two years ago. All joints are painless with normal range of motion and grip strenght. Ultrasound showed just soft tissue thickening with no signs of synovitis. Rheumatic diseases laboratorial tests were normal. According to clinical picture, results of laboratorial tests, radiological and histological findings the diagnose of pachydemodactily was made. Conclusion: Although PPD is a rare disease, probably it is very underdiagnosed. It is triggered or at least aggravated with mechanical trauma of the fingers that can be unconcious at some patients (rubbing hands or cracking knuckles). It is needed to exclude other rheumatological disease that can have similar manifestations. Make a diagnose in a short period will reduce unnecessery diagnostic tests, treatment and patient anxiety. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
M. C. Santos 1 , F. D. Kuchiki 1 , F. Arita 2 , I. Lacerda 2 , G. Oliveira 1 , C. Benavides 1 1 Pediatric Rheumatology, 2 Pediatric Neurology, Irmandade Da Santa Casa De Sao Paulo, São Paulo, Brazil Correspondence: M. C. Santos Introduction: Sjögren Syndrome is a chronic autoimmune disease characterized by inflammation of exocrine glands, mainly lacrimal and salivary exocrine glands. At least one third of patients present with systemic extraglandular manifestations like articular, pulmonary and neurological. The prevalence and clinical manifestations of neurological involvement in SS have been reported variably. Central nervous system (CNS) involvement has a variable reported frequency between 0,3 and 48%. Neuromyelitis optica spectrum disorder (NMOSD) is one of the CNS manifestations. There is a discussion about the relationship between the Sjögren syndrome and NMOS. It may be just an association or NMOSD may be a neurological manifestationof Sjögren syndrome. Objectives: The objective of this study is to present two Sjögren syndrome cases which had NMOSD asneurological manifestation. Methods: Chart review to check medical history, laboratory tests and imaging of these children with Sjogren Syndrome, according to the American College of Rheumatology SICCA Classification criteria (ACR/SICCA). An informed consent form was signed. Results: Patient, eleven years old girl, carrier of common variable immunodeficiency, admitted with history of repeated mump, paresthesia of lower limbs, headache and seizures. The laboratory test showed elevated levels of anti- Ro antibodies. Added to this, it was observed hypergamaglobulinemia, hyperproteinrrachia and increase of spinal fluid pressure. The MRI shows papilledema and multiple bilateral encephalic lesions of white and gray matter, corresponding to residual lesions of vasculitis. It was performed biopsy of salivary gland to confirm the diagnosis os pediatric Sjögren syndrome. Corticosteroid, IVIG and immunosupressives drugs were introduced and in one of them, it was necessary the use of plasmaferese and . Fews months later, patient started with headache and visual loss. A new exam showed image suggesting a diagnosis of optic neuritis. Added methylprednisolone (30 mg/kg/day for 3 days) with the medications described above and patient evaluated visual improvement, at the beginning. Patient, nine years old girl, who presented Sjögren syndrome (elevated level of anti Ro antibodies , altered sialometry and biopsy of salivary gland showing alterations compatible with Sjögren syndrome). She started with headache, evolving with a sensory motor surge in the right upper limb. Cranial magnetic resonance imaging showed areas of signal alteration in periventricular white matter, mainly in the radiated crowns, bilaterally with total improvement after pulse therapy with methylprednisolone. After 5 months, the patient9 months, he evolved with low visual acuity. Pulse therapy with methylprednisolone, rituximab and plasmapheresis were performed, with excellent response. Both patients didn’t present important symptoms of sicca syndrome. Conclusion: Conclusion: NMOSD may be a neurological manifestation in Sjögren syndrome patients, and can represent the initial manifestation of Sjögren Syndrome, even when mild complaints of SICCA symptoms are absent. Complaints like headache, visual loss, should be valued and investigated for an early diagnosis. Therefore, the detailed history, antibodies test, images and biopsy are necessary to a prompt diagnosis, stablish the appropriate treatment and avoid sequelarevisan 1 , L. Di Lenarda 2 , S. Pastore 3 , A. Saccari 3 , G. Canton 4 , U. Lucangelo 5 , L. Murena 4 , A. Taddio 3 1 Department of Medicine, Surgery and Health Science, University of Trieste, IRCCS Burlo Garofolo, 2 Department of Medicine, Surgery and Health Science, University of Trieste, 3 Institute for Maternal and Child Health, IRCCS Burlo Garofolo, 4 Orthopaedics and Traumatology Unit, Cattinara Hospital, Azienda Sanitaria Universitaria Giuliano-Isontina (ASUGI), 5 Department of Perioperative Medicine, Intensive Care and Emergency, Cattinara Hospital, Trieste, Italy Correspondence: M. Trevisan Introduction: Primary Synovial Chondromatosis (PSC) is a rare benign tumor of the synovial membrane in which cartilage metaplasia produces calcific loose bodies within the articular space. Only a few cases are reported in the pediatric population and its etiology remains unknown. This condition typically affects large weight-bearing joints with pain, swelling and decrease range of motion. Due to its slow progressions, delayed diagnosis is frequent and differential diagnosis should consider other chronic arthritis and malignancies. While arthroscopic removal of loose bodies is the current treatment up to now, the association of partial or complete synovectomy is debated. Objectives: N/A Methods: N/A Results: Case presentation: We report about a 14-year-old girl with a long-lasting right shoulder pain, especially during movements or exercise, localized tenderness and hypotonia of the glenohumeral joint. No previous trauma was mentioned. Blood exams, Mantoux test and plain radiography of the right shoulder were unremarkable. Ultrasound imaging revealed echogenic and calcified bodies stretching the glenohumeral joint and dislocating the long head of biceps tendon. Magnetic resonance showed a “rice-grain” pattern of the right shoulder. From an arthroscopic surgery, multiple loose white bodies were removed within the synovial membrane, and synovial chondromatosis was confirmed by histological analysis. At one month follow up visit, the patient completely recovered without pain. Conclusion: Synovial chondromatosis is a very uncommon cause of mono articular pain in children, especially when it affects shoulder. Pediatricians should keep in mind this condition to avoid delayed diagnosis and treatment, even in consideration of the low risk of malignant transformation. Through this case, we would highlight common diagnostic pitfalls and treatment of synovial chondromatosis. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
I. Turtsevich, S. Compeyrot-Lacassagne Great Ormond Street Hospital NHS Trust, London, United Kingdom Correspondence: I. Turtsevich Introduction: Tubulo-interstitial nephritis and uveitis (TINU) is a rare still under-recognised autoimmune condition associated with the renal and ocular involvement of unknown aetiology. Although rare in incidence, there is a hypothesis that the immune-mediated mechanisms are not limited to the renal and ocular inflammation, but may also cause multisystem involvement. Objectives: To explore other organ systems involvement and the role of the rheumatologist in the management of TINU as a multisystem entity. Methods: Patients aged 0-18 with the established diagnosis of TINU, diagnosed and treated at Great Ormond Street Hospital for Children NHS Trust between January 2010 and March 2022 were involved in the study. TIN was defined as the presence of AKI, elevated tubular markers (RBP and NAG) and confirmed by the kidney biopsy. Uveitis was confirmed by the routine ophthalmology assessment on the slit lamp examination. All the relevant data were extracted from the electronic patients records and were fully anonymised. The data analysis was carried out by using IBM SPSS, v.26. Results: A total of 19 patients were included in the study, female gender being prevalent (p=0.002). The mean age of the patient was 13.8±2.3 years old with the mean disease onset being at 11.8±2.1 years. The cohort was multi-ethnic with the prevalence of children from Black African ethnicity (p<0.001). Most of the patients were initially presented with either TIN or uveitis (47.4% and 42.1%, respectively, p=0.104) and in the majority of the cases, the trigger was not identified (0=0.021). Roughly two third of patients complained of fatigue, following weight loss, fever and abdominal/flank pain. Other symptoms also included arterial hypertension, dysuria and polyuria, cervical lymphadenopathy and polydipsia. The minority reported skin rash and arthralgia/arthritis. A large proportion of the patients were found to have bilateral anterior uveitis (78.9%, p<0.001) and the rest had unilateral anterior uveitis, retinal vasculitis, bilateral panuveitis and papilledema. Kidney biopsy was performed in 12/19 patients with TIN being confirmed in 10 patients, while 2 other patients were found to have either immune-mediated glomerulonephritis or changes were non-specific (p<0.001). Out of 19, three patients developed irreversible hearing impairment and no other extrarenal/extraocular involvement has been found. The level of inflammatory markers, including ESR and CRP were elevated in 12 patients, while creatinine was raised in all the patients. Tubular markers were measured in 17 patients with NAG being high in 84.2% and RBP in 73.7% of the children. Glucosuria was found in 68.4% of patients, whereas proteinuria and UA:UC ratio was positive in 52.6% of the TINU children. Other positive findings included microscopic haematuria and pyuria. Further laboratory analysis revealed that third of TINU patients had elevated Ig A, G and M. Furthermore, ANA was positive in 6 patients, 2 of them had hearing loss (p>0.05). ENA or ANCA were negative for all the patients. All patients received systemic GCs and Steroid eye drops. Eleven patients with active TIN were escalated to MMF, while those who remained active for uveitis had their treatment escalated to either Methotrexate (2 patients) or anti-TNF treatment (1 patient) having good response to the treatment. Conclusion: The role of the paediatric rheumatologist in the management of TINU is to identify any extrarenal/extraocular impairment, particularly hearing loss by performing an extended autoimmune screening and audiology earlier in the disease course. Further analysis is needed to explore early predictor markers for the hearing loss as one of the extrarenal/extraocular involvement and possible early escalation to steroid-sparing DMARDs. Disclosure of Interest : None declared
National Institute of Rheumatic Dieseses, Piešťany, Slovakia Correspondence: E. Vrtikova Introduction: CACP syndrome is rare condition characterized by congenital camptodactyly and early childhood onset of non-inflammatory synovial hyperplasia. This syndrome with autosomal recessive inheritance is caused by a mutation in the PRG4 gene, which is responsible for the synthesis of lubricin, a protein that lubricates the joints. In some patients only coxa vara is present, in others pericarditis is the leading sign, some have both of these symptoms. Objectives: Our patient, a 18 year old girl, was diagnosed with camptodactyly as an infant and the defect was resolved surgically by the age of 15 months. At the age of 18 months frequent swelling and “cracking” of the knee was observed. Even though the rheumatologist confirmed the presence of swelling of elbows, wrists, knees, and ankles, the mobility was intact and there was no limitation of range of motion. Ultrasonography showed a non-inflammatory synovial hyperplasia and an accumulation of the intraarticular fluid. Markers of inflammation in serum were normal, inflammation was excluded also by analysis of the synovial fluid. She was diagnosed with the CACP syndrome at the age of 30 months. Her parents were negative for consanguinity. Radiographs of the hip joints detected early signs of dysplasia. At the age of 6 years the X-ray of pelvis confirmed the presence of the coxa vara. The early course of the disease was managed by intensive physiotherapy. Over the following years there was a progression of the femoral varus deformity with pathological attitude and accentuation of lumbar lordosis, resulting to difficult walking, muscle weakness, and increased fatigue. Last year, she underwent a combined periacetabular osteotomy and proximal femoral derotation osteotomy with consequent improvement in mobility. Methods: Cese report Results: The CACP syndrome may be misdiagnosed as a juvenile idiopathic arthritis. Immunosuppressive therapy is not effective but adequate pain treatment and rehabilitation has an irreplaceable role. Musculoskeletal ultrasonography and cytological examination of synovial fluid seem to be very important for the diagnosis of the CACP syndrome. In our patient the findings of ultrasonography, together with the history of camptodactyly in infancy and negative markers of inflammation were crucial in the differential diagnosis. Conclusion: Over the following years coxa vara and limited mobility developed in our patient. Even though the disease does not affect life expectancy, its character often requires surgery and joint replacement in the early adulP416. The children’s emotional state and quality of life depends on subtypes of juvenile idiopathic arthritis P417. Exploring art therapists perceptions of using art therapy with children managing juvenile idiopathic arthritis: a vignette study 8. Survey of experienced knowledge among adolescents with JIA using the meps-questionnaire P419. Factors associated with poor medication adherence in children with rheumatic diseases P420. Present and accounted for: the workplace productivity loss for parents of children with juvenile idiopathic arthritis 21. The knowledge, attitudes and practices of non-specialist healthcare workers in Kenya towards paediatric rheumatology P422. Effects of a physical activity promotion program and workout in teenagers with juvenile idiopathic arthritis 3. Special aspects of the communicative and emotional-volitional sphere in pediatric patients with monogenic autoinflammatory diseases (FMF, CAPS, TRAPS) P424. Procedure related pain: successful reestablishment of parent-administered subcutaneous injections at home using an education program: a report of three cases P425. Prevalence and factors associated with depression and anxiety in patients with juvenile idiopathic arthritis L. Bohmat 1,2 , A. Fadieieva 1,2 , N. S. Shevchenko 1,2 1 Department of rheumatology and comorbid states, SI “Institute for Children and Adolescents Health Care of the NAMS of Ukraine”, 2 Department of pediatrics № 2, V.N.Karazin Kharkiv national university, Kharkiv, Ukraine Correspondence: A. Fadieieva Introduction: Juvenile idiopathic arthritis (JIA) is a chronic childhood disease that aggravates not only the physical component of health but also the emotional state of patients. According to modern guidelines, it is necessary both to consider the objective manifestations of the disease and to assess the subjective feelings of patients. Such actions are important for the realization of comprehensive care for patients with JIA. Objectives: The aim was to study the emotional area of quality of life (QoL) of patients with JIA with different subtypes of the disease in one week. Methods: 118 children with JIA (47 with polyarticular, 43 with oligoarticular, 28 with uveitis-associated (JIA-u) subtypes) aged from 2 to 18 years were examined, including 77 girls and 41 boys. The JADAS27 questionnaire was used to assess the disease activity, and the PedsQLTM 4.0 Generic Core, validated for Ukraine, was used for the QoL level. The general result of the questionnaire was analyzed, as well as the emotional subscale of the questionnaire. The questionnaire consists of 23 questions and has 4 age versions (for children from 2 to 18 years). The evaluation of the obtained results was performed according to the method of the Likert scale, where the result of 100 points means the best level of QoL. Disease activity was defined as high at scores above 4.2 for oligoarthritis and 8.5 for polyarthritis. Statistical processing of the material was performed using Microsoft Excel 2016. Results: The disease activity according to JADAS27 in the subgroup with polyarthritis was 6.4 ± 0.6 points, with oligoarthritis - 3.5 ± 0.5 points, with JIA-u - 2.6 ± 0.5 points. It was high in 12 patients with polyarthritis, in 10 - with oligoarthritis, and in 9 - with JIA-u. The overall rate of QoL in the group of children with JIA for 7 days of observation in the hospital was at the level of 70.9 ± 1.4, the emotional component is slightly lower - 66.3 ± 1.75. The lowest rates of total QoL were observed in the group with polyarthritis - 65.6 ± 2.9 (p≤0.01). The tendency to decrease the total QoL was also observed in the group with JIA - 69.4 ± 2.3, while children with oligoarthritis had better indicators - 77.9 ± 2.3. The assessment of the emotional component of QoL differed from the overall result, showing the worst level of emotional state in children with JIA-u - 56.6 ± 2.2 (p≤0.01). The indicators of emotional state in the groups with polyarthritis and oligoarthritis corresponded to the level of the general index of QoL (65.8 ± 3.1 and 73.3 ± 2.3, respectively). Given the importance of disease activity, the association of activity in the group as a whole with the overall QoL score and emotional component (-0.409 and -0.197, p≤0.05, respectively) was assessed. Conclusion: The most significant reduction in QoL in children with polyarticular and uveitis-associated subtypes of JIA was found. The emotional component of QoL was the lowest in patients with JIA-u and did not correlate with disease activity. Disclosure of Interest : None declared
D. Ghio, U. Toher, E. Griffiths, T. Epton ivision of Psychology and Mental Health , University of Manchester, Manchester, United Kingdom Correspondence: D. Ghio Introduction: Art Therapy, a type of psychotherapy using the creation of images and objects to improve wellbeing and facilitate communication about complex issues which is especially useful for younger populations. This type of therapy has grown in popularity to support children with long term conditions including Juvenile Idiopathic Arthritis (JIA). Managing JIA can be explored during Art Therapy using goal-setting techniques, but little is known about the processes of change occurring during therapy with different goals and what are the barriers and facilitators to getting children to participate in Art Therapy and attain their goals. Objectives: This current study has two objectives; (a) to explore the perceptions of Art Therapists on goal-based processes in Art Therapy, with children managing their JIA; (b), to identify the perceived barrier and facilitators for children when accessing the therapy and to attain their goals. Methods: Semi-structured interviews were conducted with three art therapists exploring two vignettes of hypothetical children aged 12 presenting with either physical-emotional issues (anxiety with hospital procedures and appointments and needle phobia) or social-emotional issues (social isolation from peers, lonely and low mood) in relation to JIA. These were developed from qualitative research of the psychosocial issues faced by children with JIA. The transcripts were analysed using framework analysis to identify barriers and facilitators. Results: In total there were six vignette cases discussed that explored different approaches to either physical-emotional issues or social-emotional issues. In general, there were barriers to partaking in the therapy, due to the nature of the condition e.g. flare ups of pain and fatigue, or due to the process of the referral which is either through the parents or healthcare professionals. In the latter situation, the child may need more support in setting their own goals. These goals, the Therapists explained, needed to be flexible and adaptable for the children because of fluctuations in the child’s environment that may be out of their control. Regarding the different issues there were specific barriers for example the coordination with school but also facilitators for example identifying own skills and developing problem solving skills. Conclusion: Art Therapy can be a useful for providing an outlet for exploring both physical-emotional issues and social-emotional issues empowering children with skills for problem solving in their management of JIA. The Art Therapy needs to be flexible and adaptable in how it is delivered but there is also a need to be flexible with goal setting to take into consideration the context of these behaviours that are dependent on other factors (e.g. school). Further work into children and parents’ experiences of Art Therapy will be useful to understand how children implement these skills and incorporate them in their everyday lifHaavisto Olow 1 , A.-L. Lagerkvist 1,2 1 Queen Silvia Children’s Hospital, 2 Health and Rehabilitation, Gothenburg University, Gothenburg, Sweden Correspondence: A. Haavisto Olow Introduction: As a health care team we prepare adolescents with juvenile idiopathic arthritis (JIA) for transition into adult care and promote the adolescents’ ability to self-manage. Disease education and information is an important part of this and should be adapted to the adolescents age and maturity. To know what to target in the education we need to find out how the adolescents experience their knowledge. Objectives: The objectives were to explore what knowledge adolescents with JIA have about their disease and if the knowledge differed when divided according to gender or subtype of JIA. Methods: All adolescents with JIA, aged 13-17 years, who were followed at Queen Silvia Children’s Hospital were sent the youth version of the Medical Issues, Exercise, Pain, Social Support questionnaire (MEPS). The adolescents gender, their subtype of JIA and their ongoing medical treatment were noted from patient records. Results: Thirty-seven of the 70 adolescents invited chose to participate, 73% were female. The results for the medical issues domain showed the adolescents experience an average knowledge of medical background of JIA (median 48 mm), a low knowledge of joint anatomy (median 31 mm) and of blood samples (median 18 mm). It also showed that most adolescents don’t fear giving blood sampling (median 4 mm) or getting corticosteroid injections (median 21 mm). In the exercise domain the adolescents answered that they have a high participation in both physical education in school (median 86 mm) and exercise in the spare time (median 90 mm). The pain domain showed a high self-efficacy for symptom management (median 72 mm) and pain alleviation skill (median 84 mm). In the social support domain the adolescents report a low value for exchanging experiences with other youth with JIA (median 3 mm). Further, they have low knowledge of community support measures (median 8 mm). When compared between genders the boys reported a less sufficient knowledge of signs of joint inflammation than girls (median 23 mm vs. 54 mm, p=0.023). The boys also gave lower value to JIA-contacts than girls (median 13 mm vs. 52 mm, p=0.007). Significant differences were seen in the comparison between subtypes in knowledge of medical background (p=0.016), joint inflammation (p=0.012), signs of joint inflammation (p=0.042), and value of JIA-contacts (p=0.038). A pair-wise comparison between all subtypes revealed significant differences between adolescents with enthesitis-related and polyarticular JIA, where those with enthesitis-related JIA experienced their knowledge as significantly closer to insufficient on knowledge of medical background (median 12 mm and 81 mm, p=0.017), joint inflammation (median 16 mm and 81 mm, p=0.01), and signs of joint inflammation (median 46 mm and 97 mm, p=0.035). Conclusion: This study shows that the adolescents’ knowledge of their disease differs in different domains and that there are significant differences in knowledge between girls and boys as well as between different subtypes on certain items. This information is helpful for the health care team working with the adolescents with JIA as we can use it to further develop the education in areas where the knowledge is experienced as inadequate. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
, S. Vilaiyuk, B. Lerkvaleekul Pediatric department, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand Correspondence: R. Manatpreeprem Introduction: Children with rheumatic diseases usually require long-term treatment and multiple medications, which increases the risk of noncompliance. Failure to take regular medications leads to poorer health outcomes. The Pediatric Rheumatology Adherence Questionnaire (PRAQ), a tool for assessing medication adherence in rheumatic patients, could effectively detect poor medication adherence in these individuals. Objectives: This study aimed to determine the associated factors for poor medication adherence in children with rheumatic diseases. Methods: This was a cross-sectional study design. Patients with rheumatic diseases, who had taken at least one medication and had been followed up at Pediatric Rheumatology Clinic with their caregivers, were included in the study. Patients with poor medication adherence were characterized as those who had taken less than 80% of their drugs, as determined by the pills count pharmacy refill technique. The original PRAQ was translated and validated into the Thai version and was completed by caregivers and/or literacy patients over the age of thirteen. Interviewing for additional obstacles to taking medications was conducted. Descriptive and logistic regression analyses were performed. Results: From a total of 210 patients, 52.9% had juvenile idiopathic arthritis (JIA) and 47.1% had connective tissue diseases. The patients’ mean age was 14.1±4.7 years, with a median (IQR) disease duration of 4.3 (2.1-7.0) years. With a mean total PRAQ score of 11.0±3.5, poor medication adherence was discovered in 24.8% of patients. Polyarticular JIA and enthesitis-related arthritis (ERA) were two JIA subtypes that associated with poor treatment adherence (OR 4.99 ,95%CI 1.45-17.21, and OR 4.95, 95%CI 1.56-15.71, respectively). Patients who had disease duration longer than 5 years (OR 2.88, 95%CI 1.11-7.46), PRAQ scores ≥ 10 (OR 2.88, 95%CI 1.11-7.46), and forgetting to take medications (OR 15.48, 95%CI 5.66-42.33) were the predictors of poor drug adherence. Conclusion: Poor medication adherence was found in one-fourth of children with rheumatic illnesses, particularly polyarticular JIA and ERA. Inadequate adherence was related to longer disease duration, high PRAQ scores, and forgetting to take prescriptL. Grazziotin 1 , G. R. Currie 1 , S. Cantarutti 1 , S. M. Benseler 1 , J. F. Swart 2 , M. M. Kip 3 , M. J. IJzerman 4 , M. Twilt 1 , S. J. Vastert 2 , N. M. Wulffraat 2 , R. S. Yeung 5 , D. A. Marshall 1 on behalf of on behalf of UCAN CAN-DU and UCAN Cure Consortium 1 University of Calgary, Calgary, Canada, 2 University Medical Center Utrecht, 3 University Medical Center Utrecht, Utrecht, 4 University of Twente, Enschede, Netherlands, 5 University of Toronto, Toronto, Canada Correspondence: M. M. Kip Introduction: Parents of children with juvenile idiopathic arthritis (JIA) face impacts on their time at work and daily life activities due to caregiving for their children. Work productivity impacts include both absenteeism (missing work due to their child’s JIA) and presenteeism (reduced productivity while at work). Objectives: To estimate changes in work commitment, impact on work (presenteeism and absenteeism) and usual activities for caregivers of children with JIA. Methods: Productivity loss was captured in standardized instruments, including the validated Work Productivity and Activity Impairment Questionnaire (WPAIQ): Specific Health Problems. The UCAN CANDU is an on-going prospective, multicentre study including all pediatric rheumatology clinics in Canada and the Netherlands which focused on personalized care strategies in JIA through biological monitoring systems. Caregivers participating in the UCAN CANDU study also completed questionnaires related to demographic characteristics. Results were reported using means, standard deviations, and proportions. Results: 244 caregivers were included in this study. Approximately 12% of primary parent respondents and 3% of their spouses/partners reported a change in their work commitment. The mean absenteeism and presenteeism rates reported for any reason were 15.8% and 18.1%, respectively. The mean overall work impairment reported was 28.7%, mostly due to their child’s JIA (25.8%). Conclusion: JIA creates considerable socioeconomic burden affecting paid work and usual activities for parents. Our study shows that caregivers’ work impairment can primarily be attributed to their child’s JIA, and that the productivity impact extends beyond absence from work, into impaired productivity of employees while at work. The societal and economic implications of this require further studigowa 1 , S. bernatsky 2 , A. Ngugi 3 , H. Foster 4 , P. Muriuki 5 , A. Lusambili 5 , S. Luchters 6 1 bPaediatrics and Child Health, Aga Khan University Medical College East Africa (Nairobi,Kenya), Nairobi, Kenya, 2 Rheumatology, McGill University Health Centre, Montreal, Canada, 3 Populations Health, Aga Khan University Medical College East Africa (Nairobi,Kenya), Nairobi, Kenya, 4 Paediatrics, New Castle University, New Castle, United Kingdom, 5 Population Health, 6 Institute of Human Development, Aga Khan University Medical College East Africa (Nairobi,Kenya), Nairobi, Kenya Correspondence: A. Migowa Introduction: The World Health Organization (WHO) has categorized musculoskeletal diseases into over 200 entities which are still the leading cause of disability globally(1). The prevalence of various childhood rheumatic diseases varies across different regions of the world, with the prevalence of Juvenile Idiopathic Arthritis (JIA) ranging from 0.07 to 10/1000 children and that of Systemic Lupus Erythematosus (SLE) from 0.4 to 0.6/100 000 children(2). Epidemiological data from Kenya about childhood rheumatic diseases are lacking, but on the basis of the above population data, we could estimate 300,000 children with JIA alone. Dysfunction is not only related to disease severity, but also on illness perception(1). In Sub-Sahara Africa, as a result of other competing health interests, musculoskeletal health is often not prioritized. Due to the paucity of skilled personnel to manage paediatric rheumatic diseases, efforts towards reducing this disparity need to be bolstered. Consequently, there is an urgent need to scale-up pediatric rheumatology knowledge and skills among non-specialist clinicians by tapping into digital technology to optimize remote learning due to the paucity of paediatric rheumatologists to bridge the gap in clinical care. Objectives: The objective of this study is to understand the knowledge, attitudes and practices of non-specialist clinicians towards paediatric rheumatology to help inform development of an intervention aimed at improving their clinical skills in diagnosis and management of paediatric rheumatology conditions. Methods: Online focus group discussions were conducted among first tier community health workers (clinical officers), nurses, general practitioners, and pediatricians to ascertain their knowledge, attitudes and practices towards pediatric rheumatology. The focus groups were recorded and the recordings transcribed. The data coders read the transcripts and thematic coding carried out. Results: A total of 68 participants were recruited of whom 77.9% ( 53 of 68) were female with a mean age of 36 years . Among participants involved 39.7% (27) were paediatricians, 14.7% (10) general practitioners, 17.6% (12) nurses , 16.7%(11) primary community health workers and 10.2% (7) pursued other specialities. The focused group discussions revealed a paucity of knowledge among non-specialist healthcare workers towards paediatric rheumatology. This often leads to confusion and yields an attitude of anxiety and fear upon encountering paediatric rheumatology patients, which is associated with delayed diagnosis, misdiagnosis and inappropriate management. Conclusion: Non specialist healthcare workers expressed paucity in knowledge of pediatric rheumatology which impacts negatively on their attitude and practice in this discipline. In order to improve their knowledge, attitudes and practices participants proposed that algorithms for diagnosis and management should be availed alongside continuous medical education and ongoing mentorship, either virtually or face to face. Disclosure of Interest : None declared
E. Prato 1 , M. Pirinu 1 , E. marrani 2 , I. Maccora 2 , M. V. Mastrolia 2 , I. Pagnini 2 , L. Baroni 1 , G. Simonini 2,3 1 Rehabilitation Unit, 2 Pediatric Rheumatology, Aou meyer, 3 NEUROFARBA department, University of Firenze, Firenze, Italy Correspondence: E. Prato Introduction: Despite physical activity represents a therapeutic agent for JIA[1,2] several studies report that JIA adolescents less active than healthy peers[3]. This can cause long-term negative effects, and as matter of fact inactive lifestyle is a risk factor[4,5]. Current evidence points out the need to increase the level of physical activity and limit sedentary behaviors in this population. Different approaches have been used[1], but none of them was clearly able to increase and improve daily activities and endurance of teenagers with JIA. Objectives: The study aims to assess the effect on increasing physical activity of a multifactorial protocol including an individualized program to reduce sedentary behavior in routine daily life associated with a personalized endurance enhancement program, administered through telemedicine devices. Methods: Each participant was assessed at baseline (T0), followed by a three-month training program, supervised via telemedicine by a physiotherapist associated with monthly conversations to encourage the identification of individual strategies for increasing physical activity, at the end of which they were reassessed (T3m) by a blinded researcher. Enrolled subjects were then asked to continue the program, independently, without supervision, for additional 3 months, and then again reassessed (T6m). The outcome measures selected were: Habitual Activity Estimation Scale (HAES), 10 Metres Shuttle Walk Test Modified (10MSWT), process-oriented checklist to assess Fundamental Movement Skills (FMS). In addition, two individualized goals were identified for each participant through Goal Attainment Scaling (GAS), the attainment of which was assessed at T6m. For the statistical analysis was used the Mann-Whitney test. Results: Seven teenagers with JIA were recruited (all female, median age 15 years old, range 11-18). HAES score resulted significant increased between T6m and T0 in relation to the total activity during weekdays. The 10MSWT shows a significant improvement between T3m and T0, that was kept at T6m. FMS showed a significant improvement between T6m and T0. All participants achieved the objectives defined with the GAS (Table I). Conclusion: This study reported a positive change in all the observed variables. The program appears feasible and well accepted by adolescents and their families. It allowed participants to get involved in overcoming individual obstacles, thus achieving a greater level of physical activity. It can be assumed that the proposed protocol may represent an integrative proposal in the physiotherapeutic treatment of adolescents with JIA. Further studies, in larger cohort and long-term follow-up, are needed to assess the maintenance the achieved results over tiS. Fedorov 1 , N. Y. Stepanenko 1 , S. V. Feoktistova 2 , S. Salugina 1 , V. Matkava 1 , I. E. Fedorov 2 1 Pediatrics, V.A. Nasonova Research Institute of Rheumatology, 2 Humanities Institute, Russian New University, Moscow, Russian Federation Correspondence: N. Y. Stepanenko Introduction: Monogenic autoinflammatory diseases (AIDs) are systemic diseases leading to severe functional abnormalities of various organs and systems, which may result in a malfunction in the psychological sphere. Objectives: to identify special aspects and differences in the emotional-volitional and communicative spheres in pediatric patients with monogenic AIDs. Methods: 41 children with AIDs were included into the study: FMF - 15 (boys - 8, girls - 7); CAPS - 17 (boys - 9, girls - 8); TRAPS - 9 (boys - 5, girls - 4) aged 7 to 17 years inclusive. There were no significant differences in terms of age between the studied nosologic groups. The diagnosis in all the patients was confirmed on the ground detection of the corresponding pathogenic mutations. To assess the emotional sphere, the following methods were used: a clinical conversation; emotional and communicative sphere (8-color Luscher test; CMAS (adaptation by A. Prikhozhan); Spielberger-Hanin anxiety test, drawings “non-existent animal” and “house-tree-man”). Results: TRAPS: violations in terms of all studied indicators. There were revealed communication disorders (88.9%), a tendency to form neurotic fears (66.7%), reduced social adaptation (55.5%), signs of aggression and a high level of personal anxiety (33.3%). The level of aggression and personal anxiety was higher in girls than in boys (F/M= 50%/20%). Communication disorders and a tendency to form neurotic fears were more characteristic of boys (F/M= 75%/100% and 50%/80%, respectively). FMF: high rates of communication disorders (73.3%), reduced social adaptation (46.7%), neurotic fears and a high level of personal anxiety (26.7%), signs of aggression (13.3%). A reduced level of social adaptation is characteristic of girls (F/M=57.1%/37.5%), boys are more likely to have signs of aggression (F/M=0/25%). Disorders in the communicative sphere are distributed evenly. CAPS: a relatively intact group. The highest indicators compared to other groups were detected only in terms of criterion of susceptibility to form neurotic fears (58.8%). There were also registered communication disorders (41.2%), reduced social adaptation (29.4%), a high level of personal anxiety (23.5%), signs of aggression (5.9%). Girls are more likely to have reduced social adaptation (F / M = 41% / 33.3%), susceptibility to form neurotic fears (F / M = 62.5% / 55.5%), a high level of personal anxiety (F / M =37.5% / 11.1%). Boys are more likely to have communication disorders (F/M= 25%/55.5%) and aggression (F/M= 0/11.1%). Conclusion: most often, disorders in the emotional-volitional and communicative spheres compared with other studied diseases are registered in patients with TRAPS; at that, boys are more likely to have communication disorders and form neurotic fears, while girls are characterized by high level of personal anxiety and aggression. Communication disorders and reduced social adaptation are characteristic for patients with FMF; reduced social adaptation is more common in girls. Patients with CAPS are the most intact in the studied areas, having disorders only in terms of formation of a tendency to neurotic fears, which is more common in girls. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
L. H. Weidlich, R. Hald, S. Reinholdt Paediatric Rheaumatology, Rigshospitalet, 2100 Copenhagen E., Denmark Correspondence: L. H. Weidlich Introduction: Introduction: We have experienced an increase in the number of patients for whom subcutaneous injections at home has become difficult and traumatic for both children and parents. This sometimes results in cessation of treatment and flare-up of arthritis symptoms. Since 2018 we have been developing a structured education program inspired by Julie E. Maclaren’s article “Interventions for paediatric procedure-related pain in primary care” 1 . Objectives: Objectives: To reestablish parent-administered injections at home through an education program. Methods: Methods: Case report of three cases. We used a structured education program to ensure that parents gain necessary knowledge and skills to administer and cope with subcutaneous injections at home. Children 0-18 years with juvenile idiopathic arthritis in subcutaneous treatment and their parents are the target group. The program consists of consultations facilitated by a nurse with dialog, training, and various tools to educate the patient to cope with the pain and the parents to support the child during the procedure. Results: Results: Patient 1: An eight-year-old girl. The subcutaneous injections at home take hours of negotiating and is causing the patient pain. We started with a baseline consultation uncovering the difficulties and then we made some adjustments to the methods used by changing from pen to syringe. Two additional consultations were needed with further, minor adjustments made before the family tried to home administer the medication again. At a follow-up contact three months later the family confirmed that the injections are now easily administered without any physical or psychological difficulties. Patient 2: A thirteen-year-old boy. The boy was nervous, scared, and overthinking the subcutaneous injections. At the baseline consultation we established that using local anaesthetic and a procedure agenda as a tool could be the solution for him. The procedure agenda was tested in the paediatric department successfully and the procedure lasted less than ten minutes. The boy did not find it too painful and after three additional consultations where same agenda was used, they were ready to try giving the subcutaneous injection at home again. At a follow-up contract three months later, the family confirmed that giving the subcutaneous injections still was working well at home. Patient 3: A seven-year-old boy. The boy cannot cooperate and getting medicine has become impossible. At the baseline consultation we established that using Nitrous Oxide and local anaesthetic could make a difference. The plan was successfully used in the outpatient clinic. The boy is challenged due to an attention deficit hyperactivity disorder. The family have tried giving the subcutaneous injection at home, but it did not work out, so he is still getting the subcutaneous injections in the outpatient clinic but without using Nitrous Oxide and we are working towards mowing the treatment home again. Conclusion: Conclusions: Facilitated using a structured educational program we re-established the home administered subcutaneous treatment for two of the patients and the last patient is one his waN. Yothakol 1 , S. Chantaratin 2 , M. Sukharomana 1 , S. Charuvanij 1 1 Division of Rheumatology, Department of Pediatrics, 2 Division of Child and Adolescent Psychiatry, Department of Pediatrics, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand Correspondence: N. Yothakol Introduction: Juvenile idiopathic arthritis (JIA) is chronic inflammatory arthritis in children that can cause significant physical, social functioning and psychological impairment. Common psychological problems in childhood and adolescents are depression and anxiety disorders, especially those with chronic illnesses. Objectives: The aims of study were to describe prevalence of depression and anxiety and to investigate factors associated with depression and anxiety in patients with JIA. Methods: A cross-sectional study was conducted in JIA patients, aged between 8-17 years at the Pediatric Rheumatology clinic, Faculty of Medicine Siriraj hospital, Mahidol University, Bangkok, Thailand from July 2020 to April 2021. JIA was classified by the International League of Association for Rheumatology criteria. The demographic data, JIA variable core sets and juvenile arthritis disease activity score were collected. The depression and anxiety were assessed by the Thai version of Children’s Depression Inventory (CDI) and the Screen for Child Anxiety Related Disorders (SCARED) questionnaires. The CDI score ≥ 15 was defined as significant depressive symptoms. The SCARED score of ≥ 25 indicated the clinically significant anxiety. Results: Seventy patients with JIA; 39 males (55.7%) and 31 females (44.3%) were included in this study. The mean age (SD) at the time of assessment was 12.3±2.6 years. The JIA subtypes were as follows: 26 (37.1%) patients with enthesitis-related arthritis, 19 (27.1%) patients with systemic JIA, 8 (11.4%) patients with polyarticular JIA rheumatoid factor (RF)+, 7 (10%) patients with polyarticular JIA RF-, 6 (8.6%) patients with oligoarticular JIA and 4 (5.7%) patients with undifferentiated JIA. The median follow-up time was 38 (IQR 10-67.2) months. Fifty-one (72.9%) patients had inactive disease. The anxiety symptoms were found in 35 (50%) patients; 26 (74.3%) patients had separation anxiety symptoms, 13 (34.3%) patients had social anxiety symptoms and 11 (31.4%) patients had school anxiety symptoms. SCARED score had significant positive correlation with Childhood Health Assessment Questionnaire score (r=0.311, p =0.009). Eight (11.4%) patients had depressive symptoms. Receiving biologic treatment was significantly associated with depressive symptoms at adjusted OR 18.08 (95% CI: 2.28-143.29), p =0.006 by multivariable logistic regression analysis. Additionally, CDI score had significant positive correlation with SCARED score ( r =0.526, p =<0.001). Conclusion: Half of patients with JIA had mood symptoms. Anxiety was more prevalent than depression. Psychological assessment and appropriate interventions are necessary as part of clinical care for patients with JIA. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
P426. Parent views on telemedicine in pediatric rheumatology: a survey study P427. Comparison the effectiveness of online aerobic dance exercises versus physical activity counseling in patients with childhood familial mediterranean fever P428. Is it possible to evaluate the immediate effects of postural correction and weight-bearing training on plantar pressure and balance using pedobarography in a patient with juvenile idiopathic arthritis and obstetric brachial plexus palsy? P429. Acceptability, practicality, and accuracy of the Turkish translation of video pgals in Turkish children P430. Experience of using a mobile health application as communication tool between patients and healthcare professionals P431. PGALSPLUS: a tool to facilitate assessment and identify exemplar musculoskeletal conditions P432. Paediatric Musculoskeletal Matters (PMM) - evidence-based e-resource providing the fundamentals of paediatric MSK medicine knowledge and skills for the international context Kavrul Kayaalp, F. G. Demirkan, Ö. Akgün, A. Tanatar, F. Çakmak, N. Aktay Ayaz Pediatric Rheumatology, İstanbul University, Faculty of Medicine, İstanbul, Turkey Correspondence: N. Aktay Ayaz Introduction: The expanding use of telemedicine due to the emerging needs for remote visits after the COVID-19 pandemic has led many patients with chronic diseases to seek alternative ways for follow-ups, even beyond the pandemic era. Objectives: It is aimed to investigate the demands and opinions of the parents of children with rheumatic diseases towards telemedicine applications and to examine the factors influencing telemedicine preference. Methods: A single-center, cross-sectional, web-based survey study was conducted in a university hospital. The target group was the parents of the children with rheumatic diseases of any kind. The survey included a total of 45 questions that were grouped into four different sections. Sections were on (1) sociodemographic data, (2) disease-related characteristics, (3) transportation to the clinic, (4) use of the internet, and (5) opinions on telemedicine. Results: A total of 205 parents have completed the survey. Diagnoses of the patients were periodic fever syndromes and autoinflammatory diseases (56.1%), juvenile idiopathic arthritis (28.8%), systemic connective tissue diseases (9.7%), and vasculitis (5.4%). The most common way of transportation was public transport (77%). Only 44.9 % of patients were able to arrive at the clinic in less than one hour. Of the parents, 84.9% use the internet for social media, 45.9% for the medical information, daily. An application that enables video calling was available on the smart devices of 77.5% of the parents. Only 3.8% of parents had previous telemedicine experience. Thirty percent of respondents missed a follow-up appointment at least once over the last year. The most common reasons for missing an appointment were the fear of COVID-19 exposure (29.6%), missed school days (14.8%), and unwillingness to come to the hospital for follow-up when the child had no complaints (12.3%). Of the parents, 70.7% stated that they would prefer telemedicine if it becomes available. Sixty-five percent of parents reported that telemedicine examinations can be useful for routine check-ups when their children have no complaints, 64% when they have new complaints, 49% when they want to reach their physicians to ask questions about their diseases, and 47% for a few times when it is not possible to come to the outpatient clinic. The frequency of telemedicine preference was found to be related to the education level of the parents (The preference rate increases with the level of education) (p=0.025), missing at least one outpatient clinic visit over the last year (p=0.002), and daily use of the internet for social media (p=0.035). The most common reasons for preferring telemedicine over face-to-face examinations were reducing missed school days (74.1%), reducing the risk of COVID-19 exposure (73.2%), and saving time (66.3%). Easier reach to the physician (67.3%) and more regular follow-ups without misses (66.8%) were the most commonly reported advantages. Although the telemedicine preference rate was high, the rate of believing that parents could receive the same quality of care as they were examined face-to-face remained at 36.1%. Of respondents, 38% have concerns about a wrong treatment decision, and 24.9% about the privacy of personal data during telemedicine visits. Conclusion: Telemedicine applications are demanded by the parents of children with rheumatic disease. It can be considered as an alternative option, especially in the follow-up of patients who do not come to their appointments regularly. An increase in telemedicine experience in the field of pediatric rheumatology may provide a better evaluation of the effectiveness of telemedicine methods. Disclosure of Interest : None declared
A. Albayrak 1,2 , N. Arman 3 , E. Tarakci 3 , O. Kasapcopur 4 1 PhD Program of Physiotherapy-Rehabilitation, Graduate Education Institute, Istanbul University-Cerrahpasa, 2 Department of Physiotherapy and Rehabilitation, Istanbul Kent University, Faculty of Health Science, 3 Department of Physiotherapy and Rehabilitation Cerrahpaşa Medical School, Istanbul University-Cerrahpaşa, Istanbul, Turkey Correspondence: A. Albayrak Introduction: Familial Mediterranean Fever (FMF) is the most common autoinflammatory disease that its characterized by self-limiting attacks of fever, serosal inflammation and arthritis generally developing in childhood. Symptoms associated with FMF in children and adolescent include pain, fatigue, muscle weakness, myalgia that results in activity and participation restrictions. Physical activity and exercise are key components in management of children and adolescents with rheumatism. Objectives: The aim of the study was to compare the effectiveness of the online aerobic dance exercises (OADE) and physical activity counseling (PAC) program with the PAC program in patients with childhood FMF (cFMF). Methods: 20 patients (8-18 years) with cFMF were taken account in the study. Randomly, related patients were divided as Group I (OADE and PAC), and Group II (PAC). In the Group I, the aerobic dance exercises program was performed online for 8 weeks, three days a week, and 60 minutes a day. General physical activities were recommended, and motivational interviews were organized for both groups. The functional capacity of the patients by the 6-Minute Walk Test, the physical fitness by the FitnessGram Physical Activity Test Battery, the fatigue symptoms by the PedsQL Multidimensional Fatigue Scale, pain at rest by the Visual Analog Scale, the quality of life by the Familial Mediterranean Fever Quality of Life Scale were evaluated. All assessments were performed before and after treatment. Results: Half of the patients were female and the mean age was 13.35±3.45 years. After treatment aerobic capacity, and physical fitness level improvements were observed in Group I. Additionally, the mean change in walking distance in Group I was 64±58.63 meters. Also, the improvements in physical fitness and pain scores after treatment were statistically significant in Group II. (p<0,05). In the comparison between groups, the changes in all parameters after treatment were not statistically significant, except for the increase in pain scores in Group II (p>0,05). Conclusion: This study is the first study to pay attention physical activity and exercise programs in patients with cFMF. It has been found that OADE and PAC program provide clinically significant gains in terms of functional capacity and physical fitness, and provide effective results on pain compared to only PAC program and it has been shown that online aerobic dance and physical activity counseling are applicable in the treatment of cFMF. Disclosure of Interest : None declared
N. Arman 1 , F. G. Demirkan 2 , N. Aktay Ayaz 2 1 Department of Physiotherapy and Rehabilitation, Faculty of Health Science, Istanbul University-Cerrahpasa, 2 Department of Pediatric Rheumatology, İstanbul Faculty of Medicine, İstanbul University, Istanbul, Turkey Correspondence: N. Arman Introduction: Juvenile idiopathic arthritis (JIA) is the most frequent chronic rheumatic disease of childhood with arthritis of one or more joints accompanied by various musculoskeletal problems. The literature shows that the pedobarography is useful for the examination of foot biomechanics. Objectives: The aim of this case study waspostural control using pedobarography in a patient with JIA and obstetric brachial plexus palsy (OBPP). Methods: We present a case of a 16-year-old boy, born to parents related by first-degree consanguinity and was diagnosed with polyarticular juvenile idiopathic arthritis at the age of 3 years. He has not received any medication for about 4 years. There was no history of trauma and no family history of rheumatic disease. The patient had muscle weakness and deformity in the right arm due to a fracture of the clavicle and OBPP during the delivery process. On examination, arthritis was found in the left 2nd proximal interphalangeal joint and bilateral elbows, knees, wrists and ankles. There were no significant abnormalities in other systems. It was also observed that the patient had been in a depressive mood for a while due to decreased mobilization due to deformities and pain. He can walk normally, with no further complaint of joint pain. However, he reported complaints of loss of balance and tires while walking. He cannot use his right arm in daily life activities, he prefers to keep his right hand in his pocket while walking most of the time. Besides, no arm swing and kyphotic posture were observed during walking. The patient, who had never exercised before, was given a correct posture and weight transfer exercises training including proprioceptive cues and visual imagery to increase the patient’s balance and postural control skills for 20 minutes. Pedobarographic analysis including static, dynamic and balance tests was performed before and 15 minutes after training. It was utilized for the evaluation of static analysis parameters; surface, load % and P.max for foot, dynamic loading parameters; max load %, avg. speed and duration of phase for the gait cycle as dynamic analysis, evaluation of balance parameters; sway surface, sway length and average speed mm/s for sway test. Results: The results of the two assessments were presented in Table 1. Although no significant change was obtained in the static analysis results, clinically significant results were found in the dynamic analysis and Sway Test. Conclusion: We found clinically improvements in plantar pressure and postural control as an immediate effect after exercise training in a patient with JIA and OBPP. These results may indicate that the patient learned to use his muscles correctly due to the postural awareness gained by the exercise training. Therefore, we consider that the pedobarographic analysis can be a useful tool to objectively identify patient’s problems, make a decision, choose exercises that he/she needs to do and follow the patient’s improvements for the physiotherapists, especially when the patient has complex problems in pediatric rheumatic disease. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
Z. Balik, Y. Bayindir, M. Kasap Cuceoglu, S. Sener, E. Aliyev, O. Basaran, Y. Bilginer, S. Ozen, E. D. Batu Turkey Correspondence: Z. Balik Introduction: Telemedicine practices have been adopted in most departments including pediatric rheumatology during the coronavirus pandemic. pGALS (Pediatric Gait, Arms, Leg and Spine/Pediatric Gait, Arms, Leg and Spine) is a simple and practical method developed to evaluate the musculoskeletal system of children. Video pGALS is the form of this examination performed on video to enable the follow-up of children with rheumatic diseases to continue without interruption during the pandemic restrictions. Objectives: This study aims to evaluate the validity, applicability, and accuracy of the Turkish translation of the video pGALS. Methods: Between April and May 2022, patients aged 4-18 years, who were followed up in Hacettepe University Pediatric Rheumatology Department and agreed to participate in the study, were included. The patient’s demographic data, diagnosis, complaints and physical examination findings were noted. Video pGALS was performed by a pediatric rheumatologist. A link has been created via zoom for this examination. While the doctor was connected to the link on the computer in one room, the patient’s parent was connected to this link in the other room. Normal hands-on physical examination was also performed after video PGALS, which formed the gold standard for positive findings. The acceptability (in terms of time and additional distress) of the applied examination method by the patient/patient relative was measured with a visual analog scale (VAS) created with smiling faces. Pain was evaluated with a score between 0-10, 10 being the worst. Results: A total of 102 patients, 56 girls (54.9%) with a median age of 12.41 (min-max; 4.23-17.84) years were included in the study. The median age was 12.41 (min-max; 4.23-17.84) years. Twenty-two (21.6%) patients had active complaints. The most common diagnosis was juvenile idiopathic arthritis (JIA) (25.5%), followed by familial Mediterranean fever (FMF) 19 (18.6%). The median (min-max) pain score was 4 (1-8). The median duration of pGALS examination was 7 minutes (5-13 minutes). Hands-on physical examination was abnormal in 28 (27.4%) patients, whereas video pGALS was abnormal in 25 (24.5%) patients. In 3 (2.9%) patients with abnormal physical examination, we could not detect these findings with video pGALS. These patients had scoliosis, pes planus, and striae on the thigh. All those who were abnormal in video pGALS were also abnormal on physical examination. The median (min-max) VAS score given by children and parents for acceptability of pGALS in terms of duration was 0 (0–6) for both and in terms of additional discomfort was 0 (0–5) for both. The majority of patients/parents found the duration acceptable (98%/99%, respectively) and reported that pGALS caused little or no discomfort (98%/99%, respectively). The sensitivity and specificity of video pGALS for detecting musculoskeletal abnormalities were 93.7% and 100%, respectively. Conclusion: The video pGALS tool is a quick, easy, and acceptable tool for evaluating musculoskeletal problems in children. The Turkish version of video pGALS was acceptable in terms of duration and additional discomfort. It can detect abnormalities and may be used forReferences: 1. Susan Shenoi, Kristen Hayward, Megan L Curran, et al . Telemedicine in pediatric rheumatology: this is the time for the community to embrace a new way of clinical practice. Pediatr Rheumatol Online J. 2020 Oct 31;18(1):85. 2. Ezgi Deniz Batu, Özge Keniş Coşkun, Hafize Emine Sönmez, et al . Acceptability and Practicality of the Turkish Translation of Pediatric Gait Arm Legs and Spine in Turkish Children. J Clin Rheumatol. 2017 Dec;23(8):421-424M. Doeleman, S. de Roock, G. de Joode-Smink, J. Swart, N. Wulffraat Pediatric Immunology and Rheumatology, University Medical Center Utrecht, Utrecht, Netherlands Correspondence: M. Doeleman Introduction: E-health and mobile applications are increasingly popular in paediatric rheumatic diseases. These tools can provide a way to monitor symptoms remotely and communicate with patients or healthcare professionals asynchronously. At our hospital, we have used such a mobile e-health application, Reuma2Go, during daily clinical practice since June 2016. The app allows users to track JIA-related symptoms such as joint pain, fatigue, morning stiffness and daily activities, and gives patients the ability to communicate with our healthcare team through text messages for advice or questions regarding their disease management. Objectives: Evaluate app engagement of users of the mobile application during daily clinical practice from 2018 to 2021. Assess the frequency of relevant topics in app conversations between patients and the healthcare team. Methods: Data was collected via the mobile E-health application (Reuma2Go) from January 2018 to August 2021 as pseudonymized data. App engagement was analysed using descriptive statistics. Text message analysis was performed using the quanteda package in R. Relevant topics for analysis were selected based on overall document-term frequency. Topic frequency was estimated using a dictionary search with topic-specific keywords. Results: From January 2018 to August 2021, a total of 398 unique patients with JIA created an account within the mobile application. In August 2021, 62% of JIA patients visiting the outpatient clinic were using the mobile application. The app was used to track symptoms on average 3291 times a year. User activity each year was highest from March until May and lower during winter and summer school holidays (December-January and July-August, respectively). Over the study period, a total of 1572 conversations were initiated by patients consisting of 4546 messages between patients and the healthcare team. During the first COVID lockdown in the Netherlands, the number of users increased 43% (from 204 to 292) and messaging frequency via the app doubled compared to the same period in the previous year. Topic frequency for the most prevalent conversation topics are presented in Table 1. Conclusion: These results demonstrate our experience of using a mobile health application during daily clinical practice. Topic analysis of conversations between patients and the healthcare team illustrates specific information needs of patients visiting the outpatient clinic. Frequent topics discussed with the healthcare team were JIA-related symptoms and pain, medication, appointments, and vaccination/COVID. During the pandemic, the mobile application has allowed us to monitor our patients remotely and communicate with them asynchronously through in-app text messages. This has provided a continuity of care and maintenance of disease control without the requirement of physical appointments. Furthermore, the app allows patients and caregivers to be more actively involved in their disease management. Disclosure of Interest : None declared
V. Mercer 1,2 , N. Smith 1 , S. Jandial 1,3 , H. Foster 4 1 Newcastle University, Newcastle upon Tyne, 2 South Tyneside and Sunderland NHS Foundation Trust, South Shields, 3 Paediatric Rheumatology, Great North Children’s Hospital, 4 Paediatric Rheumatology, Newcastle University, Newcastle upon Tyne, United Kingdom Correspondence: V. Mercer Introduction: Musculoskeletal (MSK) problems in children and young people (CYP) are common and often present to healthcare professionals (HCPs) in primary care and community settings who may not be trained in MSK assessment. Whilst many presentations in CYP are benign and self-limiting, HCPs need to be able to identify serious disease and have guidance on appropriate referrals to minimise delay in diagnosis. pGALS (paediatric Gait, Arms, Legs and Spine) was developed for HCPs to guide them through a simple, quick MSK assessment and has been shown to detect joint and functional problems but doesn’t provide guidance on diagnosis. We aimed to widen the scope of pGALS to support detection of a range of serious MSK conditions and support onward referral pathways Objectives: To create a pGALSplus assessment and assess its feasibility and acceptability in CYP with healthy controls (HC) and exemplar conditions to reflect a spread of MSK pathologies with development based on HCP and patient/carer feedback. Chosen exemplar conditions; Juvenile Idiopathic Arthritis (JIA), Mucopolysaccharidoses (MPS), Muscular Dystrophy (MD), Developmental Coordination Disorder (DCD) Methods: 3-phase mixed methods approach; Phase 1;scoping review of literature and qualitative interviews with expert HCPs focused on key clinical assessments used to inform diagnosis and progress. Results informed the initial ‘pGALSplus’ assessment with iterative development in Phase 2 with an expert working group (including paediatric rheumatologists and physiotherapists, neuromuscualr specialists). Phase 3; testing of pGALSplus in the exemplar condition groups by the research physiotherapist (VM) with feedback from HCPs, CYP and carers, and further validation through expert consensus (international e-survey [n=22] and a virtual dissemination event [n=13]) Results: Phase 1 data identified key additional requirements for pGALSplus through literature review and expert opinion. These were added to the pGALS examination to produce a ‘toolkit’. Phase 2; discussion with experts in MSK assessment of CYP, consensus allowed refinement and agreement on how pGALSplus would be used in practice. The agreed toolkit is colour-coded to facilitate identification of exemplar MSK conditions. Phase 3; testing of pGALSplus on 46 CYP (JIA n=10; MPS n=6; MD n=9; DCD n=10; HC n=10). Feedback from the clinician performing pGALSplus demonstrated the assessment to be achievable in the target age range (2-10 years), and quick (mean = 12.6 minutes to complete) with high levels of acceptability from patients and carers. Overall expert feedback (international e-survey; n=22 and stakeholder event; n=13) was positive and enabled iterative development of pGALSplus. Responders deemed pGALSplus to be a very useful addition to pGALS as it enables further assessment relevant to the practice of non specialists and those new to the paediatric specialty. The final pGALSplus comprises 26 manoeuvres as a toolkit with a colour coding approach and signposting to additional resources Conclusion: Our work has demonstrated pGALSplus to be a novel evidence and consensus-based toolkit to support differentiation between key MSK conditions in CYP with high acceptability and feasibility. As community-based MSK assessment in CYP becomes more established, pGALSplus aims to increasingly facilitate appropriate assessment and inform decision making about onward referral. A visual animation of pGALSplus and resources to aid HCPs to undertake the assessment are being developed and will be free and openly available on PMM (www.pmmonline.org) Patient Consent: Yes, I received consent Disclosure of Interest : None declared
N. Smith 1 , S. Jandial 2 , C. Scott 3 , H. E. Foster 4 on behalf of on behalf of the PMM Editorial Board 1 Translational and Clinical Research Institute, Newcastle University, 2 Paediatric Rheumatology, Great North Children’s Hospital, Newcastle Upon Tyne, United Kingdom, 3 Paediatric Rheumatology, Red Cross War Memorial Children’s Hospital, Cape Town, South Africa, 4 Emerita Professor Paediatric Rheumatology, Newcastle University, Newcastle Upon Tyne, United Kingdom Correspondence: N. Smith Introduction: The PMM Portfolio (www.pmmonline.org) is a free and openly available online resource encompassing the Paediatric Musculoskeletal Matters (PMM) website [1], the paediatric Gait, Arms, Legs and Spine (pGALS) app and e-learning modules (ELM) to support teaching and learning about the essentials of paediatric musculoskeletal (MSK) knowledge and skills. The target learner audiences are non-specialists in paediatric MSK medicine. Evaluation of PMM [2] demonstrated positive feedback and worldwide reach including areas with limited access to paediatric MSK specialists. PMM has recently moved to the permanent guardianship of PReS and will remain free and open to all. The transfer enables PMM to work closer with PReS and the Global Task Force to reach new prospective audiences, support educational activities and facilitate growth of paediatric rheumatology around the world. Objectives: To produce an agreed template of collated ‘Core topics’ to meet the perceived needs of target audiences for a ‘Basic Course’ in paediatric MSK medicine and use this to inform the next iteration of PMM to maximise Course support. Methods: The PMM Editorial Board was formed from health care professionals in paediatric rheumatology and orthopaedics including medical and allied health representatives from the Global Task Force. ‘Core topics’ for a paediatric MSK ‘Basic Course’ were collated from the following sources (1) programmes of previous PReS Basic Courses, and existing e-learning programmes in paediatric MSK medicine; (2) previous work assessing the level of paediatric MSK knowledge and skills needed by graduating doctors, primary care doctors and general paediatricians [3-5] and (3) feedback from the recent PMM evaluation [2]. A consensus approach with the PMM Editorial Board agreed the ‘Core Topic’ and focused on the global context and especially low resource settings. The ‘Core topic’ list informed a Mapping Exercise across the whole PMM Portfolio to identify ‘gaps’ or where updates to PMM were needed. Results: An agreed template of collated ‘Core topics’ included: (1) Epidemiology and impact of MSK conditions; (2) The importance of MSK wellness across the life course; (3) Clinical assessment and normal MSK development; (4) Approaches to clinical scenarios; (5) MSK features of red flag conditions; (6) The spectrum of Arthritis; (7) Mimics of inflammatory arthritis; (8) Multisystem conditions; (9) Orthopaedic conditions; (10) Management overview; (11) Work of the Paediatric Global MSK Taskforce; (12) Guidelines and Recommendations; (13) The WHO Essentials Medicine List; (14) The PMM Portfolio and how to use it; (15) Setting up MSK clinical services; (16) Case presentations. The Mapping Exercise informed where amends and updates are needed for PMM content to be developed by the PMM Editorial Board. Conclusion: Work is underway to launch a further iteration of PMM to support Basic Courses in paediatric MSK Medicine. PMM will continue to be a valuable e-resource to reach target audiences and support the growth of paediatric rheumatology. References [1] Smith et al. Ped Rheum 2016;14(1):1 doi.org/10.1186/s12969-015-0062-4. [2] Smith et al. Ped Rheum 2021;19(85) doi.org/10.1186/s12969-021-00567-5 [3] Goff et al. Educ Prim Care 2014;25(5):249-56 doi.org/10.1016/j.jpeds.2009.10.047. [4] Jandial et al. BMC Med Educ 2015;15:171 doi.org/10.1186/s12909-015-0449-4. [5] RCPCH 2018. www.rcpch.ac.uk/sites/default/files/2018-03/rcpch_curriculum_general_paediatrics_2018_syllabus_final.pdf433. Health-related quality of life of Uzbek children with juvenile idiopathic arthritis P434. Evaluation of functional capacity, pain, fatigue, physical activity and quality of life in children and adolescents with Familial Mediterranean FP435. The validity and reliability of the turkish version of pain catastrophizing scale-children inP436. The validity and reliability of the Turkish version of pain catastrophizing scale-parent in parents of adolescents with Familial Mediterranean Fever P437. A novel multidimensional questionnaire to monitor juvenile fibromyalgia syndrome and identify factors influencing the disease course P438. Residual complaints in juvenile idiopathic arthritis patients with inactive disease P439. Determination of gait normality through dynamic baropodometry in healthy adolescents P440. Using Virtual Reality (VR) to help children cope with joint puncture related to JIA P441. Impact of oral and subcutaneous methotrexate on the quality of life of children with juvenile idiopathic arthritis S. Akhmedova 1 , A. Ibragimov 2 , I. Kasimova 2 , K. Saipova 1 , D. Akhmedova 2 , S. Ouba 1 , A. Tomoyuki 1 , S. Aya 1 , J. Tanaka 1 1 Graduated school of biomedical and health sciences, Hiroshima university, Hiroshima, Japan, 2 Pediatric cardiology and rheumatology, Republican specialized scientific practical medical center of Pediatrics, Tashkent, Uzbekistan Correspondence: S. Akhmedova Introduction: Estimated over 2 million are currently living with (< 16 year olds) with JIA. Disease prevalence was greatest in Asia (South Asia), followed by Africa, Americas, Europe and Oceania. Due to the lack of data from Uzbekistan this study conducted in Uzbek JIA patients. As JIA influences all aspects of the child’s life and family, achievement of an optimal health-related quality of life (HRQoL) is an important goal in clinical care. HRQoL is a complex, multidimensional concept that encompasses physical, emotional, social, and behavior-related well-being and functioning. Higher health-related quality of life is important to properly administer children with Juvenile idiopathic arthritis. Objectives: Research aim is to study factors affecting HRQoL of Juvenile idiopathic arthritis (JIA) patients in Uzbekistan. Methods: The study sample includes 67 JIA patients (ages 2 –18 years) who hospitalized to the Republican specialized scientific practical medical center of Pediatrics in Tashkent, Uzbekistan between September, 2021 and January 2022. Children with JIA properly were questioned about their HRQoL using the Pediatric Quality of Life Inventory 4.0 (PedsQL). Functional ability was measured using the Childhood Health Assessment Questionnaire (CHAQ), and medical, sociodemographic parameters (number of swollen joints, visual analogue scale (VAS) activity, physician global assessment (PGA), erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP)) were assessed. Associations between the biological, clinical factors and HRQoL were analyzed by correlation and linear regression analysis in JMP 16 statistical software and GraphPad Prisme 6.0. Results: The average age of the patients is 10±4.12, and 54% are female. The median PedsQL is 68.4 (53.3-77.7), median CHAQ is 1 (0.4-1.75). Higher pain level, lower well-being, higher disability index, higher number of swollen joints were significantly associated with an unfavorable (suboptimal) HRQoL (PedsQL total < 79.3). By child total self reports 76.7% (n=46) children from 67 show suboptimal HRQOL. No significant difference were found between parent and child reports. Conclusion: Using the methods of assessing the health-related quality of life, we were convinced that children with juvenile idiopathic arthritis in Uzbekistan are not treated at the same level as other high-income countries. This kind of methods assist correct treatment and monitoring of patients. It is important to look for these risk factors in clinical practice to be able to set the course at an early stage of the disease with targeted support measures. This study needs a period of follow up to compare effectiveness of the targeted treatment over the period and inclusion of healthy peers to compare for more accurate results. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
, Istanbul University-Cerrahpaşa, Faculty of Health Science, 4 Department of Pediatric Rheumatology, Musculoskeletal findings are common in childhood Familial Mediterranean Fever (cFMF). Children and adolescents with rheumatic diseases experience significant disabilities due to pain, fatigue, decreased joint motion, muscle impairment and joint stiffness. A comprehensive and appropriate assessment is essential for planning physical activity and exercise programs in these patients. Objectives: The purpose of the study was to evaluate the functional capacity, pain, fatigue, physcial activity and quality of life in patients with cFMF. Methods: Twelve patients (aged 8-18 years) with cFMF were included in the study. 6-Minute Walk Test (6MWT) was used to evaluate the functional capacity. Visual Analog Scale (VAS) with 100 mm was used to evaluate pain intensity during rest. The PedsQL Multidimensional Fatigue Scale (PedsQL-MFS) was used to evaluate the fatigue symptoms. Physical Activity Questionnaire (PAQ) was used to evaluate physical activity levels. Familial Mediterranean Fever Quality of Life Scale (FMF-QoL) was used to evaluate the quality of life. Results: Half of the patients were female and mean age was 13.35±3.45 years. The mean of scores of 6MWT was 595.10±97.50 meters, the mean of PedsQL-MFS scores was 67.07±21.82, the mean of PAQ scores 1.97±0.89 and the mean of FMF-QoL was 29.45±15.12. When the patient’s 6MWT scores were compared with the normal values according to age and sex, %85 of the patient’s 6MWT scores were lower than the scores of the healthy children and adolescents. Only two patients reported musculoskeletal-related pain (The patients VAS scores; 25 and 57 points). Also, the physical activity levels of the patients was categorized “low-active” in %80 of them, and “sufficiently-active” in %20 of them. Conclusion: This study confirmed that the functional capacity was lower in patients with cFMF compared to their healthy peers. Besides physical inactivity, fatigue and impaired quality of life may be important factors that contribute to the worsening of their clinical condition by causing a vicious cycle in these children and adolescents. A more comprehensive evaluation of all these symptoms, especially pain may be helpful in setting a treatment plan for the patients with cFMF. Future research should focus on both physical and psychosocial factors in cFMF for understanding the vicious cycleS. Savci 4 , E. Unsal 2 1 Department of Physiotherapy and Rehabilitation, Faculty of Health Sciences, Izmir Katip Celebi University, 2 Division of Pediatric Rheumatology, Department of Pediatrics, 3 Department of Biostatistics and Informatics, 4 Faculty of Physical Therapy and Rehabilitation, Dokuz Eylul University, Izmir, Turkey Correspondence: D. Bayraktar Introduction: Familial Mediterranean Fever (FMF) is an auto-inflammatory chronic condition presenting with fever and pain attacks. Due to the unpredictable nature of the FMF, children with FMF may develop a catastrophizing behaviour towards pain. Thus, evaluating pain catastrophizing in children with FMF is important. Objectives: The aim of the present study was to investigate the validity and reliability of Turkish Pain Catastrophizing Scale-Children (PCS-C) in adolescents with FMF. Methods: PCS-C is a 13-item questionnaire (total scores range 0-52) where higher scores indicate higher levels of pain catastrophizing. PCS-C were translated into Turkish according to established guidelines. The Turkish form of PCS-C were filled up by adolescents with FMF (13-18 years). Pain levels were measured by using visual analog scales (VAS, 0-100 mm) at rest and during activity. Quality of life was evaluated by using Turkish Pediatric Quality of Life Inventory (PedsQL) 3.0 Arthritis Module. Participa65 adolescents with FMF (mean age= 15.4±1.4 years, mean body-mass index= 20.9±3.4 kg/m 2 , 36 female) were included in the study. The average time since onset of the symptoms and time since diagnosis were 100±54 months and 77±57 months, respectively. Pain levels were 30.5±30.4 mm for rest, and 47.4±34.4 mm for activity. Average PedsQL score was 80.3±13.9. Average PCS-C score was 22.5±12.9. The interval validity was excellent (Cronbach’s alpha= 0.938). Twenty-three participants (35%) responded to recalls, and test-retest reliability was good [ICC= 0.893 (95% CI= 0.766/0.953)]. Pain catastrophizing was found related to body-mass index, pain, and quality of life (p< 0.05, Table). Conclusion: Turkish version of PCS-C seems a valid and reliable tool for measuring pain catastrophizing of adolescents with FMFD. Bayraktar 1 , D. C. Sarac 1 , O. Altug Gucenmez 2 , B. Makay 2 , P. Keskinoglu 3 , N. Ilcin 4Pain catastrophizing may be a challenging problem in children with chronic pain conditions. Familial Mediterranean Fever (FMF) is a chronic disease with recurrent and unpredictable pain attacks which may lead to pain catastrophizing. The attitude of parents is important for these children who are coping with chronic pain. Thus, evaluating the views of the parents of children with FMF regarding pain catastrophizingParent (PCS-P) in the parents of the adolescents with FMF. Methods: PCS-PP were translated into Turkish according to established guidelines. The Turkish form of PCS-P were filled up by the parents of the adolescents with FMF (13-18 years). The views of the parents regarding their children’s pain levels and quality of life were measured by using visual analog scales (VAS, 0-100 mm), and Parent form of the Turkish Pediatric Quality of Life Inventory (PedsQL) 3.0 Arthritis Module, respectively. Parents were called by phone approximately a week later to evaluate the test-retest validity. Internal validity, test-retest reliability, and convergent validity were evaluated by using Cronbach’s alpha value, intra-class coefficient (ICC) value with 95% confidence intervals (95% CI), and Pearson correlation coefficients, respectively. Results: A total of 57 parents (mean age= 41.7±5.2 years, 45 mothers) were included in the study. The parents’ views regarding their children’s pain levels were 36.1±32.9mm for rest, and 49.7±36.1 mm for activity. Average Parent PedsQL score was 79.7±15.5. Average PCS-P score was 31.2±12.4. The interval validity was excellent (Cronbach’s alpha= 0.932). Twenty-three parents (40%) responded to recalls, and test-retest reliability was good [ICC= 0.887 (95% CI= 0.751/0.950)]. Parents views regarding pain catastrophizing was found related to their opinions about the pain levels and to quality of life of their children (p< 0.05, Table). Conclusion: Turkish version of PCS-P seems a valid and reliable tool for measuring the views of the parents of the adolescents with FMF regarding pain catastrophizing. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
C. Lavarello 1,2 , A. Alongi 3 , B. Mori 1 , A. Ronchetti 4 , L. Nobili 2,5 , L. Chiarella 2,5 , E. Pescio 6 , A. Ravelli 2,7 , M. Gattorno 1 , C. Malattia 1,2 1 Pediatric Clinic and Rheumatology Unit, IRCCS Giannina Gaslini, Genova, Italy, 2va, 3 Pediatrics, ARNAS Civico-Di Cristina-Benfratelli, Palermo, 4 Physical Medicine and Rehabilitation Unit, 5 Child Neuropsychiatry Unit, 6 Psychology Unit, 7 Scientific Direction, IRCCS Giannina Gaslini, Genova, Italy, Genova, Italy Correspondence: C. Lavarello Introduction: Juvenile Fibromyalgia Syndrome (JFS) is a disabling condition characterized by musculoskeletal pain, fatigue, sleep and mood disturbances. Treatment is multidisciplinary and include exercise, cognitive behavior therapy and medications as appropriate. Currently, evidence for treatment efficacy is limited due to the lack of validated outcome measures. TheJuvenile Fibromyalgia Multidimensional Assessment Report (J-FiMAR) evaluates symptoms severity (pain, fatigue, headache, sleep and mood disorders) by a numerical rating scale, quality of life and functional ability. Objectives: To explore the suitability of the J-FiMAR to monitor JFS. To identify factor influencing clinical course and predictors of treatment efficacy. Methods: We included JFS patients followed at our center between 2018 and 2022. At each 3-6 months follow-up visit, patients filled J-FiMAR. Patient’s and physician’s global assessment (PGA and PhGA) and ongoing treatment were also recorded. Agreement between PGA and PhGA was evaluated by the concordance correlation coefficient (CCC). Relationships between PGA and PhGA, patient-reported symptoms, quality of life and functional capacity were tested by using the Spearman rank-order correlations and partial correlation networks. Mixed effects logistic regression models with random subject-specific intercepts were used to assess the effect of excerice and medications on disease course (improved/not improved) and to identify predictors of treatment efficacy. Results: We included 51 patients (44 female; median disease duration 1.8 years). Patients were on regular physical activity in 83/194 (43%) follow-up visits and received drugs in 70/194 (36%).PGA and PhGA showed poor agreement (CCC 0.60, 95%Cl 0.53-0.67). A strong correlation was found between PGA and pain (r = 0.68, 95%Cl 0.59 -0.76), fatigue (r = 0.62, 95%Cl 0.52-0.71) and depression (r = 0.60, 95%Cl 0.48-0.69). Based on partial correlation networks, fatigue seems to have a key role in predicting the presence of other JFS-related symptoms. Response to treatment was observed in 29/51 (56.8%) patient. Responders showed a significant reduction in pain and depression severity. Regular aerobic exercise was the strongest independent predictor of treatment efficacy (OR 5.69, 95% Cl 1.07- 30.33, p=0.042). Higher score in the cognitive disorders domain at baseline was associated with poor response (OR 0.64, 95% Cl 0.43-0.95, p=0.027). Conclusion: The J-FiMAR allows a comprehensive evaluation of JFS course. Pain and fatigue are the main symptoms that influence PGA. Our results further support that aerobic exercise is the main cornerstones in the JFS treatment and emphasize the importance of addressing cognitive disorders in adolescents with JFS as well. To identify clinical domain that affect PGA and treatment efficacy is essential for a more individualized management strategy. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
G. Gestler, A. M. Rocha, C. A. Len, A. Araujo, D. S. Jesus, R. D. Gestler, M. T. Terreri s, Universidade Federal de São Paulo, São Paulo, Brazil Juvenile idiopathic arthritis (JIA) is an inflammatory disease. In addition to physical impairment, it compromises the patient’s quality of life with conditions that can extend into adulthood, such as limitations, noninflammatory pain, fatigue, short stature, in addition to social, emotional and educational issues. Approximately 20% to 30% of patients with JIA treated properly may have limited daily activities and impaired quality of life and health. Objectives: To identify the main residual complaints of inactive JIA patients followed up in a tertiary pediatric rheumatology outpatient clinic. Methods: We studied medical records of all patients with a definitive diagnosis of JIA and who were under 18 years old. From these charts, we selected all those related to patients with inactive disease (clinical and laboratory parameters) for at least 6 months, with or without the use of medication. In addition, we applied a questionnaire on possible residual complaints and also questionnaires PedsQL 4.0 and the PedsQL Fatigue Module. Results: From a total of 503 electronic medical records, we identified the medical records of 117 patients with inactive disease. Among them, 76 patients (65%; 95%CI [53.9%; 76.1%]) presented no complaints and 41 (35%; ( 95% CI [23.9%; 46.1%]) presented some residual complaint, such as arthralgia (17.9%), joint limited range of motion (1.7%) or emotional problems (4.3%). Regarding the PedsQL 4. And the PedsQL Fatigue Module, we observed general tiredness in 24%, (95% CI [14%; 33.9%]); sleep-related fatigue in 31% of patients (95%CI [20.2%; 41.7%]); mental tiredness in 37%, (95% CI [25.4%; 47.8%]); physical disability in 29%, (95% CI [29.4%; 52.3%]); emotional problems in 62% (95% CI [50.7%; 73.3%]); socialization problems in 15% (95% CI [7.1%; 23.9%]) and school problems in 82% of the patients (95% CI [72.7%; 90.7%]). Conclusion: We observed a higher-than-expected prevalence of residual complaints in JIA patients with inactive disease. We also observed lower scores in the health-related quality of life questionnaires (PedsQL 4.0 and PedsQL-Fatigue Module). Our data reinforce the importance of the multidisciplinary team in the care of JIA patients, since these patients can present multiple needs even in the phases of apparently controlled disease. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
C. A. Len 1 , M. C. Costa 1 , E. S. Lima 2 , J. Natour 2 , M. T. Terreri 1 1 Pediatrics, 2 Medicine, Universidade Federal de São Paulo, São Paulo, Brazil Correspondence: C. A. Len Introduction: In our clinic we work in a line of research on the gait of children with rheumatic diseases and idiopathic musculoskeletal pain. Our aim is to develop a multiprofessional treatment program based on rehabilitation and to help patients with some measures that can improve the quality of life of them. However, there are no studies in the literature on the standardization of gait in healthy children and adolescents. Objectives: 1) To determine the parameters of normal gait in healthy adolescents, through the use of dynamic baropodometry, 2) To correlate gait parameters with functional capacity, lifestyle and the quality of life. Methods: The sample consisted of healthy adolescents between 14 and 17 years old of both sexes, selected among those enrolled in a recreation center and friends of these students. All subjects were evaluated in two steps: 1) Health related questionnaires (Childhood Health Assessment Questionnaire – CHAQ and Assessment of Physical Activity and Sedentary Lifestyle in Children and Adolescents – IPAQ), and 2) gait evaluation - dynamic baropodometry (walking track FootWalk Pro, AMcube®, France) with the Footwork Pro software (IST Informatique – Intelligence Service et Tecnic, France). We measured gait speed, cadence and step time in all subjects. Results: We included 102 healthy adolescents aged between 14 and 17 years. Of these, 55 were aged between 14 and 15 years and 47 aged between 16 and 17 years. Regarding sex, 54 were female. All participants had normal physical capacity according to the CHAQ. Regarding the IPAQ questionnaire for adolescents, we observed that no individual was sedentary: 18.6% were irregularly active, 45.5% were active and 35.5% were very active. Regarding the dynamic baropodometry parameters, we observed an average gait speed on the right foot of 318.335 mm/s, an average gait speed on the left foot of 319.268 mm/s, a cadence (average) of 1.457 steps/second, a right foot step time of 747.661 seconds and a left foot step time of 738.331 seconds. We did not observe any correlation between the dynamic baropodometry parameters and the results obtained in the CHAQ and IPAQ questionnaires (p > 0.05). Conclusion: In our study, we established the normality of the gait patterns of healthy adolescents, according to dynamic baropodometry, a method commonly used to study the gait of children and adolescents with health problems, including neurological and rheumatological problems, among others. As expected, we did not observe any correlation between the dynamic baropodometry parameters and the results of the CHAQ and IPAQ questionnaires, since all individuals had normal functional capacity. These data will be useful for the continuation of our line of research, since gait may be compromised in chronic rheumatic patients. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
A. Pieler, H. K. Ipsen H.C. Andersen Childrens Hospital, Odense, Denmark Correspondence: A. Pieler Introduction: Virtual Reality goggles became an option as a resource to help children overcome pain related procedures at the H.C. Andersens Children’s Hospital. When steroid injections related to treatment is necessary in our outpatient clinic, we can offer local anesthesia or N 2 O. If general anesthesia is needed, the patient must come back another day. Objectives: To determine if VR can be used as distraction during joint puncture as an alternative to N 2 O and general anesthesia. Methods: VR was offered to some patients based on the health professionals assessment of the childrens cooperation before the joint puncture. On a tablet connected to the goggles it was possible to see, what the child was seeing. That way it was possible to guide the child or comment on what they were doing and seeing. Results: 6 children aged 12-14 agreed to try VR. Two of them had earlier tried N 2 O. Both of them preferred VR to N 2 O. The child could use the VR how they felt like it. Trying games and movies and shifting between them. All procedures carried out with VR was successful. One child removed the goggles prior to the injection, as he needed to watch, but reported feeling VR helped cope with nerves up to. The children felt helped by VR and would like to use them again, should there be a need for it. Conclusion: It is possible to use VR to help children cope with joint puncture and it is now an offer in our outpatient clinic alongside general anesthesia and N 2 O. It is an individual assessment by health professionals what is suitable in each case. Disclosure of Interest : None declared
M. Couto 1 , M. J. Santos 2 , S. N. Sousa 2 1 Faculdade de Medicina da Universidade de Lisboa, 2 Serviço de Reumatologia, Hospital Garcia de Orta, Lisboa, Portugal Correspondence: S. N. Sousa Introduction: Methotrexate (MTX), administered either orally or subcutaneously, remains the mainstay of Juvenile Idiopathic Arthritis (JIA) treatment due to its effectiveness and acceptable safety profile. Even though, less than half of patients remain in treatment beyond two years. Both routes of administration are effective, but a greater bioavailability with subcutaneous route, especially at higher doses, has been reported. Despite this, fear of injections and other concerns, can compromise the quality of life (QoL) and hamper medication adherence. Objectives: To evaluate the impact of orally and subcutaneously administered MTX on the QoL of children and adolescents with JIA, as well as the effectiveness and side effects with both routes of administration. Methods: Children and adolescents followed at a Pediatric Rheumatology Centre were included. The QoL was evaluated at last appointment using the EQ-5D-Y questionnaire (0–1; higher scores indicate better QoL) and the QoL section and the well-being VAS of Juvenile Arthritis Multidimensional Assessment Report (JAMAR) (0–30, higher scores indicate worse QoL); satisfaction with treatment was measured on a Numeric Rating Scale (0-10). Effectiveness was assessed by the improvement in disease activity and function at 6 and 12 months after starting MTX. Side effects that led to change of dose or route of administration or MTX discontinuation were also collected. Results: A total of 26 children and adolescents with JIA were evaluated. Of those, 15 were receiving MTX at last appointment, 9 orally and 6 subcutaneously (Table). 17 treatment courses with subcutaneous and 29 with oral MTX were analyzed. Quality of life was similar for both routes of MTX administration. The median scores of EQ-5D-Y were 0.85 and 1 (p=0.864) and the median scores of JAMAR (children) were 4 and 3.5 (p=0.818) for subcutaneous and oral routes of administration, respectively. The results for physical subdimension of JAMAR were 2.5 and 1 (p=0.937), for subcutaneous and oral routes, respectively and for psychosocial subdimension were 1 (p=0.937) for both routes and the reported well-being on the VAS scale of JAMAR (0 corresponding to very well and 10 to very poor) was 0 (p=0.818) for both routes of administration. Similar results were reported by parents. Regarding satisfaction with medication, the median value was 10 (min 5 – max 10). Subcutaneous MTX was superior to oral MTX, in terms of JADAS (p=0.02) and ESR (p=0.015) reduction at 6 months and reduction of pain at 6 (p=0.046) and 12 (p=0.047) months. However, the dosage of subcutaneous MTX was significantly higher (SC 0.58 ± 0.031; Oral 0.45 ± 0.03; p=0.008). Both subcutaneous and oral MTX were well tolerated. Conclusion: Conclusion: In this cohort of children with well-controlled JIA, the route of MTX administration does not appear to have a negative impact on QoL. MTX was effective and well tolerated regardless of the route of administration. Disclosure of Interest : None declared
P442. ThinkJIA: A campaign to raise awareness that children and young people get arthritis, to enable families and frontline health professionals to recognise symptoms and refer to specialist services 43. Parliamentary inquiry into childhood rheumatic disease: a description of the inquiry process as a novel form of advocacy, and of preliminary inquiry outcomes 4. A parent guide to rasing a child with juvenile arthritis R. Beesley 1,2 1 European Network for Children with Arthritis, Geneva, Switzerland, 2 Juvenile Arthritis Research, Tonbridge, United Kingdom Correspondence: R. Beesley Introduction: Ais low within the general population and amongst non-rheumatology front-line health professionals. This can lead to delays in diagnosis and reduced access to treatment, and consequential risk of joint damage and permanent eye complications through undiagnosed JIA-associated uveitis. Objectives: To develop an awareness campaign to enable frontline health professionals and families to remember that CYP can develop arthritis, to be aware of the main signs and symptoms, and to pursue early referral to speciality services. Methods: An awareness-raising campaign was developed by UK charity Juvenile Arthritis Research working with parents of CYP with JIA, adults with JIA, teachers, campaigners, paediatric rheumatologists, ophthalmologists, and other interested lay and professional individuals. The campaign, called #ThinkJIA, includes postcards with key messages and a supporting website, www.thinkjia.org Resources are aimed at both the general population, to help improve recognition of the signs and symptoms and encourage early attendance at primary care health services; and at non-rheumatology front-line health professionals to support recognition of JIA symptoms and the need for prompt referral. Whilst the key messaging is common to both audiences, the language used differs to reflect the lay and medical professional expectations. Once draft campaign resources were developed, they were shared with clinical professionals and tested by parents and school teachers. Results: Of the pilot group of parents and teachers receiving draft resources, 100% of parents and 68% of teachers agreed that they would feel confident in seeking medical attention if their child, or a child in their school, started showing signs of JIA. Following feedback from recipients in the pilot group resources have been updated with clearer imagery. These are also supported by posters for use in health clinics and general circulation. Conclusion: The development of the #ThinkJIA awareness-raising resources has helped ensure frontline health professionals and the general population can have access to information about JIA. This can help facilitate early referral and improved access to treatment. The broad rollout of awareness resources is essential to appropriately support CYP with JIA and ensure they receive the treatment they require promptly. [Acknowledgements: Thanks to Prof Helen Foster for her vital role in the initial development of these resources, and to all those involved in the development and pilot testing]R. A. James 1 , G. Murray 2 , W. Renton 3 , G. Tiller 3 1 Rheumatology, Queensland Children’s Hospital, Brisbane, 2 Rheumatology, Women’s and Children’s Hospital, Adelaide, 3 Rheumatology, Royal Children’s Hospital, Melbourne, Australia Correspondence: R. A. James Introduction: In December 2021, following advocacy from the Juvenile Arthritis Foundation of Australia (JAFA) and the Australian Paediatric Rheumatology Group (APRG), the Australian Parliament called an Inquiry into childhood rheumatic diseases. Objectives: To describe how a patient advocacy group, with the support of healthcare professionals, successfully advocated for a Parliamentary Inquiry into childhood rheumatic diseases, as well as the process and early outcomes of that inquiry. Methods: Terms of reference of the Inquiry centred around 5 key themes: research; disease impacts; access to care; best practice care and professional awareness. Submissions were invited over a two month period and were followed by public hearings at which JAFA, the APRG and others were invited to present. The APRG submission was structured as a collaborative writing effort by a multidisciplinary team of 16 volunteers, with a core group of 4 editors. Virtual meetings were conducted on a weekly basis, and more frequently for the editing group. Consumer input from JAFA was sought around content and strategy. Both JAFA and the APRG actively promoted the Inquiry to a broad spectrum of interested parties, inviting further submissions. Results: 127 submissions were received: 34 from named individuals, 30 from organisations and 63 as de-identified or confidential submissions. This reflects a strong response compared to previous inquiries into other conditions (TABLE 1). Submitting organisations included patient advocacy groups, professional organisations, health services, pharmaceutical companies and others. Submissions were received from across Australia and internationally. The vast majority were from patients and caregivers. Common themes amongst these submissions included the mental health impacts of childhood rheumatic disease; impacts on family and household members; medication access; difficulties with accessing care in rural or remote areas; the desire for care to be repatriated to local services; and workforce shortages. In March 2022 the Committee tabled an interim report in the Australian Parliament which made 15 key recommendations. These included tripling the Australian workforce over an 8 year period; the creation of Centres of Excellence in major cities; a hub-and-spoke model for outreach to regional areas; improved access to community-based therapies and social security supports; priority funding for key areas of workforce need; improved access to medications; and the creation of a disease registry. The Australian Government will formally respond to this report after the May 2022 election. In the interim, concrete and emergent outcomes continue to be observed including streamlined methods for prescribing biologic medications; the development of new Australian Standards of Care for JIA; attention and interest of hospital executives resulting in increased clinical staffing in some centres; matured institutional relationships and priorities; and greater collaboration between patient and professional organisations over existing funding streams. Conclusion: An Australian Parliamentary Inquiry into childhood rheumatic disease was well-received and attracted many submissions from interested parties. The resulting report made sound recommendations across a number of areas of recognised service shortfalls. Concrete and emergent outcomes from this inquiry are continuing, and will lead to improvements for patients, families and healthcare professionals in this fieldcCormack 1 , G. Faller 2 , P. Ambaram 3 , K. Webb 4,5 , B. Mistry 6 , A. Eckstein 1 , L. Tomasella 7 1 Arthritis Kids South Africa, 2 Paediatric rheumatology, WITS Gordon Medical Centre, 3 Paediatric rheumatology and Honorary Lecturer, University of the Witwatersrand, Johannesburg, 4 Paediatric rheumatology, University of Cape Town, Cape Town, South Africa, 5 Crick African Network, Francis Crick Institute, London, United Kingdom, 6 Paediatric rheumatology, Chris Hani Baragwanath Academic Hospital, 7 Global Rheumatology Alliance, Johannesburg, South Africa Correspondence: C. McCormack Introduction: South African parents and caregivers (PaC) of children with juvenile idiopathic arthritis (JIA) face numerous challenges in accessing credible information about their child’s condition, including language barriers, disparate education levels, poor literacy rates, limited access to affordable internet, financial barriers, and cultural beliefs about this disease. Objectives: Arthritis Kids South Africa’s (AKSA) objective is to ensure PaC of children with JIA can access credible information and support about this disease regardless of socioeconomic status, language, level of basic education, and literacy rates. We also want to ensure that cultural beliefs do not hamper efforts to achieve disease management. Methods: Relevant topics to be included were identified informally in workshops with paediatric rheumatologists, patients, parents, and allied healthcare professionals (HCP). With the support of this team, individual articles were written for each topic. Certain portions were translated to three of the eleven official South African languages, namely, isiZulu, Sepedi and Afrikaans. Thereafter, an informal qualitative process took place in two paediatric rheumatology clinics to test PaC understanding of concepts included. Results: The resulting collection of articles, including a host of interviews with relevant allied HCP, patients and parents, was made available to PaC via the AKSA website and, where funding allowed, in printed form. The collection precipitated the development of the first parent guide for JIA in South Africa in two South African languages. Topics include ‘My child has JIA, what’s next?’, ‘When to call your child’s doctor?’, ‘Symptoms and flares’, ‘Living with JIA’, ‘Stress and anxiety’, ‘What about food?’, ‘Exercise lowers inflammation, ‘Alternative therapies’, and ‘Frequently asked questions’. The booklet includes information on the immune system, autoimmunity, nutrition, specific school issues and the prognosis for JIA in accessible language and with images. It includes a section where a parent may record treatment notes, record growth, track symptoms and ask their health team to write a plan for a flare or an infection. It was recognised that translations alone would not adequately address the language barrier in South Africa due to differing literacy rates in both own and English languages. Access to the internet and financial barriers also prevent the satisfactory dissemination of the support material. To overcome these challenges, we created a combination of audio and video supplements to the written library in the form of an animated video and podcasts. These are made available in up to five of the eleven South African languages. Conclusion: We have created the first South African ‘Parent Guide to JIA’ with supplemental video and podcast content to assist paediatric rheumatologists, nurses and other relevant HCP to better explain concepts related to the diagnosis, treatment and long term management of JIA. Parents can share these tools with extended family members, schools, and broader social circles. We anticipate that this will improve parents’ experience, empower them as partners in their children’s care and ultimately improve the outcomes of their children with JIA. Patient Consent: Not applicable (there are no patient data) Disclosure of Interest : C. McCormack Grant / Research Support with: AKSA received a grant from Roche SA to translate materials in the support library. Janssen Pharmaceutica SA paid for an advert in the printed guide which funded printing costs., G. Faller: None declared, P. Ambaram: None declared, K. Webb: None declared, B. Mistry: None declared, A. Eckstein: None declared, L. Tomasella: None declared
P445. Azerbaijani data on Multisystem Inflammatory Syndrome in Children (MIS-C) P446. SARS-COV-2 infection in children and adolescents with rheumatological diseases, the largest tertiary center experience from Saudi Arabia 7. SARS-COV-2 infection and vaccination in children with rheumatic diseases:an Italian experience P448. MIS-C and Kawasaki disease: a comparison based on our experience P449. Gastrointestinal involvement in multisystem inflammatory syndrome associated with COVID-19 in children P450. When to use biologic drugs in the treatment of Multisystem Inflammatory Syndrome in Children (MIS-C): a single center cohort study P451. The characteristics of patients with COVID-19-associated pediatric vasculitis: an international, multicenter study P452. Cardiac MRI in long term follow-up of MIS-C: a retrospective multicentre study 53. Spontaneous nlrp3 inflammasome-driven IL-1b secretion is induced in severe COVID-19 patients and responds to anakinra treatment P454. High incidence of MIS-C and other autoimmune diseases after SARS-COV-2 infection compared to COVID-19 vaccination in pediatric population from South Central Europe P455. Comparison of demographics, presentation and short-term outcomes in mis-c across different variants of SARS-COV-2 in Cape town, South Africa P456. An update of the international registry on COVID-19 related hyperinflammation in children and young adults (hyperped-covid) P457. MIS-C and severe COVID-19 in children from the amazonian intensive care unit: poor outcome and high mortality rate P458. The role of SARS-COV-2 vaccination and SARS-COV-2 reinfection in children with previous multisystem inflammatory syndrome (MIS-C) P459. The cytokine profile of cerebrospinal fluid in multi-system inflammatory syndrome in children P460. Determination of factors to distinguish Multisystem Inflammatory Syndrome (MIS-C) from other acute febrile illness in the emergency department P461. Prospective cohort of patients with pediatric multisystem inflammatory syndrome temporally associated with SARS-COV-2 infection followed up in a tertiary hospital P462. Patients with systemic juvenile idiopathic arthritis treated with IL-1 inhibitors: what happens if SARS-COV2 infection occurs? P463. COVID-19-associated vasculitis/vasculopathy P464. Inflammatory markers linked to cardiovascular involvement in Multisystemic Inflammatory Syndrome in Children (MIS-C) among the paediatric population of a third level hospital at Northeast Mexico P465. Multisystem Inflammatory Syndrome in Children (MIS-C) with rapid progression: a case report P466. Multisystem inflammatory syndrome in children: a year and a half of experience of university children’s hospital, Sofia, Bulgaria P467. Immunogenicity and safety of SARS-COV-2 vaccine in patients with juvenile idiopathic arthritis P468. Clinical and paraclinical characteristics of pediatric patients with multisystemic inflammatory syndrome associated to COVID-19 and Kawasaki disease in a tertiary public healthcare center in Mexico Northeast 69. Multysystem infalmmatory syndrome– not every crown is COVID-19 related 70. Monitoring of SARS-COV-2 antibody levels in children and late adolescents with inflammatory rheumatic diseases P471. Safety and humoral response following the second and third doseP472. The risk factors for a severe disease course in multisystem inflammatory syndrome in children - a multicenter retrospective study P473. A case of Juvenile Idiopathic Arthritis (JIA) deterioration after BNT162B2 booster vaccination (3rd vaccination) P474. MIS-C/PIMS-TS: clinical manifestations and therapy 5. Capillary leak syndrome as a new, common phenomenon in children with MIS-C - a retrospective, population-based cohort from Southern Sweden P476. Epidemiology of Multisystem Inflammatory Syndrome in Children (MIS-C) in Southern Italy P477. Cardiac magnetic resonance to evaluate cardiovascular involvement in children with covid-19 temporally related Multisystem Inflammatory Syndrome (MIS-C): can we arrive to the heart of the problem? P478. The impact of COVID-19 pandemic on children with juvenile idiopathic arthritis under immunosuppressive and biological therapy: a single-center experience P479. Current approach to covid-19 and other vaccinations in children with previous Multisystem Inflammatory Syndrome (MIS-C): an international survey P480. Analysis of the dynamics of changes in laboratory parameters in patients with PIMS-TS - one-year follow-up, single-center case-control study, preliminary results P481. COVID-19 and children: complications and late outcomes at G. Zhvania academic clinic of pediatry of TSMU P482. Safety of the COVID-19 vaccination in children with juvenile idiopathic arthritis – a questionnaire study from two pediatric rheumatology centers in Poland P483. Relapse of acral chilblain-like lesions during covid-19 waves in a cohort of children and adolescents P484. COVID-19 and autoimmunity (antiphospholipid antibodies syndrome) P485. Observational study on COVID-19 vaccination in paediatric patients with rheumatic diseases P486. COVID-19 infection and auto-inflammatory diseases in children P487. Painful cervical involvement and febrile torticollis: a frequent symptom of PIMS P488. Relapse of JIA-related anterior uveitis: an observational single-centre study during SARS-COV2 vaccination campaign P489. Differences between mis-c presenting with Kawasaki-like symptoms and classical Kawasaki disease 490. Disease relapse and protective antibodies after sars-cov-2 infection compared to vaccination against SARS-COV-2 in children with rheumatic diseases P491. IL-27 Expression is up-regulated in in south african patients with multisystem inflammatory syndrome in children P492. Usefulness of anakinra in multisystem inflammatory syndrome in children: results from an Italian survey P493. No differences found in humoral and cellular immune response to mrna SARS-COV-2 BNT162B2 vaccine in teenagers with rheumatic diseases and healthy peers 94. Multisystem inflammatory syndrome in children related with SARS-COV 2 (MIS-C) multicenter study in Mexico P495. COVID-19 in children with juvenile idiopathic arthritis on DMARDs treatment P496. Parental social media habits and its effect on children vaccination against COVID-19 P497. Effectiveness of the BNT162B2 MRNA COVID-19 vaccine in adolescents with juvenile-onset autoimmune inflammatory rheumatic diseases liyeva 1,2 , N. Rahimova 3 , L. Gulmammedova 3 , S. Nesirova 3 , L. Mirsalayeva 3 , K. Eshrefova 3 , E. Rahimov 4 , A. Musayev 5 on behalf of This Working Group members consist of 2 working place that I have mentioned above, investigation lead and organize by Dr. Aytan Alieva. 1 Pediatric reumatology, Baku Medical Plaza, 2 Pediatric reumatology, 3 Pediatric, Baku/Azerbaijan Scientific Research Institute of Pediatrics, 4 Pediatric, Baku Medical Plaza, 5 Pediatric Surgeon, Baku/Azerbaijan Scientific Research Institute of Pediatrics, Baku, Azerbaijan Correspondence: A. Aliyeva Introduction: Children’s Multisystem Inflammatory Syndrome (MIS-C) is hyperinflammatory response that occurs following infection with COVID-19 and can result in serious consequences Objectives: The aim of this study was to analyze the demographic, clinical laboratory characteristics and treatment modalities of MIS-C patients from Azerbaijan Methods: Methods: The trial comprised patients under the age of 18 who were diagnosed with MIS-C and treated at two locations ( 1 Baku/Azerbaijan Scientific Research Institute of Pediatrics 2 Baku/Azerbaijan Baku Medikal Plaza) between December 2020 and March 2022. The patients’ information was gathered retrospectively from their medical records. The following data were collected: age, gender, admission symptoms, laboratory and clinical findings, and complications. Results: Results: The study included 80 patients, 60%of whom were male (n:48) and 40% were female (n:32). The median age at the time of diagnosis was 6 years (1-14). Six individuals (7.5%) had PFAPA syndrome, two (2.5%) had juvenile idiopathic arthritis (JIA), one (1.25%) had biotinidase deficiency, one (1.25%) had diabetes mellitus, and one (1.25%) had leukemia. Among them, 85% (n=68) patients were followed at pediatric inpatient department, while 15% (n=12) were treated at intensive care unit. As a consequence, 74 patients (92.5%) fully recovered, 5 patients (6.25%) were unresponsive, so they required more aggressive treatment, and 1 patient (1.25%) died. The median duration of hospitalization was 9 (5-27) days. Involvement of the heart was found in 81.25 % (n=65) of the patients: in10.7% (n=7) of patients coronary aneurysm, in 57.5 % (n=46) mitral and tricuspid insufficiency, in 31.25 % (n=25) pericarditis. Gastrointestinal abnormalities, stomach pain, and lymphoadenopathy were found in 12.5% (n=10) of the participants. As a result, 74 patients (92.5%) received complete treatment. The therapy was repeated in 5 patients (6.25%) due to recurrence of symptoms, and 1 patient (1.25%) was lost. 61 patients (76.25%) received 2g/kg IVIG, whereas 19 patients (23.75%) were unable to acquire IVIG. All patients received steroid medication, with 30 patients (37.5%) receiving pulse steroid, 80 patients (100%) receiving antibiotic therapy, and 10 patients (8%) receiving anakinra treatment. All of the patients were given low-dose aspirin, and 50 (62.5%) of them were also given heparin. Conclusion: Conclusion: One of the most serious consequences seen during the COVID-19 epidemic is MIS-C. Serious complications, such as heart involvement and coronary aneurysm, might occur if left untreated. In terms of prognosis, early diagnosis and treatment are critical. Keywords: COVID-19, hyperinflammation, MIS-C, Patient Consent: Yes, I received consent Disclosure of Interest : None declared
J. Alqanatish 1,2,3 , A. Ahmed 2,4,5 , A. Alfadhel 2,4,5 , A. Albelali 2,4,5 , S. Alghnam 2s, King Saud bin Abdulaziz University for Health Sciences, 5 King Abdullah Specialist Children’s Hospital, National Guard Health Affairs., Riyadh, Saudi Arabia Correspondence: J. Alqanatish Introduction: Pediatric population are considered at a lower risk of contracting SARS-CoV-2 infection. Only about 10% of SARS-CoV-2 cases were among children, however; they may suffer severe complications such as multisystem inflammatory syndrome in children (MIS-C). Data in adults with rheumatological diseases showed favorable clinical outcome of COVID-19 but pediatric data are limited. Objectives: We report on the clinical characteristics of SARS-COV2 infection in children and adolescents with Rheumatological diseases. To our knowledge, this is the largest experience reported from a tertiary center in Saudi Arabia. Methods: We reviewed the medical records of children and adolescents who were diagnosed with a rheumatological diseases with or without COVID-19 infection and visited rheumatology clinic or day care unit at King Abdullah Specialized Children Hospital (KASCH), Riyadh, during pandemic. Moreover, we conducted phone calls to patients’ families to verify and add to the information in medical records. We described these patients’ clinical characteristics analyzed the factors associated with SARS-CoV-2 infection and associated complications, as well as clinical outcome. Results: Among 496 patients included in the study, 117 (23.6%) contracted COVID-19. Gender, biologics, conventional or targeted DMARDs and comorbidities were not associated with the likelihood of infection; however, those who had COVID-19 infection were younger (average age=11.1 vs. 13.8 years; P<0.01). The most common symptom among COVID-19 patients was fever (n=82,70%).Overall, 38 (7.8%) patients suffered any complication, but that varied with COVID-19 status. Of patients who had COVID-19, 66% suffered complications or death, compared with only 0.76% in patients who did not have COVID-19 (P<0.01). Conclusion: In our cohort, younger, compared to older, children with Rheumatological diseases have more commonly contracted COVID-19 infection. Nevertheless, no other clear associations were shown between patients’ demographics or comorbidities and the likelihood of infection. Although, only few patients suffered any complication but that was still more common in patients with COVID19 infeF. Anselmi, B. Macri, E. Barbieri, R. Ragucci, V. Aiello, R. Naddei, M. Alessio, Naples, Italy Correspondence: F. Anselmi Introduction: Since the spread of SARS-CoV-2 infection, a great concern has been addressed to rheumatic patients, especially given that they often require immunosuppressive therapies. So far few studies have described clinical course of SARS-CoV-2infection in children affected by rheumatic diseases and their response to COVID19 vaccination. Objectives: to evaluate the impact of SARS-CoV-2 infection and COVID19 vaccination on our patients with chronic rheumatic diseases. Methods: In this single-centre,retrospective,cohort study,we analyzed 78 patients(M30;age11±4,1y) who developed SARS-CoV-2 infection between 2020 and 2022.A survey was submitted to their families to investigate the childrens’ vaccination status and course of infection (duration,symptoms/severity,time to negative test). Data were analyzed through SPSS2019. Results: Clinical data of our cohort are shown in Table 1.COVID19 infection resulted significantly more frequent in the age group0-11 compared to the age group12-18(75,6%vs51,5%,p<0,05). No significant differences in symptoms were observed between age,sex or diagnosis groups,except for sore throat(47,9%vs20%,p<0,05) and ageusia(16,7%vs0%,p<0,02) which resulted more frequent in females. No significative correlation was found between vaccination status and development or duration of symptoms. Neverthless,23(59%)vaccinated patients vs 7(17,9%)unvaccinated showed a negative swab in less than 10 days(p<0.0001),while 14 unvaccinated patients(35,9%)vs 4 vaccinated(10,3%)resulted negative after more than 14 days(p<0.0001). Likewise, patients who contracted COVID19 before vaccination showed a longer time to negative test conversion compared to those who contracted it before vaccination (respectively 25,5% vs 63% tested negative in less than 10 days, p<0.005;31,4% vs 7,4%tested negative in more than 14days,p<0.005);Moreover,100%(4) of patients that relapsed after the infection were unvaccinated . Among patients under double immunosuppressive therapy (methotrexate+etanercept) fever ad arthromyalgia were significantly less frequent(2.5%vs18,4%,p <0.05). Conclusion: In our cohort most patients with rheumatological diseases developed paucisymptomatic/asymptomatic COVID19 infection, regardless their vaccination status,consistently with general pediatric population reports. Nevertheless our vaccinated children showed a significantly shorter time to negative PCRtest compared to unvaccinated patients. Only 4patients relapsed after infection and all of them were not vaccinated. Therefore in our experience the antiSarsCov2 vaccination helped reducing the duration of the infection and possibly the risk of disease relapsing. Infection was more frequent in children under 12years,but this may be due to the fact that vaccination in children>12years started earlier. Further studies on larger case series are needed to evaluate the effects of COVID19infectionand vaccine on children with rheumatological diseases. Disclosure of Interest : None declared
R. Asaro 1 , A. Cilona 2 , C. Alizzi 2 , G. Corsello 3 1 Dipartimento di scienze per la promozione della salute materno infantile G D’Alessandro Università degli studi di Palermo , 2Palermo, Italy, 3 dipartimento di scienze per la promozione della salute materno infantile “G. D’Alessandro” Università degli studi di Palermo, Italy , Palermo, Italy Correspondence: R. Asaro Introduction: Following the onset of the COVID-19 pandemic, there has been an increase in the incidence of autoinflammatory syndromes in pediatric age. In addition to the already known Kawasaki disease (KD), a new pathological picture has been characterized: the multisystem inflammatory syndrome in children (MIS-C). Usually MIS-C affects children> 5 years of age and adolescents, KD younger children. They are characterized by persistent fever, conjunctival hyperemia, skin rash, edema of the hands and feet, stomatitis but in MIS-C is common the finding of organ dysfunction and inflammatory markers are higher. Differential diagnosis is based on SARS-CoV-2 serology and exposure history Objectives: In 2021 and 2022 at our O.U. of General Pediatrics of Hospital “G. Di Cristina”, a significant incidence of MIS-C and KD was recorded. The purpose of this work is to describe the similarities and differences between the two groups of patients using the following table. Methods: Children diagnosed with MIS-C had an average age of 7 and IgG positive for Sars-Cov2.All presented with high fever, conjunctival hyperaemia, diffuse maculopapular rash and increased inflammation indices. They all had pulmonary involvement on chest CT. Three of them had also a cardiac involvement. All of them showed improvement in clinical conditions after therapy with IV Ig, corticosteroids and ASA. Patients with KD had an average age of 13 months and IgG negative for SarsCov2.In addition to the same symptoms as MIS-C, two of them had edema of the hands and feet and stomatitis. None showed pulmonary or cardiac involvement on imaging, but only an increase in cardiac enzymes. High-risk patients (male, age <1 year, high inflammation indices) were treated following the specific protocol with intravenous Ig at 2g/kg, corticosteroids at 2 mg/kg and ASA at 50 mg/kg with benefit Results: Conclusion: In accordance with the international scientific literature, in the last two years our O.U. reported an increase in the incidence of KD and placed numerous diagnoses of MIS-C following the spread of COVID-19 infection. Our experience suggest that Sars-Cov2 may act as a superantigen by triggering an abnormal immune system response in predisposed children. Disclosure of Interest : None declared
I. Avrusin 1 , K. Belozerov 1 , L. Bregel 2,3 , O. Efremova 3 , E. Dondurei 4,5 , A. Vilnits 6,7 , T. Kornishina 1 , V. Masalova 1 , T. Burtseva 8,9 , O. Kalashnikova 10 , V. Chasnyk 1 , M. Kostik 1 1 Hospital Pediatrics, Saint Petersburg State Pediatric Medical University, Saint Petersburg, 2 Irkutsk State Medical Academy of Postgraduate Education, branch of Russian Medical Academy of Continuous Professional Education, 3 Irkutsk Regional Children’s Clinical Hospital, Irkutsk, 4 Scientific Research Institute of Influenza n.a. A.A. Smorodintsev, 5 Children’s City Clinical Hospital # 5 n.a. N.F. Filatov, 6 Pediatric Research and Clinical Center for Infection Diseases, 7 Saint Petersburg State Pediatric Medical University, Saint Petersburg, 8 Pediatrics and Pediatric Surgery, North-Eastern Federal University (NEFU), 9 Yakut Research Center of Complex Medical Problems, Yakutsk, 10 Hospital Pediatrics, Saint Petersburg State Pediatric Medical University, Saint Petersbug, Russian Federation Correspondence: M. Kostik Introduction: COVID-19 in children is often asymptomatic or with only mild symptoms. However, since April 2020 there are many reports that the new coronavirus infection might be associated with pediatric hyperinflammatory condition, that fully or partially meets the criteria for Kawasaki disease (KD). This phenomenon was later called multisystem inflammatory syndrome in children (MIS-C). Objectives: Our study aimed to evaluate main clinical and laboratorial features and course of MIS-C and find factors associated with gastrointestinal (GI) involvement. Methods: The retrospective study included 162 children (96 male, 66 female), aged from 4 months to 17 years (median 8.2 years), who met the WHO criteria for MIS-C. Results: GI involvement is one of the most frequent early manifestations and main features that helps to distinguish MIS-C from Kawasaki disease. Main GI symptoms were abdominal pain, vomiting, ans diarrhea. Also, in 3 patients there were peritoneal symptoms that required diagnostic laparoscopy and appendix removal (1.8%). Patients with MIS-C were divided into two groups: with (n=125, 77.2%) and without GI involvement (n=37, 22.8%). The predominance of boys (64.8%) was noted in the group of patients with GI involvement compared to the group without it (40.5%, p=0.008). Differences in the incidence of facial edema (56.5% vs. 30.8%, p=0.022), hepatomegaly (69.6% vs. 47.1%, p=0.016), splenomegaly (46% vs. 23.5%, p=0.02), neurological symptoms (51.6% vs. 33.3%, p=0.053), hypotension/shock (51.2% vs. 18.9%, p=0.0005), signs of myocardial damage (35% vs. 19.4%, p=0.078) compared to children without GI involvement were found, respectively. Patients with GI involvement had higher level of CRP (157.7 mg/l vs. 106.1 mg/l, p=0.077) and troponin (7 pg/ml vs. 1 pg/ml, p= 0.065). Signs associated with GI damage, their sensitivity, specificity and odds ratio (OR) are presented in the table. Of the initial 6 predictors included in the multifactorial regression model, only 2 variables were significantly associated with GI damage: male gender, and hypotension/shock. Conclusion: GI involvement is an important early predictor of the severity of MIS-C, requiring careful clinical and laboratory monitoring. GI disorders are significantly more common in males, and also associated with such characteristics of MIS-C as face swelling, CNS lesion, hepatomegaly and splenomegaly, hypotension / shock. Acknowledgements : This work was supported by the RSF grant № 20-45-01005 Disclosure of Interest : None declared
O. Basaran 1 , E. D. Batu 1 , U. Kaya Akca 1 , E. Atalay 1 , M. Kasap Cuceoglu 1 , S. Sener 1 , Z. Balik 1 , S. Kesici 2 , T. Karagoz 3 , Y. Ozsurekci 4 , Y. Bilginer 1 , A. B. Cengiz 4 , S. Ozen 1 1 Department of Pediatric Rheumatology, 2 Department of Pediatric Intensive Care Unit, 3 Department of Pediatric Cardiology, 4 Department of Pediatric Infectious Diseases, Hacettepe University, Ankara, Turkey Correspondence: E. D. Batu Introduction: Multisystem inflammatory syndrome in children (MIS-C) is a newly identified and life-threatening condition associated with SARS-CoV-2 infection. Although our understanding of COVID-19 and MIS-C has been evolving, optimal treatment for MIS-C remains unclear. intravenous immune globulin (IVIG)±corticosteroids is widely accepted as the first step management in MIS-C. Objectives: To evaluate and compare the demographic and clinical features of MIS-C patients who had IVIG plus steroid and who had additional biologic drugs, to analyze the indications for biologic drugs in MIS-C treatment. Methods: This retrospective cohort study included 79 MIS-C patients from a tertiary-care pediatric hospital who were followed up between July 2020 to November 2021. We used univariate and multivariate regression analysis for finding out the variables predicting biologic use. The Receiver Operating Characteristics (ROC) curve was used to demonstrate the best performing cut-off value for CRP levels. Results: Of 79 patients (median age 8.51 years, 33 girls), 51 (64.5%) received steroid plus IVIG plus biologic agents, while 28 (35.5%) had only steroid and IVIG. Compared with patients who had IVIG and steroid, patients having additional biologic therapy were more likely to have hypotension (6.9% vs. 93.1%, p<0.001) and pulmonary infiltration (0% vs. 100%, p<0.001), and need more inotropic agents (5.0% vs. 100%, p<0.001) and required O 2 support (0% vs. 100%, p<0.001). Patients who received biological agents had significantly lower hemoglobin levels (p=0.01), thrombocyte (p<0.001) and lymphocyte counts (p=0.02), albumin (p<0.001) levels, and higher C-reactive protein (CRP) (p<0.001), ferritin (p<0.001), brain natriuretic peptide (BNP) (p<0.001), d-dimer (p=0.02), troponin (p<0.001) levels and longer median inpatient stay (p<0.001) and duration of fever (p=0.02). All patients were discharged with healing and none of the patients had an exitus. The multivariate regression analysis disclosed only CRP level as being independently associated with biologic use in MIS-C treatment. In the ROC analysis, the area under curve was 0.709 with a standard error of 0.064 and 95% confidence interval of 0.585-0.834 (p=0.002). CRP≥12 mg/dl was the best performing cut-off value with a sensitivity of 80.4% and specificity of 60.7% for predicting biologic use. Conclusion: We suggest that the CRP level at MIS-C diagnosis may help to decide on the indication of biologic treatment. Identifying the MIS-C patients who require biologic treatment in the acute disease period would improve the outcome of these patients. Patient Consent: No, I have not receive consent Disclosure of Interest : None declared
G. Ozomay Baykal 2 , E. Arslanoglu Aydin 3 , S. Ozdel 3 , A. Gagro 4 , F. G. Demirkan 5 , E. Esen 6 , N. Akpinar Tekgoz 7 , K. Ozturk 8 , O. Vougiouka 9 , H. E. Sonmez 10 , M. Heshin-Bekenstein 11 , M. C. Maggio 12 , U. Kaya Akca 13 , M. Jelusic 14 , A. Pac Kisaarslan 6 , B. Celikel Acar 7 , N. Aktay Ayaz 5 , B. Sozeri 2 , S. Ozen 1 on behalf of PReS Vasculitis Working Party 1 Hacettepe University Faculty of Medicine, Ankara, 2 Umraniye Research and Training HospitalTurkey, 4 Children’s Hospital Zagreb, Zagreb, Croatia, 5 Istanbul University Faculty of Medicine, Istanbul, 6 Erciyes University Faculty of Medicine, Kayseri, 7 Ankara City Hospital, Ankara, 8 Istanbul Medeniyet University, Goztepe Research and Training Hospital, Istanbul, Turkey, 9 Athens University School of Medicine, Athens, Greece, 10 Kocaeli University Faculty of Medicine, Kocaeli, Turkey, 11 Dana Dwek Children’s Hospital, Tel Aviv Medical Center, Tel Aviv, Israel, 12 University of Palermo, Palermo, Italy, 13 Aydın Obstetrics and Pediatrics Hospital, Aydın, Turkey, 14 University of Zagreb, School of Medicine, Zagreb, Croatia Correspondence: E. D. Batu Introduction: COVID-19 has been associated with some rheumatic manifestations such as arthritis and vasculitis. Kawasaki disease (KD)-like vasculitis in MIS-C (multisystem inflammatory syndrome in children) forms the majority of COVID-19 associated pediatric vasculitides. Non-MIS-C COVID-19 associated pediatric vasculitis is very rare and only isolated case reports and case series have been reported in the literature to date. Objectives: To analyze the characteristics, treatment and outcome in COVID-19-associated pediatric vasculitis other than KD-like vasculitis in MIS-C. Methods: The inclusion criteria were as follows: 1) <18 years old; 2) evidence of SARS-CoV-2 exposure (history of COVID-19 or contact OR positive PCR OR serology); 3) evidence of vasculitis diagnosis (angiographic or histopathological evidence of vasculitis OR meeting diagnostic/classification criteria for vasculitis). Patients with MISC were excluded. Results: A total of 42 patients (median age at vasculitis onset 9 years; M/F=1) were included from 14 centers and six countries. The most frequent phenotype was IgA vasculitis (IgAV) (n=28) followed by skin vasculitis (n=4) and Takayasu arteritis (n=3). Other vasculitis subtypes were polyarteritis nodosa (n=1), granulomatous polyangiitis (n=1), and unclassified vasculitis (n=5). The history of COVID-19 was present in 28 (66.7%) patients. SARS-CoV-2 PCR was positive in 16 out of 37 patients (43.2%) and SARS-CoV-2 serology was positive in all 24 of 24 (100%) patients. The median duration between exposure to SARS-CoV-2 and onset of vasculitis-associated symptoms was 15 days. The median duration of follow-up was 3 months. Skin (90.5%%) and musculoskeletal (73.8%) involvements were the most common manifestations of vasculitis. Gastrointestinal system and renal involvements were present in 59.5% and 42.9% of the patients, respectively. The majority of patients received corticosteroids (73.8%), while 21.4% used immunosuppressive drugs. 8 (19%) patients received antiplatelet or anticoagulant therapy. Outcome data was available for 40 patients (2 were recently diagnosed). Complete and partial remission was achieved in 80% and 17.5% of patients while one patient had refractory disease course. None of the patients died. Regarding IgA vasculitis (M/F=1.8; median age at vasculitis onset 9.8 years), all patients had skin manifestations while 18 (64.3%) and 12 (42.9%) had gastrointestinal system and renal involvement, respectively. 17 (60.7%) patients received NSAIDs and corticosteroids were used in the treatment of 18 (64.3%) patients. Remission was achieved in all but one who had refractory disease course with severe gastrointestinal and renal vasculitis. Conclusion: Although COVID-19 associated pediatric vasculitis is very rare, analyzing its characteristic features is important to raise awareness among physicians. COVID-19-associated IgAV seems similar to non-COVID-19 associated IgAV. However, the disease course may be slightly more severe. The temporal association of SARS-CoV-2 and vasculitis is similar to what is observed in MIS-C which suggests an immune reaction triggered by the virusS. Benvenuto 1 , G. Simonini 2 , S. Della Paolera 3 , S. Abu-Rumeileh 2 , M. V. Mastrolia 2 , A. Manerba 4 , D. Chicco 3 , T. Caiffa 3 , M. Cattalini 4 , A. Taddio 1 1 University of Trieste, Trieste, 2 Rheumatology Unit, Meyer Children’s Hospital, University of Florence, Florence, 3 Institute for Maternal and Child Health, IRCCS “Burlo Garofolo”, Trieste, 4 ASST Spedali Civili di Brescia, Brescia, Italy Correspondence: S. Benvenuto Introduction: Heart involvement is a common feature of multisystem inflammatory syndrome in children (MIS-C), but exhaustive data on long-term cardiac recovery are lacking. Objectives: To evaluate the long-term outcome of cardiac involvement in children affected by MIS-C, assessed through cardiac MRI. Methods: Children referring to three Italian tertiary pediatric centers (Institute for Maternal and Child Health, IRCCS “Burlo Garofolo”, Trieste; ASST Spedali Civili, Brescia; Meyer Children’s University Hospital, Florence) between February 2020 and November 2021 with a diagnosis of MIS-C, who underwent cardiac MRI during a follow-up visit were enrolled. Demographic, clinical, laboratory, treatment and outcome data were collected in an anonymized database. Results: Twenty mostly male (75%) MIS-C patients, aged 9-17 (median 12), were included. Most common clinical features at onset were fever (100%), gastrointestinal (70%) and/or mucocutaneous (70%) manifestations, hypotension and/or shock (55%). All presented laboratory evidence of myocardial involvement, either as elevated troponin or NT-proBNP, and most (15/18 assessed, 83%) showed reduced left ventricular ejection fraction (LVEF) on echocardiography on admission. Half of them required admission to pediatric intensive care unit, while 40% required inotrope agents; a nonfatal cardiac arrest was observed. A good clinical, laboratory and echocardiographic response to treatment was observed in all of the patients, with normal (15/19 assessed, 79%) or improved echocardiography at discharge. Cardiac MRI was performed after a median interval of 3 months from discharge, with a maximum of 9 months. Mild residual left ventricular dysfunction was found in 20%, all showing normal LVEF on echocardiography at discharge. A thin layer of pericardial effusion or myocardial edema were found in 5% of patients, in line with previous similar studies 1 . Finally, in contrast to all of the previous observations, late gadolinium enhancement (LGE) as a sign of minimal myocardial scars was found in 25% of our patients. Persisting myocardial scars are typical findings of “classic” viral myocarditis 2 , which has however been associated with a worse cardiac outcome when compared to MIS-C myocarditis 3 . Of note, one patient in our cohort who had been evaluated at two consecutive time points showed partial resolution of a myocardial scar after 7 months from its first finding. Conclusion: Despite the severity of heart involvement in the acute MIS-C phase, the long-term cardiac outcome is good. Larger longitudinal studies are needed in order to confirm this positive evolution and to establish the effective need and duration for physical activity restrictions or specific cardioprotective treatment. References 1. Bartoszek M, et al. Cardiac Magnetic Resonance Follow-Up of Children After Pediatric Inflammatory Multisystem Syndrome Temporally Associated With SARS-CoV-2 With Initial Cardiac Involvement. J Magn Reson Imaging. 2022 Mar;55(3):883-891 2. Malek LA, et al. Children With Acute Myocarditis Often Have Persistent Subclinical Changes as Revealed by Cardiac Magnetic Resonance. J Magn Reson Imaging. 2020 Aug;52(2):488-496 3. Patel T, et al. Comparison of Multisystem Inflammatory Syndrome in Children-Related Myocarditis, Classic Viral Myocarditis, and COVID-19 Vaccine-Related Myocarditis in Children. J Am Heart Assoc. 2022 May 3;11(9):e024393Bertoni 1,2 , F. Penco 1 , H. Mollica 1 , P. Bocca 1 , I. Prigione 1 , A. Corcione 1 , D. Cangelosi 3 , F. Schena 1 , G. Del Zotto 4 , A. Amaro 5 , N. Paladino 1 , E. Pontali 6 , M. Feasi 6 , S. Signa 1 , M. Bustaffa 1 , R. Caorsi 1 , R. De Palma 7 , U. Pfeffer 5 , P. Uva 3 , A. Rubartelli 1 , M. Gattorno 1 , S. Volpi 1 1 Centro Malattie Autoinfiammatorie ed Immunodeficienze, IRCCS Istituto Giannina Gaslini, 2 DINOGMI, Università degli Studi di Genova, 3 Clinical Bioinformatics Unit, 4 Department of Research and Diagnostics, IRCCS Istituto Giannina Gaslini, 5 IRCCS Ospedale Policlinico San Martino, 6 Ente Ospedaliero Ospedale Galliera, 7 Department of Internal Medicine, IRCCS Ospedale Policlinico San Martino, Genova, Italy Correspondence: A. Bertoni Introduction: Coronavirus disease 2019 (COVID-19) is an acute respiratory illness caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). COVID-19 severe pneumonia has been associated to systemic inflammation. Recently several reports have highlighted the role of innate immune cells, particularly monocytes, and of monocyte-derived cytokines such as IL-1b, in COVID-19 and showed a correlation between the degree of involvement of innate immunity and disease severity and outcome. Stimulation of peripheral blood mononuclear cells (PBMC) from patients with severe COVID-19 have shown an increased secretion of IL-1β. In support of a key role of IL-1β in patients with severe COVID-19 we and others have recently reported the potential efficacy of blocking IL-1β by using anti-IL1 drugs. NLRP3, activated by inflammatory signals, oligomerizes and recruits pro-caspase-1 and the adaptor molecule apoptotic speck like protein-containing a CARD (ASC), resulting in activation of caspase-1, processing of pro-IL-1β and secretion of the mature cytokine. Recently NLRP3 activation and ASC speck formation in peripheral blood cells and CD14+ cells from autoptic lung tissue of patients with COVID-19 has been reported. However, the mechanism underlying this activation is unknown. Objectives: To dissect the mechanisms underlying SARS-CoV-2 associated inflammation in severe COVID-19 patients, which is reminiscent of a so-called cytokine storm syndrome, we analyzed the involvement of IL-1β, a pivotal cytokine driving inflammatory phenotypes, whose maturation and secretion are regulated by NLRP3 inflammasome. Methods: We analyzed NLRP3 activations using confocal microscopy, plasma cytokines levels, cytokines secretion following in vitro stimulation of blood circulating monocytes and whole blood RNA sequencing. The role of ORF3a SARS-CoV2 protein was assessed by confocal microscopy analysis after nucleofection of a monocytic cell line. Results: We observed that circulating monocytes isolated from COVID-19 patients revealed ASC specks formation, signal of NLRP3 activation. Furthermore this cells spontaneously secrete IL-1β in vitro. This spontaneous activation reverts following patient’s treatment with the IL-1 receptor antagonist anakinra. Transfection of THP-1, a monocytic cell line, with cDNA coding for the ORF3a SARS-CoV2 protein, leading to ASC speck formation. Conclusion: These results provide further evidence that NLRP3 and IL-1β targeting could represent an effective strategy in this disease and suggest a mechanistic explanation for the strong inflammatory manifestations associated to COVID-19. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
M. Bizjak 1 , N. Emeršič 1 , M. Zajc Avramovič 1 , T. Vesel Tajnšek 1 , G. Markelj 1 , V. Berce 2 , N. Toplak 1,3 , A. Taddio 4,5 , T. Avčin 1,3 1 Department of Allergology, Rheumatology and Clinical Immunology, Children’s Hospital, University Medical Centre Ljubljana, Ljubljana, 2 Division of Pediatrics, University Medical Centre Maribor, Maribor, 3 Department of Pediatrics, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia, 4 Institute for Maternal and Child Health, IRCCS “Burlo Garofolo”, 5 University of Trieste, Trieste, Italy Correspondence: N. Emeršič Introduction: Children are less likely than adults to develop serious disease upon infection with SARS-CoV-2 but are at increased risk for inflammatory and autoimmune diseases linked to the virus. The reported incidence of multisystem inflammatory syndrome in children (MIS-C) varied from 0.2 to 11.4/100,000 persons under 21 years. It is yet unknown whether MIS-C can recur after SARS-CoV-2 reinfection or COVID-19 vaccination. Objectives: To estimate the incidence and describe the spectrum of inflammatory and autoimmune diseases linked to SARS-CoV-2 infection and COVID-19 vaccination in pediatric patients from two neighbouring South Central European countries and regions, Slovenia and Friuli Venezia Giulia (FVG), Italy. Methods: We performed a multi-centre prospective cohort study of all children (under 18 years) newly diagnosed with MIS-C or other inflammatory/autoimmune diseases linked to SARS-CoV-2 infection, who were admitted to the pediatric tertiary care hospitals in Slovenia or FVG, Italy during the period from January 1, 2020, to December 31, 2021. These hospitals serve a combined population of 546,032 children. Only patients with positive anti-SARS-CoV-2 antibodies and/or positive SARS-CoV-2 PCR test within 3 months prior to disease onset were considered for estimating the disease incidence. We obtained the number of patients with serious adverse events (SAE) after COVID-19 vaccination and with severe COVID-19 in the same population. This study was conducted as a part of the EU interregional Italy-Slovenia project CATTEDRA (Cross border cooperation for innovative diagnosis of rare diseases in paediatrics). Results: 192 children were diagnosed with inflammatory and autoimmune diseases linked to SARS-CoV-2. The most common disease was MIS-C in 112 patients, followed by vasculitis in 74 patients of whom the vast majority presented with chilblain-like lesions. The neurological cases included patients with a rare Guillain-Barré syndrome variant with predominant cranial nerve involvement, peripheral facial palsy, autoimmune encephalitis and long COVID. One patient presented with acute myocarditis and one with acute pericarditis. Median age at diagnosis was 11.9 years (IQR 7.6 -14.7). All included patients were White. Incidence of MIS-C was one in 860 children after SARS-CoV-2 infection and one in 5460 of all children. Cumulative incidence of MIS-C since the start of the pandemic was 18.3/100,000 children. Until December 31, 2021, 95,191 children (17 %) received at least one dose of COVID-19 vaccine. Two patients with MIS-C and one patient with myositis presented after COVID-19 vaccination. All 3 had evidence of recent SARS-CoV-2 infection in form of positive anti-N SARS-CoV-2 antibodies. In the same period, 15 children were hospitalised with severe COVID-19. Eight patients from our cohort were vaccinated against COVID-19 median 8 months after MIS-C and further 20 patients had a SARS-CoV-2 reinfection 1.2-15 months after MIS-C. None of them experienced SAE or recurrence of MIS-C. Conclusion: MIS-C was the most common manifestation and its incidence in this predominantly white population was higher than previously reported. Based on our limited experience, MIS-C does not seem to recur after SARS-CoV-2 reinfection or COVID-19 vaccination. Autoimmune diseases were much more common after SARS-CoV-2 infection than after COVID-19 vaccination. Hospitalisations due to MIS-C were seven times as frequent as hospitalisations due to severe COVID-19 in children. Disclosure of Interest : None declared
C. Butters 1,2 , D. R. Abraham 3 , T. Spracklen 2 , J. Day 2 , H. Rabie 3 , C. Scott 2 , K. Webb 2 1 Pathology, 2 Paediatrics and Child Health, University of Cape Town, 3 Tygerberg Hospital, Stellenbosch University, Cape Town, South Africa Correspondence: J. Day Introduction: The impact of the variants of concern (VOC) of SARS-CoV-2 on Multisystem Inflammatory Syndrome in Children (MIS-C) on disease phenotype and severity is not yet understood. Objectives: Describe the demographic and clinical features of MIS-C across the four variant specific SARS-CoV-2 waves. Methods: Children with MIS-C admitted to the Red Cross War Memorial Children’s Hospital (RXH) and Tygerberg Hospital (TBH) between 22 June 2020 and 27 March 2022 were recruited. Demographic, clinical and outcome data were recorded. Results: There were four SARS-CoV-2 waves in Cape Town, South Africa during the time period driven by the wildtype, beta, delta and omicron variants respectively. During the time period, 129 children had confirmed MIS-C. There was no significant difference in age across the four waves, however children in the fourth wave tended to be lower (Table 1). There was no difference in sex distribution or the number of children with comorbidities. The presence of clinical signs and symptoms remained relatively constant with only diarrhoea being less prevalent in the wave driven by Omicron (p=0.028). Blood levels of inflammatory, coagulation and cardiac markers, markers, immune cells, and sodium were unchanged throughout the four waves. Abnormal ECHO findings were common, but no more so in one wave than another. However, there was a trend towards an increase in the presence of coronary artery involvement in the second and third waves (p=0.056) and reduced ejection fraction in the first and fourth waves (p=0.065). There was no difference in the need for ICU admission, inotropic support or length of hospital stay. There was only one death in the wave driven by the Omicron variant. Conclusion: Overall, there does not appear to be a difference in the demographic, clinical or outcomes of MIS-C across the four waves of SARS-CoV-2 infection in Cape Town, South Africa. In this setting MIS-C remains a severe disease with a stable phenotype. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
R. Caorsi 1 , A. Consolaro 1,2 , C. Bracaglia 3 , F. Minoia 4 , M. Cattalini 5 , P. Brogan 6 , C. Wouters 7 , A. Taddio 8 , F. Candotti 9 , I. Meyts 10 , F. De Benedetti 3 , N. Ruperto 11 , M. Gattorno 1 on behalf of on behalf of the Steering Committee for HyperPED-COVID registry for RITA-ERN, ISSAID, PRES, ESID, PRINTO 1 Clinica pediatrica e reumatologia, Gaslini Institute, Genoa, Italy, 2 Dinogmi, Università degli studi di Genova, Genova, 3 Division of Rheumatology, IRCCS Ospedale Pediatrico Bambino Gesù, Roma, 4 Pediatric rheumatology, Fondazione IRCCS Ca’ Granda-Ospedale Maggiore Policlinico, Milano, 5 Pediatrics Clinic, University of Brescia and ASST Spedali Civili di Brescia, Brescia, Italy, 6 UCL GOS Institute of Child Health, London, United Kingdom, 7 Pediatric Rheumatology and Immune-inflammatory diseases, University of Leuven, Leuven, Belgium, 8 Institute for Maternal and Child Health IRCCS “Burlo Garofolo” and Univeristy of Trieste, Trieste, Italy, 9 Division of Immunology and Allergy, Laboratory of Inherited Immune Disorders, CHUV, Lausanne, Switzerland, 10 Department of Microbiology and Immunology, University of Leuven, Leuven, Belgium, 11 Printo, IRCCS Istituto Giannina Gaslini, Genova, Italy Correspondence: R. Caorsi Introduction: The Multisystem Inflammatory Syndrome in Children (MIS-C), is a serious inflammatory condition characterized by a; the clinical and laboratory features are similar to those usually observed in Kawasaki disease, cytokine storm syndrome and macrophage activation syndrome. Objectives: to create an International multicenter collection of patients with MIS-C involving the main pediatric networks committed in the care of patients with hyperinflammatory conditions. Primary endpoint of the project is to collect information on clinical presentation, laboratory parameters, clinical outcome and response to treatment of patients with MIS-C. Methods: a steering committee constituted by representatives of ERN-RITA, PRES, ESID and ISSAID and with the coordination of PRINTO developed a shared form to collect. The registry is available online on PRINTO and ESID websites (www.printo.it , www.ESID.org). Results: Currently, more than 1000 patients from 44 centers of 20 countries worldwide have been included in the study; completed data are available for 688 patients. At the time of admission, 56 (8%) patients were younger than 2 years, 173 (25%) between 2 and 6 years, 279 (41%) between 6 and 12 years, while 180 (26%) patients were older than 12 years. 234 (34%) patients required ICU admission; 51 (7.4%) patients presented long term sequaele and 7 (1%) patients died. Mucocutaneous manifestation were observed in 586 patients (85.2 %), hematological in 583 (84.7%), gastrointestinal in 556 (80.8%), cardiovascular in 360 (52.3%), lymphoid organ in 356 (51.7%), respiratory in 238 (34.6%), musculoskeletal in 217 (31.5%), neurological in 131 (19%), genito-urinary in 75 (10.9%). Most common treatment were Ig infusion (576 patients, 84%) and corticosteroids (572 patients, 83%); 78 patients (11%) were treated with biologics. Conclusion: The first analysis of the registry confirms that MISC is a severe inflammatory condition, requiring anti-inflammatory treatment. Even if the mortality and the complication rate is low, one third of patients required intensive care unit admission. Further analysis will be performed in order to identify predictors of outcome. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
E. Farias 1 , G. Clemente 2 , M. T. Terreri 2 , M. Pavão Junior 1 , S. Sales 1 , L. Nascimento 1 , D. Pavão 1 , V. Santos 1 , A. Pinheiro 1 , M. Alves 1 , P. Carvalho 3 1 Pediatrics, Centro Universitário Metropolitano da Amazônia, Belém, 2 Pediatrics, UNIVERSIDADE FEDERAL DE SÃO PAULO, São Paulo, 3 Pediatrics, Fundação Hospital das Clínicas Gaspar Viana, Belém, Brazil Correspondence: G. Clemente Introduction: Some children and adolescents may develop critical disease related to SARS-CoV-2, such as severe coronavirus disease (COVID-19) or multisystem inflammatory syndrome in children (MIS- C). Objectives: Our aim was to study the outcome of patients with critical disease related to SARS-CoV-2 hospitalized in Paediatric Intensive Care Units in the Brazilian Amazonian region. Methods: This multicentre cohort included children and adolescents with critical disease related to SARS-CoV-2 current or recent infection who were admitted into three Amazonian PICUs between April 2020 and January 2022. Exclusion criteria were patients who had positive microbiology other than SARS-CoV-2, had no criteria for PICU admission, or were immunocompromised. Results: A total of 192 patients were included: 65 (33.9%) from the MIS-C group and 127 (66.1%) from the severe COVID-19 group. The total median age was 3.5 (0.8-8.6) years; 4.4 (1.0-9.3) years in MIS-C and 2.3 (0.75-8.4) years in severe COVID-19 group (p=0.241). Previous history of complex chronic condition was present in 123 (64.1%) patients. Almost half of the patients (94 [49%]) required mechanical ventilation support. The median mechanical ventilation time was 4 (3-9) days, and median length of hospital stay was 14 (10-20) days. Death occurred in 35 (18.2%) patients, 16 (24.6%) in MIS-C group and 19 (15%) in severe COVID-19 group, with no statistical difference. The results of SARS-CoV-2 tests and outcomes are described in Table 1. Conclusion: In the Brazilian Amazonian region, we observed elevated requirement for mechanical ventilation support, long length of hospital stay, and high number of deaths in paediatric patients with critical disease related to SARS-CoV-2. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
F. Corona 1 , F. Burlo 1 , S. Pastore 2 , M. V. Mastrolia 3 , F. Ricci 4,5 , S. A. Rumeileh 3 , M. Cattalini 4,5 , G. Simonini 3 , A. Taddio 1,2 1 Department of Medicine, Surgery, and Health Sciences, University of Trieste, 2 Institute for Maternal and Child Health - IRCCS “Burlo Garofolo” , Trieste, 3 Pediatric Rheumatology Unit, Meyer Children’s University Hospital, School of Human Health Science, Florence, 4 Pediatrics Clinic, University of Brescia, 5 ASST Spedali Civili, Brescia, Italy Correspondence: A. Taddio Introduction: Multisystem inflammatory syndrome in children (MIS-C) is a rare hyperinflammatory and life-threatening complication that usually occurs 2–6 weeks after SARS-CoV-2 infection. Objectives: Many studies have investigated the short and long-term consequences of this condition, but the potential risks of SARS-CoV2 vaccination or reinfection in those children who have previously been hospitalized for MIS-C are still lacking. Methods: This is a multicenter retrospective study which investigated the rate and even the consequences of SARS-CoV-2 vaccination or reinfection in children who were hospitalized for MIS-C at Institute for Maternal and Child Health Burlo Garofolo in Trieste, at Meyer Children’s University Hospital in Firenze, and at the Paediatric Clinic of ASST Spedali Civili in Brescia, Italy. The parents of these children were asked if they were vaccinated or if they were reinfected with SARS-CoV-2 after MIS-C diagnosis. Results: Overall, 69 children were investigated. The median age was 10 years (range: 1-19). There were 43 males and 26 females. They developed MIS-C from April 2020 to April 2022. Afterwards, 23 patients were vaccinated against SARS-CoV2 infection. Ten patients could not be vaccinated because younger than 5 years-old. The median time from MIS-C to the first dose of vaccine was ten months (range: 6-15). Just 7 patients complained mild symptoms soon after the vaccination, such as headache, fatigue or pain at the injection site. Moreover, 9 patients overall got another Covid infection after MIS-C. The median time from MIS-C to reinfection was 13 months (2-18). Two of them were hospitalized for fever and gastrointestinal or upper airway symptoms respectively and were discharges 2 days after, 1 was asymptomatic, 7 developed mild symptoms such as sore throat, cough and mild fever, and 2 of the latter were vaccinated against SARS-CoV2. No one reported a new MIS-C diagnosis. Conclusion: This study reports that MIS-C was not a risk factor for severe complications in case of SARS-CoV2 reinfection or vaccination. All the symptoms complained by children in case of reinfection or vaccination were mild. In particular, no new MIS-C cases were reported in this cohort of patients. Disclosure of Interest : None declared
J. Day 1 , U. Rohlwink 2 , T. Spracklen 1 , C. Butters 1 , R. Stander 1 , H. Facey-Thomas 1 , A. Davis 3 , C. Scott 1 , L. Zühlke 4 , K. Webb 1 1 Paediatric Rheumatology, 2 Neuroscience, University of Cape Town, Cape Town, South Africa, 3 School of Life and Medical Sciences, UCL, London, United Kingdom, 4 Paediatrics, University of Cape Town, Cape Town, South Africa Correspondence: J. Day Introduction: Multi-inflammatory syndrome in children (MIS-C) is characterized by hyperinflammation following infection or exposure to SARS-CoV-2 in children and young people. Approximately a third of MIS-C cases have central nervous system manifestations, the pathogenesis of which is unknown. Here, we characterize the cytokine profile of the cerebrospinal fluid (CSF) of a small cohort of children with MISC with neurological manifestations. Objectives: To characterise the cytokine milieu of cerebrospinal fluid in those with MIS-C compared to controls. Methods: CSF was acquired via lumbar puncture, as part of in-hospital clinical care, from 3 children who met the criteria for MIS-C with neurological manifestations and 3 control patients presenting for procedures requiring CSF; such as revision of a blocked ventriculoperitoneal shunt. Cytokine concentrations were measured via chemiluminescence and analyzed using SPSS statistical software. Results: Conclusion: In a small number of patients, we show that certain inflammatory cytokines are raised in the CSF of children with MIS-C and neurological manifestations. Understanding the specific cytokine profile of this condition may be key in efforts to understand pathogenesis, and identify potential treatment targets. These data underpin the need for further studies investigating the mechanism of CNS disease in MIS-C. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
M. M. N. F. Soares, A. L. C. Bessa, L. R. M. P. Crivelenti, V. P. Baldão, D. C. Aragon, M. C. Cervi, A. K. Matsuno, R. A. P. de La OssaUntil the moment, Multisystem Inflammatory Syndrome (MIS-C), temporally associated with SARS-CoV-2 infection is a challenging diagnosis in pediatric practice. As the signs and symptoms associated with the syndrome are very common to other serious febrile illness, the confirmation of the previous infection by RT-PCR or serology for SARS-CoV-2, is not enough in some cases, given the co-infection cases and coincidental relationship. More sensitive and specific criteria for syndrome classification are necessary. Objectives: To establish clinical and laboratory criteria to differentiate, at admission, patients with MIS-C from patients with other febrile syndromes unrelated to SARS-CoV-2 infection. Methods: Cross-sectional observational study of children and adolescents under 18 years of age with febrile syndromes who manifested with signs and symptoms characteristic of MIS-C, attended at the Clinical Hospital of the Ribeirão Preto Medical School - São Paulo University, in the period of March of 2020 to October 2021. The diagnosis of MIS-C was performed based on CDC and/or WHO criteria. Data from patients with MIS-C and other febrile syndromes not associated with SARS-CoV-2 at admission were compared using Fisher’s exact and Wilcoxon tests, with a level of significance of 5%. The measure of association used was the relative risk (RR) and the adjusted relative risk (RRaj) for the variables that were significantly different between the groups with 95% CI. The Hospital Ethics and Research Committee approved the study. Results: During the study period, 44 children and adolescents under 18 years of age presented any of the clinical and laboratory criteria for MIS-C at admission. MIS-C temporally associated with SARS-Cov-2 infection was confirmed in 23 patients (group 1). The final diagnosis of the others febrile syndromes (group 2) were: acute viral gastroenteritis, atypical Kawasaki, acute viral bronchiolitis, viral myocarditis, mononucleosis-like syndrome, pneumonia, post-streptococcal glomerulonephritis, mesenteric adenitis, type 1 diabetes mellitus and staphylococcal shock. The groups were clinical and laboratory similar, except for older age, presence of skin rash, previous antibiotic use, hyponatremia and increase in C-reactive protein (CRP) which were more frequent in the group 1. CRP above 6.4 mg/dl increased the risk of the patient being in the MIS-C group at 3 times, even when the value was adjusted for previous antibiotic use. Conclusion: The clinical and laboratory criteria associated with MIS-C are similar to other diseases. In our study, the CRP above 6.4 mg/dl at admission was the main risk factor associated with MIS-C. Disclosure of Interest : None declared
A. L. C. Bessa 1 , M. M. N. F. Soares 1 , L. R. M. P. Crivelenti 1 , W. A. D. O. Borghi 1 , P. H. F. Muccillo 1 , M. C. Cervi 1 , R. A. P. de La Ossa 1 , L. M. De Carvalho 1 Pediatrics, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, Brazil Correspondence: L. M. De Carvalho Introduction: Multisystem inflammatory syndrome in children (MIS-C) is a potentially severe pediatric hyperinflammation disease associated with SARS-CoV-2 infection. The acute manifestations of the disease have been extensively described but the evolution profile studies are still limited. Objectives: To describe the medium-term evolution profile of a cohort of MIS-C patients followed in a tertiary care clinic. Methods: Prospective cohort study of 12 months follow-up of patients under 18 years of age in a specialized multidisciplinary outpatient clinic in a Brazilian hospital, diagnosed as MIS-C between March 2020 and December 2021. Assessments of anthropometric data, clinical complaints, biochemical markers, and imaging tests were performed at a mean of 1, 3, 6, 9 and, 12 months after admission. For continuous data comparison were used Whitney test (2 groups) or the Kruskal-Wallis test (3 or more groups). A significance level of 5% was established. Results: Twenty-two patients met CDC and/or WHO criteria for MIS-C and had laboratory evidence of SARS-CoV-2 infection. Patients generally had a good outcome during the follow-up period. The average length of stay of patients was 8 days, with an average of 4 days in an intensive care unit. The mean time of outpatient follow-up was nine months, with 9 patients followed up for one year. There were no deaths. One patient evolved with pulmonary pneumatocele. Thirty-two percent of patients had cardiovascular changes at presentation, including left ventricular systolic dysfunction, valvular insufficiency, pericardial effusion, and hypokinesis of the cardiac wall. Only one patient maintained cardiac chamber enlargement during follow-up, without clinical repercussions. Coronary changes were not described. One of patients developed stage 1 arterial hypertension. Fourteen percent of the patients had transient renal changes. Gastrointestinal symptoms were reported in 82% of the patients and 9% had abdominal pain after 6 months of follow-up. One of patients developed chronic arthritis during follow-up. All patients had elevated markers of systemic inflammation and troponin and NT-pro-BNP concentrations at baseline, with normalization of these tests within one month, except for 2 patients who maintained elevated VHS for 6 months. Conclusion: This study supports the idea of an optimistic short and medium-term outcome for MIS-C, despite the severe initial presentation. Disclosure of Interest : None declared
A. De Matteis 1 , M. Pardeo 1 , G. Marucci 1 , F. De Benedetti 1 , C. Bracaglia 1 Division of Rheumatology, IRCCS Ospedale Pediatrico Bambino Gesù, Roma, Italy, Roma, Italy Correspondence: C. Bracaglia Introduction: Systemic juvenile idiopathic arthritis (sJIA) is a polygenic autoinflammatory disease in which inflammatory cytokines, such as IL-1 and IL-6, play a pivotal role. Macrophage activation syndrome (MAS) is the most severe potentially life-threatening complication of sJIA and it can be triggered by infections. Only few reports describe the clinical course of SARS-CoV-2 infection in sJIA patients during treatment with cytokine inhibitors. Moreover, the role of IL-1 inhibitors in preventing the development of MIS-C is not known. Objectives: We report 9 cases of SARS-CoV-2 infections occurred in sJIA patients during IL-1 inhibition treatment. Methods: Demographic, clinical and laboratory features of the 9 patients (7 female, median age 7 years) were summarized in table 1. Results: All patients were in clinical inactive disease (CID) at time of SARS-CoV-2 infection and the infection was pauci-symptomatic. Two patients were hospitalized to be monitored. No episodes of MAS, MIS-C nor lung involvement occurred. Three patients were in treatment with canakinumab and 6 with anakinra; 2 patients were also on low dose of prednisone. Five out of 9 patients continued IL-1 inhibitors at the same dose, 3 patients increased the dose, while one patient shifted from canakinumab to anakinra (table 1). Conclusion: In our sJIA patients treated with IL-1 inhibitors, SARS-CoV-2 infection was not associated with disease flare or with worse clinical outcome. SARS-CoV-2 infection did not trigger MAS in any of those patients and none of them developed MIS-C. Considering the role of IL-1 in the pathogenesis of the cytokine storm syndrome, we may hypothesize the preventing role of IL-1 inhibitors in the disease flare during SARS-CoV-2 infection. References: Demir F, Ulu K, Çağlayan Ş, Coşkuner T, Sözeri B. Clinical course of COVID-19 in children with rheumatic disease under biologic therapy. Clin Exp Rheumatol. 2021 de Carvalho JF. COVID-19 in a pregnant ASIA syndrome patient. Eur Rev Med Pharmacol Sci. 2021 Calvo C, Udaondo C; Rheumatic Diseases EPICO-AEP Working Group. COVID-19 in Children With Rheumatic Diseases in the Spanish National Cohort EPICO-AEP. J Rheumatol. 2021 Patient Consent: Yes, I received consent Disclosure of Interest : A. De Matteis: None declared, M. Pardeo: None declared, G. MarucciN. Emeršič 1 , M. Debeljak 2,3 , A. Koren Jeverica 1 , G. Markelj 1 , N. Toplak 1,3 , T. Vesel Tajnšek 1 , M. Zajc Avramovič 1 , T. Avčin 1,3 1 Department of Allergology, Rheumatology and Clinical Immunology, University Medical Centre Ljubljana, Children’s Hospital, 2 Clinical Institute for Special Laboratory Diagnostics, University Children’s Hospital, University Medical Centre Ljubljana, 3 Department of Pediatrics, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia Correspondence: N. Emeršič Introduction: Since the onset of the 2019 coronavirus disease pandemic (COVID-19) caused by the severe acute respiratory syndrome virus, coronavirus type 2 (SARS-CoV-2), many articles have been published describing non-infectious manifestations associated with the infection, including COVID-19-associated vasculitis/vasculopathy. In children, most of these vascular complications are chilblain-like lesions. Objectives: To determine possible risk factors, which contribute to the development of vasculitis/vasculopathy in children, to determine the role of genetics, immunological profile of the patients and possible endothelial cells changes, to evaluate long term outcome of these complications and to offer possible treatment options. Methods: In our prospective cohort study, we included children up to 18 years of age with COVID-associated vasculitis/vasculopathy, who were examined at Children’s hospital University Medical centre Ljubljana, Slovenia from March 2020 until May 2022. Data collection included epidemiological, clinical information, associated diseases, extended blood analyses and genetic information, which were sistematically evaluated and compared. All children were tested for specific anti-SARS-CoV-2 immunoglobulin antibodies and had genetic analyses for TREX1, type I interferonophaties and monogenic vasculitides. Changes on the skin were dermatoscopically observed and evaluated with thermographic camera. We searched for similarities and differences from known vasculitidies and systemic connective tissue diseases with emphasis on clinical and immunological profile of the patients. Approval from the institutional Ethics Committee has been obtained. Results: 45 children were diagnosed with COVID-19-associated vasculitis/vasculopathy. The estimated incidence was one in 1522 children after SARS-CoV-2 infection. All included patients were Caucasian. The mean age at diagnosis was 12 years (2 months-17,5 years). 29/45 patients (64%) were female. The most common changes were chilblain-like lesions. 20/44 (45%) patients were tested positive for SARS-CoV-2 antibodies. In 24/45 (53%) patients, only lower extremities were affected, in 6/45 (13 %) patients only upper extremities and in 10/45 (22%) patients upper and lower extremities were affected, one girl had changes only on her face and another on the other parts of the body. The skin changes were usually mildly painful, some of the patients complained of pruritis. Vasculitis was more severe in younger children, where other organs, not just skin, may have been involved. A 1-year old girl developed necrotizing stomatitis, vasculitic rash, skin desquamation on the fingers and toes, and persistent hypertension. A 2-month-old boy developed skin and severe gastrointestinal vasculitis. Both patients with severe vasculitis had negative genetic analysis for TREX1, type I interferonophaties and monogenic vasculitides. A 13-years old boy developed Raynaud’s phenomenon after the second dose of SARS-CoV-2 vaccination. In 10 patients the problems recurred. In majority of patients there were some dilated capillary loops along fingernails. The affected parts of the limbs photographed with thermographic camera showed elevated temperature compared to unaffected areas. Conclusion: In children, COVID-19-associated vasculitis is the second most common autoimmune manifestation after SARS-CoV-2 infection. Most patients have uneventful disease course with complete resolution. 4,4% (2/45 patients) had severe vasculitis with internal organ involvement or mutilating skin and musculoskeletal lesions. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
J. M. Galindo Hayashi 1 , K. González Moctezuma 1 , L. F. González González 1 , G. R. Ayala Villegas 1 , J. G. Rodriguez Chong 1 , A. V. Villarreal Treviño 2 1 Pediatrics, 2 Pediatric Rheumathology, Programa Multicentro TecSalud, Monterrey, Mexico Correspondence: J. M. Galindo Hayashi Introduction: Cardiovascular involvement has been found in 86.5% of patients with multisystem inflammatory syndrome in children (MIS-c) (1). One of MIS-c´s diagnostic criteria are elevation of inflammatory markers (2). Objectives: To analyse inflammatory markers to determine which of them are linked with cardiovascular involvement in MIS-c among the paediatric population of a third level hospital in northeast Mexico. Methods: The researchers of this study conducted a cross-sectional retrospective study. The World Health Organization (WHO) MIS-c criteria were used to choose the eligible patients to analyse. Cardiovascular involvement was confirmed by transthoracic echocardiography (TTE). The researchers dichotomize MIS-c patients in two groups, those with and without cardiovascular involvement. Descriptive statistics were performed by calculating the mean and standard deviation (SD) for each group. To test stastitical significance between inflammatory markers in the two groups we used the Mann-Whitney U test ( U ). The following markers were analysed: CRP, ERS, Ferritin, D-dimer, albumin, CK, CK-MB, neutrophils, lymphocytes, DHL, leukocytes, platelets, and fibrinogen. Results: 70 patients met the eligibility criteria, six were excluded for not having an TTE, and four for having an incomplete medical record. From the 60 patients left, 58.2% had cardiovascular involvement confirmed by TTE (n=25). We found higher prevalence of cardiovascular involvement in males (56%) and the mean age was 7 years and 1 month. The mean, standard deviation (SD) and p values are represented in the following table (Table 1). Conclusion: Markers such as BNP and IL-6 have shown association to cardiovascular involvement in MIS-c (3). Nevertheless, these markers aren’t commonly available in health centres in third world countries. A panel of specialists with expertise in MIS-C, concluded that some patients with mild symptoms may require only close monitoring without immunomodulatory treatment (4). These could set ground of exclusive use of IVIG and/or steroids in patients with suggestive inflammatory markers of cardiovascular involvement. This study found that there is suggestive evidence that platelets and neutrophils could be used as markers of risk of cardiovascular involvement in MIS-c. The results of this study could serve as precedent to a prospective randomized experimental trial to evaluate if a series of inflammatory markers and clinical features could predict accurately cardiovascular involvement in MIS-c. With the final purpose of prognosticating which patients would benefit the most from IVIG and/or steroid therapy in centres with limited resources. Trial registration identifying number: Not applicable. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
G. A. Turrubiates-Hernández 1 , A. V. Villarreal-Treviño 2 , J. M. Galindo-Hayashi 1 1 Pediatrics, 2 Rheumatology, Tecnologico de Monterrey, Escuela de Medicina y Ciencias de la Salud, Monterrey, Mexico Correspondence: J. M. Galindo-Hayashi Introduction: MIS-C is a novel disease reported for the first-time weeks after the first cases of COVID-19 in 2020 were known. It is a heterogeneous vasculitis which can be difficult to diagnose since it can overlap to other entities. Objectives: NA Methods: NA Results: A previously healthy 8-year-old female presented to the ER with a history of 5 days of cervicalvolume increase, diarrhea, gastric content vomit and a 24hour subjective report of temperature rise with diffuse colic abdominal pain, hyporexia, myalgias, arthralgias, non-purulent conjunctivitis, hands and feet edema and malaise. Hours before admission, she presented palpebral hyperemia, pruriginous maculopapular wheal-like lesions on the neck, trunk, abdomen, legs and genitals as well as cracked injected lips. Three weeks prior, she presented with a mild upper respiratory tract infection. History of recent contact with patients COVID-19 positive was denied. On arrival, she was tachypneic (35 bpm), oxygen saturation of 95% under room air, tachycardic (136 bpm), hypotense (77/32) and with a 37°C temperature. Initial blood tests revealed kidney and liver dysfunction, leukocytosis, neutrophilia, lymphopenia, thrombocytopenia, elevated CRP, ferritin, and D-dimer, with a negative SARS COV 2 rapid antigen test (Table 1). Abdominal ultrasound revealed increased echogenicity of liver and kidneys. Echocardiography showed mild pericardial effusion and pericarditis with ejection fraction of 60%. Treatment was initiated with methylprednisolone (30mg/kg/dose), norepinephrine (0.05mcg/kg/min), spironolactone (12.5mg q12h) and rivaroxaban (10mg/day), she was transferred to the PICU after subspecialists evaluation. On day 7 of illness kidney dysfunction showed recovery and IVIG at 2g/kg/dose was started. On day 8 noradrenaline was withdrawn and oral feeding was started. The 3-day methylprednisolone bolus course ended and the shift to 1.5mg/kg/day with following weaning was made. On day 10 hand and feet edema subsided and abdominal pain disappeared. On day 11 the patient was transferred from the ICU to the general ward. Fever was never reported. She demonstrated a good clinical course and was diagnosed with MIS-C based on CDC criteria. On day 19 she was discharged with normal liver, kidney, and cardiac function. Treatment with antibiotics was not started since impaired consciousness, high fever, and livedo reticularis, which are usually noted in sepsis, were not present. The principal differential diagnosis was Kawasaki disease but, since 5 day- fever was not present, the patients age was not in the typical period, the history of an upper respiratory tract infection within the previous month, and blood test results, mild hyponatremia and elevated accute phase reactants, plus no other apparent cause of symptoms, MIS-C was suspected and the patient was treated as such. In patients with MIS-C diagnosis fever lasting more than 48 hours is nearly present in 100%, interestingly, in our patient subjective temperature rise was reported just one time and never again during her hospitalization, furthermore the clinical course progressed fast towards shock, which was rapidly reversed with immunomodulatory treatment. Conclusion: Our case study illustrates the importance of suspecting MIS-C when caring for patients under 21 years of age presenting with shock, even without clearly documented fever. Trial registration identifying number: NA Patient Consent: Yes, I received consent Disclosure of Interest : None declared
M. Ganeva, A. Teltcharova, A. Dimitrova, D. Hristova, K. Temelkova, T. Vasilev, V. Kostova, S. Stefanov 1 Department of Pediatric Rheumatology and Cardiology, University Children’s Hospital, Medical University Sofia, Sofia, Bulgaria Correspondence: M. Ganeva Introduction: The novel coronavirus disease 2019 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) faced us with a great challenge - multisystem inflammatory syndrome in children (MIS-C). A patient is diagnosed with MIS-C based on the presence of fever, involvement of 2 or more organ systems, laboratory evidence of inflammation and evidence of SARS-CoV-2 infection or exposure and when other plausible diagnosis are excluded. Objectives: To describe the demographic, epidemiological, clinical and laboratory parameters in patients diagnosed with MIS-C. Methods: Demographic and clinical parameters of MIS-C patients admitted between November 2020 and May 2022 were recorded. Blood samples of MIS-C patients were collected at the time of admission to the Department of pediatric rheumatology and cardiology, Sofia, Bulgaria. Heart ultrasound was performed in all patients. Results: Thirty-one MIS-C patients (15 girls and 16 boys) with a mean age of 7.35 years (1-17 years) were admitted for a period of 18 months. Nine patients had a history of household contact. Eight children had a history of SARS-CoV-2 illness. In all of the patients IgG SARS-CoV-2 antibodies were detected. Eighteen MIS-C patients were positive for IgM SARS-CoV-2 antibodies. Fever was observed in all children with a mean duration before admission of 5.67 days (3-10 days). Skin rash was noticed in 25 (80.6%) children. Kawasaki-like features were detected with 25 (80.6%) of the children having conjunctivitis. In ten MIS-C patients edema of the hands was noticed and twelve had erythema of the palms and soles. Eight of the children experienced cervical lymphadenitis. Periorbital edema and erythema were detected in ten children. Twenty-two patients (70.9%) had gastrointestinal symptoms - vomitting was observed in nine children, diarrhea in 5 patients and eight suffered from both. Myocarditis was diagnosed in nineteen (61.3%) children with eleven of them requiring inotropic support. Serositis was detected in sixteen (51.6%) patients. Neck pain was observed among eight (29%) children. Acute kidney injury occurred in two (6.4%) patients. All children had elevated CRP and ferritin at the time of admission with mean value of accordingly 193.47 mg/l (14.75-387.95 mg/l) and 1124.34 ng/ml (182.7-5708 ng/ml). ESR was not elevated in four of the patients. Lymphopenia was noted in twenty (64%) children. Seventeen (54.8%) patients had low platelets (92.4x10 9 /l (27-149 x10 9 /l)) at the time of admission. Hypoalbuminemia was detected in twenty-three children. Intravenous immunoglobulin treatment was performed in all but four children. Corticosteroids with an initial dose of 1.76 mg/kg were used in all MIS-C patients. Pulse steroid therapy was performed in one child. Low-molecular-weight heparin was injected subcutaneously in twenty-one children. Antiplatelet therapy was prescribed to twenty-six MIS-C patients. The mean hospital stay was 11.3 (6-23) days. All patients with myocarditis had favourable outcome and complete recovery. Conclusion: Our data demonstrate that mucocutaneous and gastrointestinal symptoms are most commonly observed in MIS-C patients with over half of the children being diagnosed with myocarditis in our study cohort. Favourable outcome and complete recovery was observed in all myocarditis patients. Acute kidney injury was observed in two patients. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
F. G. Abbate, A. Amodio, E. Miraglia del Giudice, A. N. Olivieri 1Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) continues to be an important health problem since December 2019 but the management of the infection and the pandemic trend considerably improved thanks to mass vaccination. The vaccination against SARS-CoV2 in patients with autoimmune inflammatory rheumatic disease is very important because these patients have a higher risk to get the infection and even more with a worse course. The two vaccines approved for them are the mRNA based BNT162b2 (BioNTech-Pfizer) and mRNA-1273(Moderna). Since May 2021 these vaccines have been approved also for children aged 12 and older, but there is still no data enough regarding their use in patients affected from rheumatological diseases as Juvenile Idiopathic Arthritis. Objectives: To evaluate the immunogenicity and safety of vaccination against SARS-CoV2 infection in JIA patients. Methods: We performed a retrospective observational study comparing the immunogenicity and safety of SARS-CoV-2 mRNA vaccine in JIA patients compared with healthy controls. We enrolled 36 patients and 33 healthy controls. For each patient we recorded JIA subtype, disease activity (according to JADAS-10), pharmacological treatment. According to EULAR/PRES recommendations all patients were in remission during vaccination and no one suspended pharmacological treatment. For both patients and controls we evaluated the side effects due to vaccination with telephone surveys. IgG antibodies against SARS-CoV-2 were quantified by CLIA method. Blood samples for the evaluation of Sars-Cov-2 antibodies were collected from all the subjects at the time of enrolment as well as one month after the second vaccination. Results: In table are reported baseline characteristic, antibodies levels and side effects after immunization with Sars-Cov-2 vaccines among children affected by JIA compared with controls. In our cohort we recorded: 15 oligoarthritis, 13 polyarthritis (11RF negative, 2 RF positive polyarthritis), 4 enthesis’s related arthritis, 4 systemic arthritis. JIA treatments was assigned according to ACR recommendations, in particular 6 patients were in treatment with NSAIDs, 7 with methotrexate, 9 with methotrexate and biological drugs, 14 with only biological drug. Regarding antibodies levels we did not found any statistically significant differences comparing the average level of antibodies between patients and controls. Evaluating JIA subtype we found that patients with systemic JIA produced lower levels of antibodies compared with patients with oligoarthritis (p = 0.05) polyarthritis (p = 0.03) and entesitis related arthritis (p 0.02). Investigating the influence of treatments on Sars-Cov-2 antibodies production, no statistically significant difference was detected between the different treatment arms. No disease flare was noted based on evaluation of the JADAS-10 at baseline (before first vaccination) as well as in follow-up visits. As for side effects, local pain and need of NSAIDs were more frequent in healthy control than patients, the other symptoms were comparable in both groups. Conclusion: In this study we did not find any difference in safety and immunogenicity of Sars-Cov-2 vaccines between JIA patients and healthy controls. Although our cohort is small it may be concluded that the vaccine has an adequate safety and tolerability profile. Further research is needed to investigate if the differences we observed effect the long-term protection offered by vaccine Patient Consent: Yes, I received consent Disclosure of Interest : None declared
K. González-Moctezuma 1 , A. V. Villarreal-Teviño 2 , M. M. Rangel-Fuentes 2 , J. E. Mares-Gil 2 , O. Tamez-Rivera 1,2 , B. D. J. Fortuna-Reyna 2 , A. Rojas-Martínez 3 , G. Vargas-Duarte 1 1 Pediatrics, Tecnológico de Monterrey, Escuela de Medicina y Ciencias de la Salud, 2 Pediatrics, Hospital Regional Materno Infantil, Secretaria de Salud Nuevo León, 3 Escuela de Medicina y Ciencias de la Salud, Tecnológico de Monterrey, Monterrey, Mexico Correspondence: K. González-Moctezuma Introduction: The precise risk factors which predisposed children to present Multisystemic Inflammatory Syndrome associated to COVID-19 (MIS-C) are certainly unknown; likewise its wide spectrum of clinical presentations and complications. Objectives: To analyze and describe the different phenotypes of clinical, paraclinical and sociodemographic presentation in pediatric patients with diagnosis of MIS-C and Kawasaki disease (KD) in our center. Methods: This was a cross-sectional descriptive, observational and retrospective study. Criteria inclusion were patients who met World Health Organization definition of MIS-C and KD who were diagnosed and hospitalized between 0-15 years old from June 2020 to November 2021. Patients with incomplete medical records or incomplete diagnosis criteria were excluded. Information was collected by clinical record review and a data base was created using clinical and paraclinical variables which were then separated in groups according to their phenotype of presentation: Classic MIS-C: Main gastrointestinal involvement with abdominal pain, vomit or diarrhea; Kawasaki like: Main mucocutaneous involvement with dermatosis and conjunctival injection; Lethal MIS-C:Hemodynamic compromise with refractory shock, vasoactive agents and pediatric intensive care unit admission; Kawasaki disease (KD): Diagnosis criteria for KD and absent history of COVID-19 disease or contact (negative PCR-SARS-CoV-2, negative antigenic and serologic tests). Results: n=63 patients with MIS-C (classic=20, KW-like=25, lethal=18) and n=14 patients with KD. Male gender predisposition was observed in lethal group with 72%(n=13). The median age at Kawasaki-like phenotype were children under 5 years old (IQR:2-7), in classic phenotype was 7 years (IQR:5-11) and lethal phenotype was 10 years (IQR:7-14). 61 patients were previously healthy children. Overweight or obesity was present in 15% (n=3) of MIS-C, 16% (n=4) of KW-like and 33% (n=6) of lethal phenotype. 6 patients from the lethal phenotype died, having all of them seizures previous to their death and representing a global mortality rate of 10%. The median number of inflammatory markers was higher in lethal phenotype, while median number of platelet count was the lowest in this group, expected thrombocytosis was observed in KD. Biological results, median and IQR values are described on the following table (table 1). Conclusion: Malnutrition, male gender and adolescent group of age were related to severity. The higher number of inflammatory markers at their admission to the hospital was directly proportional to severe form of disease; unlike platelet count which was inversely proportional presenting severe thrombocytopenia and bleeding in the lethal phenotype group. Seizures were highly related to mortality. It is fundamental to stay alert and create affordable strategies for early prevention, diagnosis, treatment and management of complications to support our children while we continue fighting against COVID-19 and this life threatening conditionGotloib 1,2 , A. ziv 1,2 , R. haviv 1,2 , Y. uziel 1,2 1 Pediatric rheumatology, Meir hospital, kfar saba, 2 Sackler School of Medicine , Tel Aviv, Israel Correspondence: V. Gotloib Introduction: Following the Coronavirus Disease 19 (COVID-19) pandemic, a new syndrome was described in April 2020, of a hyperinflammatory condition termed Multisystem Inflammatory Syndrome in Children (MIS-C). This life-threating, multi-organ failure can present as a severe disease that requires urgent diagnosis and therapy. As in many rheumatologic diseases, no single parameter or analysis can be used to diagnose this syndrome; rather, it requires a combination of clinical and laboratory criteria along with the elimination of other etiologies, mainly infectious. Objectives: To introduce four cases that at presentation were diagnosed as MIS-C, but after broader analysis were found to be different illnesses. Methods: we collected data regarding demographics, diagnosis, evaluation, treatment and follow up from the patients’ electronic medical records. Results: Case 1 : A 16-year-old boy presented with high fever, headache, new onset of psychosis, increased inflammatory markers and cytopenia 3 weeks after exposure to a patient infected with COVID-19, Empiric treatment with doxycycline was initiated due to suspicion of an atypical infectious agent. After severe clinical deterioration occurred with severe shock, MIS-C was suspected and he was treated accordingly with steroids and supportive therapy. The final diagnosis was Borreliosis with Jarisch-Herxheimers reaction. Case 2 : A 7-year-old boy, presented with fever, gastrointestinal symptoms, valvulitis diagnosed by echocardiography, serositis, cytopenia, elevated inflammatory markers, acute renal failure, with hematuria and proteinuria, 2 weeks after COVID-19 infection. Improvement under steroid therapy was noted. The final diagnosis was systemic lupus erythematosus. Case 3 : A 14-year-old boy presented with high fever, chills, headache, rash, tachycardia, elevated inflammatory markers and elevated coagulation marker, 1 month after recovering from COVID-19. Corticosteroid and antibiotic therapy led to improvement. The final diagnosis was rickettsiosis. Case 4 : A 4-year-old girl, presented with high fever, strawberry tongue, skin rash, abdominal and chest pain, pleural effusion, serositis, hemodynamic shock and elevated inflammatory markers, 3 weeks after recovering from COVID-19. The final diagnosis was gas empyema and streptococcal toxic shock syndrome. Conclusion: In many cases, anamnesis of a recent COVID-19 infection brings us to think of a post-COVID-19 syndrome, but as these cases illustrate, it is very important to maintain a broad differential diagnosis with emphasis on so-called “older” diseases, that can “mimic” MIS-CHaslak 1 , D. Ozbey 2 , M. Yildiz 1 , A. Gunalp 1 , A. Adrovic 1 , S. Sahin 1 , O. Koker 1 , A. Aliyeva 1 , V. Guliyeva 1 , G. Yalcin 1 , G. Inanli 1 , B. S. Kocazeybek 2 , O. Kasapcopur 1 , K. Barut 1 1 Pediatric Rheumatology, 2 Department of MicrobiM. Yildiz Introduction: Although current studies mostly indicate that the presence of inflammatory rheumatic disease (IRD) in childhood is not a significant risk factor for severe coronavirus disease-2019 (COVID-19), data on monitoring the seropositivity status of this patient group are limited. Chronic inflammation and immunosuppressive medications may affect the serological response in children with IRD, and considering the vaccination needs of these patients, it is seen that there is a need for studies that monitor their serological status. Objectives: We aimed to monitor the Severe Acute Coronavirus-2 (SARS-CoV-2) antibody levels of patients with IRD and healthy children who were previously found seropositive. Methods: In addition to the participants who were found to be seropositive in one of our previous studies, patients under 21 found seropositive between March 2020 and October 2020 were included in the study. Antibody levels of all the subjects were measured again at the third and sixth months by ELISA method. In this process, their symptoms were also questioned in terms of COVID-19. Results: The study included 35 participants (female/male: 1.69) (Healthy control group: 10, patient group not receiving biological therapy: 19, patient group receiving biological therapy: 6). Their mean age was 14.27 ± 5.49 years. Of the participants, 13 (37.1%) had a history of symptomatic infection, and 4 (11.4%) had a history of hospitalization. At the end of the six-month observation, a significant decrease was found in the immunoglobulin (Ig) G levels of the participants (p=0.002). While no significant decrease was observed in the IgG levels of the participants from the beginning to the 3rd month (p=0.085), a significant decrease was observed from the 3rd to the 6th month (p<0.001). Age, gender, presence of IRD and use of biological agents did not affect this decrease. Conclusion: It has been shown that antibodies acquired by infection in both healthy children and children with IRD were at an acceptable level in the first 3 months but decreased rapidly in the second trimester. This data can be used to schedule vaccination programs. Although it has been shown that IRD and biologic drugs do not affect the decrease in antibody levels, it indicates that no additional precautions are required in terms of vaccination in this patient group, but due to the limited number of patients, the data of our study should be confirmed with studies involving a larger number of patients. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
M. Heshin-Bekenstein 1,2 , A. Ziv 3 , N. Toplak 4 , S. Lazauskas 5 , D. Kadishevich 6 , E. Ben Nun 6 , A. Miller 7 , Y. Aviel Butbul 7 , E. Saiag 8 , G. Shefer 9 , S. Pel 10 , O. Elkayam 1,10 , Y. Uziel 1,3 1 Sackler School of Medicine, Tel Aviv University, 2 Pediatric Rheumatology, TEL AVIV MEDICAL CENTER, Tel Aviv, 3 Pediatric Rheumatology Unit, Meir Medical Center, Kfar Saba, Israel, 4niversity Medical Center Ljubljana, Ljubljana, Slovenia, 5 Tel Aviv Medical Center, Tel Aviv, 6 Pediatrics, Meir Medical Center, Kfar Saba, 7 Pediatric Rheumatology Unit, Rambam Medical Center, Haifa, 8 Hospital Management, Information and Operation Branc, 9 Department of Endocrinology Metabolism and Hypertension, 10 Rheumatology Department, Tel Aviv Medical Center, Tel Aviv, Israel Correspondence: M. Heshin-Bekenstein Introduction: Long term data on the safety and dynamics of the immune response to the BNT162b2 mRNA COVID-19 vaccine in adolescents with juvenile-onset autoimmune inflammatory rheumatic diseases (AIIRD) are limited. Objectives: To explore the safety profile and immune response over time of the second and third doses of the BNT162b2 mRNA vaccine in adolescents with juvenile-onset AIIRD and in comparison to healthy controls. Methods: This international prospective longitudinal multicenter study included adolescents with AIIRD (N=121) and healthy controls (N=49), with mean ages of 15.4 and 13.4 years, respectively. 60% of the AIIRD patients are on chronic immunomodulatory therapy. Participants were vaccinated with either a 2-dose mRNA vaccine: AIIRD n=93 (77.5%); controls n=19 (39%), or a 3-dose vaccine AIIRD n=27 (22.5%); controls n=30 (61%) and were followed to monitor for vaccine side effects, disease activity and medication change from baseline to time-points post-vaccinations. In addition, antibody titers were measured serially for part of the adolescents from both cohorts, with serum anti-spike protein S1/S2 IgG titers measured at three-time points: 2-9 weeks following the second vaccine, six-month time point and prior to the third vaccine, and 2-9 weeks following the third vaccine dose. Seropositivity was defined as an anti-S1/S2 IgG titer ≥ 15 BAU/ml. Results: The safety profile was good, with the vast majority of patients reporting mild or no side effects. The rheumatic disease has stayed stable at 97% and 100% after the second and third vaccines, with no worsening of the disease activity. The two-dose-vaccine induced similar, high immunogenicity rates among adolescent patients and controls, with seropositivity rates of 93% vs. 100%, respectively (p=0.55). S1/S2 IgG titers were significantly higher in the controls compared with AIIRD patients (377±78 vs. 246±137 BAU/ml). As expected, at the 6-month measurement, before the third vaccine dose, the AIIRD cohort seropositivity rates were lower compared with the healthy controls, 86.7% vs. 100%, respectively (p=0.3), with similar S1/S2 titers (221±159 vs. 220±93 BAU/ml, p=0.98). Following the third vaccine, the seropositivity rate increased to 100% in both AIIRD and control groups, with similar S1/S2 IgG titers among AIIRD patients compared with controls, 398±6 vs. 400±0 BAU/ml (p=0.35). Conclusion: This study reports the long-term safety and immune response induced by the two- and three-dose BNTb262 mRNA COVID-19 vaccine in adolescents with AIIRD compared with healthy controls. The safety profile in both cohorts was good following all three vaccine doses, with an adequate immunogenic response, though adolescents with AIIRD had lower seropositivity rates prior to the third vaccine as compared to the healthy adolescents (86.7% vs 100%). The third vaccine dose restored the immune response in both AIIRD and healthy adolescents. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
K. Kaidar 1 , Y. Dizitzer 2 , P. J. Hashkes 3 , L. Wagner-Weiner 4 , M. Tesher 4 , Y. Butbul Aviel 5 , I. Kanteman 5 , Y. Berkun 6 , E. M. Eisenstein 7 , M. H. Saied 8 , O. Goldzweig 9 , M. Heshin-Bekenstein 10 , E. Ling 11 , M. Feldon 12 , Y. Levinsky 13 , R. Tal 14 , L. Harel 15 , G. Amarilyo 16 1 pediatric rheumatology, 2 ward C, SCHNEIDER MEDICAL HOSPITAL, Petach TIkva, 3 pediatric rheumatology, Shaare Zedek Medical Centre, Jerusalem, Israel, 4 pediatric rheumatology, University of Chicago Medical Centre, Chicago , United States, 5 pediatric rheumatology, Meyer Children’s Hospital of Haifa, Haifa, 6 pediatrics, Hadassah-Hebrew University Medical Center, Mount Scopus, , 7 pediatrics, Hadassah-Hebrew University Medical Center, Mount Scopus , Jerusalem, 8 Pediatric Rheumatology, Carmel Medical Centre, Haifa, 9 pediatric Rheumatology, Kaplan Medical Center, Rehovot, 10 pediatric rheumatology, Dana Children’s Hospital of Tel Aviv Medical Center , Tel Aviv, 11 Pediatrics Department B, Saban Pediatric Medical Center, Beer Sheva, 12 Pediatric rheumatology, Shamir medical center, Rehovot, 13 pediatric Rheumatology, Schneider Children’s Medical Center, lPetach Tikva, 14 Pediatric Rheumatology, Schneider Children’s Medical Center, Petach Tikvah, 15 pediatric Rheumatology, Schneider Children’s Medical Center , Petach Tikva, 16 pediatric rheumatology, Schneider Children’s Medical Centre, Petach Tikvah, Israel Correspondence: K. Kaidar Introduction: Multisystem Inflammatory Syndrome in Children (MIS- C) associated with COVID-19, presents as a cytokine storm with features of Kawasaki disease. It is characterized by protracted fevers, gastrointestinal, cardiac and neurological symptoms. Laboratory tests are characterized by neutrophilia, lymphopenia, hypoalbuminemia and high inflammatory and myocardial damage markers. Many cases present with shock and require intensive care admission. Objectives: To identify predictors of a severe clinical course of multisystem inflammatory syndrome in children (MIS-C), as defined by the need for ionotropic support. Methods: This retrospective study included patients diagnosed with MIS-C in nine Israeli and one US medical centre. Univariate and multivariate regression models assessed odds ratio (OR) of demographic, clinical, laboratory and imaging variables during admission and hospitalization for severe disease. Results: Of 100 patients, 61 (61%) were male; mean age 9.65±4.48 years. Sixty-five patients were hypotensive, 44 required ionotropic support. Eleven fulfilled Kawasaki disease diagnostic criteria; 87 had gastrointestinal symptoms on admission. Echocardiographic evaluation showed 10 patients with acute coronary ectasia or aneurism, and 37 with left ventricular dysfunction. In a univariate model, left ventricular dysfunction was associated with severe disease (OR 4.178 [95%CI 1.760-9.917], while conjunctivitis (OR 0.403 [95%CI 0.173-0.938]) and mucosal changes (OR 0.333 [95%CI 0.119-0.931]) at admission were protective. Laboratory markers for a severe disease course were low values of haemoglobin, platelets, albumin and potassium; and high leukocytes, neutrophils, troponin and brain natriuretic peptide. In multivariate analysis, central nervous system involvement and fever >39.5°C were associated with severe disease. Mucosal involvement showed 6.2-fold lower risk for severe disease. Low haemoglobin and platelet count, and elevated C-reactive protein and troponin levels were identified as risk factors. Conclusion: Key clinical and laboratory parameters of MIS-C were identified as risk factors for severe disease; and may prompt earlier, more aggressive treatment decisions. Patients presenting with a Kawasaki-like phenotype were less likely to require ionotropic support. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
im Pediatrics, Hanyang University Myongji Hospital, Goyang-si, Korea, Republic Of Correspondence: K. Kim Introduction: Vaccine is an effective public health measurement to control the global COVID-19 pandemic. Patients with rheumatoid arthritis (RA) are twofold more vulnerable to infections that result in hospitalization and impaired quality of life. With consideration to the benefits of mRNA vaccination outweighing the risks, EULAR recommends that patients with RA should receive COVID-19 vaccines without needing major adjustment to their ongoing treatment regimens. Objectives: However, concerns about the safety of the vaccines are a major hurdle to widespread vaccination to the adolescent and children. Here, we experienced a JIA flare-up more than 5 months, the day after receiving the booster shot of BNT162b2 mRNA vaccine. Methods: In 2012, at the age of 10 years of boy, he was diagnosed with oligo JIA and received DMARD, but his symptoms gradually worsened, and in 2014, it changed to an extended type of oligo JIA. The symptoms improved after add on the Etenercept SC injection. All medications were discontinued in 2017 because no more symptoms occurred. However, it relapsed in 2019 and resumed DMARD plus Etenercept. After that, he has reached remission until he was COVID-19 mRNA vaccinated. After the first and second COVID-19 mRNA vaccinations, there was no adverse reaction, so he was vaccinated for the booster shot (3 rd vaccination) in Dec 2021, but the Right knee swelled the next day. He is on medication state, MTX 3tab weekly, Folic Acid 1tab daily and Enbrel 1.0 twice/week since May 2019. He was given the third vaccination on Dec. 16, 2021. The next day after the vaccination, his right knee was swollen and felt pain on Dec. 17, 2021. The swollen joint was not improved with NSAID. So, prednisolone 15mg (3tab) was added on Jan. 3, 2022. Nevertheless, the swollen right joint worsened. The US on January 5 th showed large amount of joint effusion on right knee and US guided aspiration and US guided mixed fluid (triamcinolone acetonide + ropivacaine + saline) injection was done. It worked for the first seven days, but after that, the right joint was swollen again and complaining of pain. These symptoms continued for more than 5 months after mRNA booster vaccination. Results: Conclusion: We experienced that after full vaccination (3 rd booster vaccination) with BNT162b2 patient with JIA, showed an increased risk of possible arthritis flare-up. So, To understand the association between arthritis flare and vaccination is important to overcome vaccine hesitancy. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
B. Koren, V. Berce clinic, University Medical Centre , Maribor, Slovenia Koren Introduction: In children, coronavirus disease 2019 (COVID-19) is usually mild. However, in rare cases, children can be severely affected and develop a significant systemic inflammatory response, which has been termed multisystem inflammatory syndrome in children (MIS-C) or pediatric inflammatory multisystem syndrome temporally associated with severe acute respiratory syndrome (SARS) associated coronavirus 2 (SARS-CoV-2) (PIMS-TS). Objectives: To evaluate clinical manifestations and therapy in pediatric patients diagnosed with MIS-C. Methods: A single small center study was performed. Included were patients with MIS-C treated in inpatient service of our pediatric clinic from November 2020 till March 2022. WHO/CDC diagnostic criteria for MIS-C were used. Data was collected retrospectively from patients’ medical records. Results: A total of 12 patients were included. The mean age of all patients was 8.9 years (range 1-15 years). Female and male patients were equally affected (ratio 1:1). 3 patients (25%) had positive PCR swab for COVID-19 and 10 patients (83.3%) had positive serology to COVID-19. The clinical characteristics of patients with MIS-C are presented in Table 1. In addition to fever and myocardial dysfunction, which were present in all patients, gastrointestinal involvement was the most common clinical manifestation. 3 boys aged 4, 5 and 15 years also fulfilled diagnostic criteria for typical and 1 girl aged 1 year fulfilled diagnostic criteria for atypical Kawasaki disease. All of our patients were treated with intravenous immune globulin (IVIG) and aspirin. 9 patients (75%) were put on glucocorticosteroids. Moreover, among them 3 (25%) needed pulse doses. Anticoagulation with a low- molecular- weight heparin (LMWH) was used in 11 patients (92%). As MIS-C can present with signs and symptoms that mimic those of septic shock and toxic shock syndrome, 3 patients (25%) were put on antibiotic therapy. 1 girl who presented with COVID-19 pneumonia received antiviral therapy with remdesivir. We didn’t use biologics in any of our patients. All 4 patients (33.3%) presenting with shock required pediatric intensive care treatment. Conclusion: The results of our study are consistent with data from the literature. All of our patients with MIS-C achieved full recovery. Because the disease can present as a severe shock- like illness early diagnosis and prompt treatment are of significant importance. Long- term follow- up data is limited, but the prognosis looks favourable as most children have a full clinical recovery. References : 1. Riphagen S, Gomez X, Gonzalez- Martinez C, et al. Hyperinflammatory shock in children during COVID-19 pandemic. Lancet 2020; 395:1607. 2. Son MBF, Friedman K. COVID-19: Multisystem inflammatory syndrome in children (MIS-C) clinical features, evaluation and diagnosis. Available at: https://www.uotodate.com/contents/covid-19-multisystem-inflammatory-syndrome-in-children-mis-c-clinical-features-evaluation-amd-diagnosis (Accessed on April 8, 2022). 3. Licciardi F, Pruccoli G, Demina M, et al. SARS-CoV-2- Induced Kawasaki- like hyperinflammatory syndrome: A novel COVID phenotype in children. Pediatrics 2020; 146P. Król 1,2 , M. Mossberg 1 , B. Månsson 3 , R. Kahn 1,2 1 Department of Pediatrics, 2 Wallenberg Centre for Molecular Medicine, 3 Department of Rheumatology, Lund University, Lund, Sweden Correspondence: P. Król Introduction: Multisystem Inflammatory Syndrome in Children (MIS-C) is a rare entity caused by a previous, often inapparent or mild, COVID-19 infection. The pathogenesis of MIS-C is still discussed, but a few theories has been suggested (1). One could be endothelial dysfunction, that might lead to systemic capillary leak syndrome (CLS), a feature of MIS-C. Objectives: The aim of this study was to compare clinical characteristics and laboratory parameters between MIS-C patients with and without CLS symptoms. Methods: This retrospective, population-based cohort study from southern Sweden includes all individuals between 0-19 years diagnosed with MIS-C from the beginning of pandemic in 2020 to end of July 2021. Inclusion criteria were: 1) diagnosis according to the WHO case definition for MIS-C diagnosis (2), 2) patient and families agreed to be registered in the National Pediatric Rheumatology Register by signing the informed consent, 3) Living in the study area at time of diagnosis. Exclusion criteria were that patient/family moved to another region or country. Clinical variables included MIS-C symptoms, admission to standard ward or ICU/PICU, length of stay and treatment (IvIg or IvIg + corticosteroids or IvIg + corticosteroids + anakinra). Laboratory parameters (CRP, leukocyte- and lymphocyte count, platelets, proBNP and albumin) were measured daily. CLS was defined as having at least two of four symptoms; 1) oedema; 2) treatment-required hypotension; 3) hemoconcentration and 4) hypoalbuminemia. Results: In total, 31 children with MIS-C were identified (39% females), and 24 out of these met the criteria for CLS. The median age at diagnosis was 10,6 years. Children were hospitalized for a median of 10 days (range 4-48). Six children were treated at ICU/PICU, all with CLS. The clinical characteristics are shown in Table 1. Children in CLS group had higher CRP and proBNP and lower platelets and albumin levels the first days of hospitalization as compared to the non-CLS group. Leukocyte and lymphocyte count was similar in both groups. 58% of patients from CLS group were treated with IvIg + steroids and remaining 42 % with IvIg + steroids + anakinra. Six of seven patients from non-CLS group were treated with IvIg + steroids and one patient with only IvIg. No patient from the non-CLS group were treated with IvIg + steroid + anakinra. Conclusion: CLS is a severe and common feature of MIS-C. Seventy seven percent of our MIS-C patients developed this complication during the first days of hospitalization. In our cohort patients with CLS compared to non CLS patients had more severe disease course, higher inflammatory parameters, lower platelet count and need of more aggressive anti-inflammatory treatment (42% patients with triple-combination, compared to none). It is important to identify and treat CLS early and we suggest that MIS-C children with low platelet count and severe inflammatory phenotype might be at higher risk to develop CLS. References. 1. McMurray JC, May JW, Cunningham MW, Jones OY. Multisystem Inflammatory Syndrome in Children (MIS-C), a Post-viral Myocarditis and Systemic Vasculitis-A Critical Review of Its Pathogenesis and Treatment. Front Pediatr. 2020 Dec; 8: 626182 2. World Health Organization. Multisystem inflammatory syndrome in children and adolescents temporally related to COVID-29. World Health Organization; 2020. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
M. P. Elicio 2 , V. A. Monno 2 , M. Chironna 3 , A. Campanozzi 4 , F. Moramarco 5 , A. Civino 6 , V. Cecinati 7 , U. Vairo 8 , L. Milella 9 , D. Loconsole 3 , V. Mastrorilli 2 , F. Cardinale 2 1 Department of Pediatrics, Giovanni XXIII Pediatric Hospital, University of Bari, 2 Department of Pediatrics, Giovanni XXIII Pediatric Hospital, University of Bari, 3 Department of Interdisciplinary Medicine, Hygiene Section, University of Bari, Bari, 4 Department of Medical and Surgical Sciences, Pediatric Section, University of Foggia, Foggia, 5 Department of Pediatrics, Antonio Perrino Hospital, Brindisi, 6 Division of Pediatric Rheumatology and Immunology, Vito Fazzi Hospital, Lecce, 7 Department of Pediatrics , SS Annunziata Hospital , Taranto, 8 Division of Pediatric Cardiology, 9 Division of Intensive Care, Giovanni XXIII Pediatric Hospital, Bari, Italy Correspondence: F. La Torre Introduction: Multisystem inflammatory syndrome in children (MIS-C) is a newly identified clinical entity not yet very well known in terms of epidemiology, pathogenesis and long-term outcome. So far very few studies worldwide have investigated the incidence of MIS-C. Most studies published come from US, reflecting mainly north-American racial and genetic backgrounds. Data about the incidence of MIS-C in Europe are lacking. Objectives: The aim of the study was to gain insight into the epidemiology of MIS-C in Apulia region in southern Italy. Our primary goal was to estimate the incidence of newly identified cases of MIS-C in children 0-18 years of age, during a period of six months, encompassing the second pandemic wave. We also evaluated the percentage of children with previous SARS-CoV2 infection who developed MIS-C in the following weeks, the incidence rate of MIS-C in terms of cases/month/year, and the cumulative incidence per 100,000 residents. Methods: An observatory network between all pediatric departments in Apulia was created since October 2020. Data from all cases of MIS-C, between 0 and 18 years, hospitalized in all regional pediatric departments, in a period of six months, spanning from November 1, 2020 to April 30, 2021, were collected. The data collected were analyzed using STATA MP17 software. Continuous variables were expressed as mean ± standard deviation and range, categorical variables as proportions. The data source used was the “Infections Regional Informative System (IRIS) - Apulia. The number of residents aged 0-18 years in Apulia relating to the 2020-2021 was obtained from the ISTAT (Italian National Statistics Institute) registry. Results: During the time frame considered, 22 new cases of MIS-C were admitted to five community pediatric departments in Apulia (Bari, Foggia, Brindisi, Lecce and Taranto). The cumulative incidence of MIS-C was 3.27 per 100,000 residents between 0 and 18 years of age and the incidence rate of MIS-C among pediatric subjects with previous SARS-CoV-2 infection was 0.07%. The age group most affected was between 6 and 10 years with a cumulative incidence of 5.67 per 100,000 residents between 0 and 18 years. The Table show the cumulative incidence of multisystem inflammatory syndrome in children (MIS-C) by the age groups: Most newly diagnosed cases of MIS-C followed the peak of SARS-CoV2 infections after 2-6 weeks and in particular a greater number of cases was registered in December 2020 and between late February and early April 2021 with a higher incidence rate per month, expressed in cases/month/year, on March 2021. Conclusion: Our paper represents the first epidemiological study of MIS-C in Europe. The strength of our study was the creation of a network among all the pediatric departments in a well defined geographical region which allowed us to catch all cases of MIS-C that occurred in the examined period. This makes our epidemiological data more accurate as possible and it confirms in Europe the US data Patient Consent: Yes, I received consent Disclosure of Interest : None declared
M. C. Maggio 1 , A. Lembo 2 , F. Finazzo 3 , A. Alaimo 4 , M. C. Failla 5 , S. Giordano 5 , G. Corsello 2 1 PROMISE “G. D’Alessandro”, 2 University Department PROMISE “G. D’Alessandro”, University of Palermo, 3 U.O.C. of Paediatric Radiology, Children Hospital “G. Di Cristina”, 4 U.O.C. of Paediatric Cardiologic Division, Children Hospital “G. Di Cristina”, 5 U.O.C. of Paediatric Infectious Diseases, Children Hospital “G. Di Cristina”, ARNAS, Palermo, Italy Correspondence: M. C. Maggio Introduction: MIS-C is a hyperinflammatory syndrome following SARS-CoV-2 exposure. A transitory myocardial impairment as myocarditis, occurs with full recovery in most of them. Coronary lesions, valvular insufficiency and pericardial effusion are variable associated. The pathogenesis of the heart involvement is still object of study, linked to cytokines storm. Objectives: To evaluate the usefulness of cardiac magnetic resonance (CMR) in highlighting heart involvement in children affected by MIS-C. Methods: We describe a case series of 18 children (age:1-14 years;11F;7M), admitted to a tertiary care Children Hospital with diagnostic criteria of MIS-C. All the patients were followed by ECG and echocardiogram. CMR was performed during the hospitalization or after the discharge, with a 1,5 Tesla scanner. The CMR study was not performed during the acute phase in some patients, clinically unstable and needing general sedation. The protocol included, before intravenous contrast media injection, retrospective ECG-Gated fiesta cine sequences (short axis, 4,3,2 chamber views), sequences for edema, nd hyperemia T2 -short tau inversion recovery (Stir). Myocardial edema was evaluated following the Lake Louise criteria. Because poor references for normal value in native T1 mapping and T2 relaxation time in children, myocardial edema was defined by increased signal intensity on T2-weighted imaging and myocardial damage by non-ischemic patterns late gadolinium enhancement. The study for evaluating myocyte necrosis and fibrosis considered late gadolinium-enhanced 2D inversion recovery sequences performed at 6 min following intravenous contrast medium administration. Results: ECG evidenced arrythmia in 8 patients. All resolved arrythmia in the subacute phase. Transthoracic echocardiography demonstrated in all the patients signs of cardiac involvement: 2 showed coronaritis without aneurisms; 4 showed pericarditis; 12 showed valvular insufficiency: 7 Mitral insufficiency; 1 Tricuspid insufficiency; 2 Mitral and Tricuspid insufficiency; 2 Mitral and Aortic insufficiency. In 16/18 patients, T2-Stir sequences didn’t show myocardial edema and/or hyperemia and/or fibrosis. In 2 patients CMR showed late gadolinium enhancement in a non-ischemic pattern. 1 patient was studied in subacute phase, with evidence of ventricular myocardial oedema, persisting as delayed enhancement during the follow-up. 1 patient was studied 1 month after the hospitalization, showing myocardial fibrosis. 2 children showed pericardial effusion; during the follow-up, pericardial effusion resolved. 1 patient showed a persistent aortic insufficiency. In the other patients, cardiac lesions resolved during the follow-up. Conclusion: CMR is an excellent noninvasive diagnostic tool for the diagnosis and follow-up of heart involvement. Furthermore, CMR can predict prognosis and recognize children at risk to develop arrhythmias and unfavorable events. Above all, CMR is a standardized imaging highlight some features of myocardial damages: edema, inflammation, pericardial effusion, contractile scar impairment and necrosis. Most of CMR exams didn’t detect myocardial edema, probably because the CMR was performed during the clinical recovery phase. In 2 patients CMR showed late gadolinium-enhanced in a non-ischemic pattern. CMR was an accurate and reliable method to follow children with cardiac involvement in MIS-C and guide the targeted therapeutic strategy. Disclosure of Interest : None declared
Manojlovic 1 , J. Vojinovic 1,2 1 Paediatric Clinic, Department of Rheumatology, University Clinical Center of Nis, 2 University of Nis, Medical Faculty, Niš, Serbia Correspondence: M. Manojlovic Introduction: During COVID-19 pandemic it was observed that, despite the use of immunosuppressive and biological disease-modifying antirheumatic drugs (bDMARDs), children with paediatric rheumatic diseases are not susceptible to infections. Objectives: The aim of the research was to further broaden current knowledge of whether COVID-19 affects the course of juvenile idiopathic arthritis (JIA) in children under immunosuppressive treatment. In addition, we analysed epidemiological risk and exposure to COVID-19, as well as patient interest in COVID-19 vaccination. Methods: The survey was conducted by interviewing parents and patients. The questionnaire consisted of 4 domains: first, regarding COVID-19 infection or exposition; Second, assessment of underlying disease-related information (disease duration, length of the treatment, current medication, disease status at the last visit, comorbidity, and concomitant drugs) and COVID-19 influence on clinical course; And in last domain, possible interest in COVID-19 vaccination was evaluated. Results: Patients who were under immunosuppressive therapy and followed up at the Paediatric Rheumatology Department were asked to provide one-on-one communication and informed about the research. Patients with incomplete medical data and those whose parents refused to participate were excluded from the study. In our cohort (n=68), the mean age was 11±4.82 years, and 69% (n=47) of the patients were female. The patients were categorised into two groups: bDMARDs (n=45) and nonbiological DMARDs (n=23). In bDMARDs group 60% were tested positive for COVID-19, and five of them (11,1%) had disease deterioration after COVID-19 infection, requiring therapy change. Two of them had their drugs changed, and one had drug dose increased. There was one case (4,3%) with exacerbation of underlying disease due to not responding to regular check-ups and self-initiated breaks in therapy, thus dose of the drug was increased. Similarly, in group of children treated with nonbiological DMARDs, 65% were diagnosed with COVID-19 and worsening of the underlying disease has been reported in one case (4,3%), after second reinfection, requiring drug change. There was no statistically significant difference between those two groups (p<0,05). None of the patients diagnosed with COVID-19, had any severe symptoms or required hospitalisation. In bDMARDs group, parents of three children were motivated for COVID-19 vaccination, while one child was vaccinated (2,2%). The rest of the parents (91,1%) had negative attitude regarding vaccination. In nonbiological DMARDs group one child (4,5%) was vaccinated, while remaining (95,6%) had negative attitude towards vaccination. Conclusion: Children with JIA taking immune-modulatory drugs in COVID-19 era should be recommended to maintain the chronic therapy aiming optimal control of active disease. In our cochort most of patients under both biological and nonbiological DMARDs did not have disease exacerbation, however, some of them required therapy change. Disclosure of Interest : None declared
F. Minoia 1 , F. Lucioni 1 , M. Heshin-Bekenstein 2 , S. Vastert 3 , C. Kessel 4 , Y. Uziel 5 , L. Lamot 6 , N. Ruperto 7 , M. Gattorno 7 , C. Bracaglia 8 , N. Toplak 9 on behalf of the MAS/sJIA and Vaccination WPs 1 Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy, 2 Dana Dwek Children’s Hospital, Tel Aviv Medical Center, Tel Aviv, Israel, 3 University Medical Center, Utrecht, Netherlands, 4 University Children’s Hospital Muenster, Muenster, Germany, 5 Meir Medical Center; Sackler School of medicine, Tel Aviv University, Kfar Saba, Israel, 6 University Hospital Center, Zagreb, Croatia, 7 IRCCS Istituto Giannina Gaslini, Genoa, 8 IRCCS Ospedale Pediatrico Bambino Gesù, Rome, Italy, 9 University Children’s Hospital, University Medical Centre, MF, UL, Ljubljana, Slovenia Correspondence: F. Minoia Introduction: Following the Coronavirus Disease-19 (COVID-19) pandemic outbreaks, the hyperinflammatory condition termed Multisystem Inflammatory Syndrome in Children (MIS-C) has become a healthcare issue worldwide. Since December 2020 the mRNA vaccine against SARS-CoV-2 has become available with a good safety profile. However, evidence regarding safety and vaccination strategies in children with previous MIS-C is still lacking Objectives: To investigate the current approach in different international centres to COVID-19 and other vaccinations among children with previous MIS-C Methods: Physician all around the globe who take care of patients with MIS-C worldwide were invited to anonymously complete a 15-question web-based survey. The survey was open from October 6 th to December 31 st 2021. Centre, country and specialty of the participants were collected. Participants were asked to describe their current vaccination strategy for MIS-C patients and to provide the most important variables affecting their decision-making process Results: A total of 290 replies from 237 centres in 61 countries were collected. Most respondents (86%) were pediatric rheumatologists. The anti-COVID-19 vaccine was available in 85% of the countries covered by the survey. Sixty-seven centres (28%) from 22 countries had already vaccinated MIS-C patients (< 5 patients: 52%; 5-10 patients: 29%; > 10 patients: 20%), without adverse reactions in most cases (89%). Six centres reported complications after the anti-COVID-19 vaccine: 2 unknown, 3 mild symptoms (fever, sore arm) and only 1 centre reported a MIS-C like reaction. MIS-C patients re-infected by SARS-CoV-2 were seen in 15% of centres and 2 (8%) reported a MIS-C flair. Most centres (84%) were in favour of vaccinating MIS-C patients against SARS-CoV-2, waiting 3-6 months (40%), 6-12 months (52%) or > 12 months (8%) after a MIS-C episode. The variables with the greatest impact on the decision not to vaccinate MIS-C patients were current lack of evidence (51%), patient/parent decision (40%), fear of MIS-C relapse (36%), history of severe MIS-C with myocarditis (35%) and excessive proximity to MIS-C episode (33%). The most relevant parameters in the vaccination strategy were time from MIS-C episode (78%), ongoing immunosuppressive treatment (35%), SARS-CoV-2 serologic status (32%) and MIS-C features (31%). Almost all centres favoured continuing regular vaccination with non-live (99%) and live (93%) vaccines, waiting 3-6 months and 6-12 months, respectively Conclusion: When vaccinating MIS-C patients against SARS-CoV-2, the experience reported by the international pediatric rheumatology community to date, is overall reassuring. However, lack of evidence still affects the vaccination strategy of many centres worldwide. Large, prospective studies are needed to properly evaluate the safety of anti-COVID-19 vaccination among MIS-C patients Patient Consent: Not applicable (there are no patient data) Disclosure of Interest : F. Minoia Consultant with: SOBI, F. Lucioni: None declared, M. Heshin-Bekenstein: None declared, S. Vastert: None declared, C. Kessel Grant / Research Support with: Novartis, Consultant with: SOBI and Novartis, Y. Uziel: None declared, L. Lamot: None declared, N. Ruperto: None declared, M. Gattorno: None declared, C. Bracaglia Consultant with: Novartis and SOBI, N. Toplak: None declared
V. Opoka-Winiarska 1 , E. Grywalska 2 , I. Morawska 3 , K. Gosik 2 , O. Kądziołka 4 , J. Roliński 3 1 Department of Paediatric Pulmonology and Rheumatology, 2 Department of Experimental Immunology, 3 Department of Clinical Immunology and Immunotherapy, Medical University of Lublin, 4 Department of Pediatrics, Pulmonary Diseases and Rheumatology, University Children’s Hospital in Lublin, Lublin, Poland Correspondence: I. Morawska Introduction: The pediatric inflammatory syndrome temporarily associated with SARS-CoV-2 (PIMS-TS) is a severe disease entity with an incompletely established pathogenesis. Typically, the symptoms develop 4-6 weeks after exposure to the SARS-CoV-2 virus, but the clinical course is heterogeneous. The pathogenesis and the course of the disease is still incompletely established and long follow-up of patients after illness is still carefully followed. Programmed cell death-1 protein (PD-1 or CD279) is a member of CD28 superfamily and expressed on CD4+ and CD8+ T cells, NK cells, B cells and activated monocytes. Binding to the ligands PD-L1 and PD-L2 on T cells, B cells, dendritic cells (DCs) and macrophages, PD-1 conducts its inhibitory function to regulate T cell activation, tolerance, and immune-mediated tissue damage. The aim of the study was to evaluate the expression of selected suppressive molecules by assessing the PD-1 serum concentration in PIMS-TS patients in relation to laboratory parameters at the time of diagnosis and annual follow-up. Objectives: Our study aimed to analyze expression of the PD-1 receptor on CD8+, CD4+ and CD19+ lymphocytes, in patients diagnosed with PIMS-TS, diagnosed at the Department of Lung Diseases and Pediatric Rheumatology at the Medical University of Lublin from January 2021 to April 2022. The study was approved by the bioethics committee no KE-0254/236/2020. Methods: The study included 31 patients diagnosed with PIMS-TS and 36 healthy volunteers (HV), from whom, after obtaining consent, whole blood was collected, and appropriate laboratory tests were performed immediately. The observation period was one year. The tests were performed at diagnosis (21 tests), then 6 weeks (31 tests), 3 months (29 tests), 6 months (21 tests) and a year (9 tests) after diagnosis. Laboratory and immunological parameters were assessed using standard methods by a hospital laboratory, and flow cytometry was used to assess PD-1 receptor expression. Statistical analysis of the results was performed using GraphPad Prism 8 and the following tests: ANOVA Kruskal-Wallis and multiple comparisons with multiple comparison correction: two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli were used to determine statistical significance (p<0.05). Results: The highest % of PD1+CD4+ (median% 6.62 vs% 3.97 control) was observed in patients before treatment initiation, and the highest % of PD1 +CD8+ (median% 8.79 vs% 5.42 control) was observed 6 weeks after treatment initiation, however, these relationships are not statistically significant. PIMS-TS patients were characterized by an increased level of leukocytes compared to the healthy group (p=0.0029) and lower values of lymphocytes (p=0.0006); both parameters normalized up to 6 weeks after the onset of the disease. We did not find statistical differences in the levels of IgG and IgM anti-SARS-CoV-2 between the groups of patients in particular follow-up periods or between patients and controls. Conclusion: Our research showed interesting finding the overexpression of the PD-1 receptor on T lymphocytes and the change of its expression over time. We believe this topic requires in-depth statistical analyzes and evaluation of the usefulness of this parameter as an additional diagnostic factor. We showed no differences in the serological response to the SARS-CoV-2 virus between individual groups of patients and the control group. This allows for a hypothesis about the existence of factors predisposing to the development of PIMS-TS. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
E. Nakhutsrishvili 1 , T. Kutubidze 2 1 Pediatric Rheumaatology, TSMU G. Zhvania’s Pediatric Academy Clinic, Tbilisi, Georgia, 2 Tbilisi State Medical University, Tbilisi, Georgia Correspondence: E. NakhutsrishviliThey might require hospitalization, intensive care. Objectives: In 2020-2021 at the Academic Pediatric Clinic named after G. Zhvania of Tbilisi State Medical University, we observed 60 children with post-covid complications and late Covid syndrome. More than half (32 children-53.3%) were under 5 years of age, with a predominance of boys (33 children-55%) who had had a Covid-19 infection 1.5-2 months before contacting us with a positive antibody reaction. Most of them (51 children-85%) were healthy before the disease. vasculopathy, immune thrombocytopenia, iron deficiency anemia, coagulopathy, pneumonia-atelectasis, primary diabetes, exacerbation of the underlying disease - arthralgia, arthritis, and abnormal manifestations of sleep disturbance, general weakness and dizziness were noted. Separately, it is necessary to highlight the multisystem inflammatory syndrome in chirdren - MIS-C (8 children - 13%) proceeding with clinical signs of Kawasaki disease (mucocutaneous-lymphatic syndrome) with hectic temperature, polyserositis, hepatosplenomegaly, high rates of inflammation markers, a tendency to hypercoagulability. One patient had a coronary artery aneurysm. In 3 cases, the ANA and ANF titer was increased (up to 1: 640) and also nucleic cytoplasmic fluorescence with linear fibrils (actin-like fluorescence), which indicates immune reactions in Covid infection, which can explain the positive effect of corticosteroid therapy in the treatment of these patients. Only 22 patients (36%) were hospitalized, the rest were observed on an outpatient basis. Methods: We studied 60 patients with post-Covid19 complications and “long Covid” syndrome. Results: Majority of patients had no pre-existing diseases. MIS-C, arthritis/arthralgia, diabetis type 1, exacerbation of preexisting diseases, pneumonia/atelectasis, vasculopathy, ITP, Iron deficiency anemia, coagulopathy, sleep disturbances etc Conclusion: The evidence that COVID-19 can have long-term impact children as well, including those with asymptomatic/symptomatic COVID-19. There is a need for more high-quality pediatric SARS-CoV-2 research and observation in dynamics. Disclosure of Interest : None declared
V. Opoka-Winiarska 1 , J. Lipińska 2 , K. Orczyk 2 , A. Michalak 3,4 , J. Burzyński 4 , O. Kądziołka 1 , E. Smolewska 2 1 Department of Paediatric Pulmonology and Rheumatology, Medical Universiy of Lublin, Poland, Lublin, 2 Department of Pediatric Cardiology and Rheumatology, Medical University of Lodz, Poland, Łódź, 3 Department of Pediatrics, Diabetology, Endocrinology and Nephrology, 4 Department of Biostatistics and Translational Medicine, Medical University of Lodz, Poland, Lodz, Poland Correspondence: V. Opoka-Winiarska Introduction: The safety of COVID-19 vaccines in children with JIA with long-term treatment has been drawing the researchers’ interest in the current pandemic reality. Objectives: Therefore the principal objective of the study is to assess how often and what adverse events after COVID-19 vaccines develop the patients with juvenile idiopathic arthritis (JIA) and how these vaccines influence the disease activity. Methods: The study project involved a 3-month follow-up of 43 patients aged 12.8 to 16.9 years (median -14.6 years), 17 boys and 26 girls, already diagnosed with JIA, who have been admitted to one of two pediatric rheumatology centers in Poland (Lodz and Lublin) for a regular check-up visit. Majority of the study group children (32.56%) had ERA-JIA subtype, polyarticular or oligoarticular JIA had equal 27.91%, systemic JIA – 6.98% and other (not classified) JIA subtype – 2.7% children. The patients’ history regarding SARS-CoV-2 infection, exposure to these virus and current vaccination record was collected using appropriate validated questionnaire. All study group received Pfizer COVID-19 vaccine. Furthermore, each patient was evaluated in terms of disease activity using Visual Activity Scale (VAS) and Juvenile Arthritis Disease Activity Score 27-Joint Count (JADAS27). The study group was subdivided into two age groups: 5-11 years and 12-18 years. Results: Whole study group have been vaccinated for the obligatory vaccines according to polish mandatory vaccination calendar and more than 1/3 have got one or more additional recommended vaccinations. All parents of study group children and the majority of siblings have been vaccinated for COVID-19. The HLA-B27 antigen presence was confirmed in 27.91% (12/43) of children with JIA. Ten out of 43 (22.72%) children with JIA had COVID-19 infection before getting COVID-19 vaccine. More than a half (24/43 - 55.81%) of the children with JIA were treated with classical DMARDs (methotrexate, sulfasalazine), 3/43 (6.98%) - cyclosporine A. Eleven out of 43 (25.58%) had biological treatment – 2 with etanercept, 6- with adalimumab and 3 – tocilizumab. After first dose of COVID-19 vaccine 26/43 (58.14%) children presented typical adverse events: arm pain or oedema at the application side or weakness. The same number of children had side effects after second dose of COVID-19 vaccine, however the spectrum of the symptoms was wider (arm pain or oedema at the application side, weakness, headache, fever, lymphadenopathy, arrhythmia). The number of patients with active disease according to JADAS-27 was even lower after COVID-19 vaccination than before, however it was not a statistically significant difference (p>0.05). Furthermore, the grade of JADAS-27 activity (0-3) was higher in study group before COVID-19 vaccination (p=0.47). Conclusion: The analysis indicated that children with JIA with remission without treatment or on the long-term treatment – classical or even biological, may be safely vaccinated for COVID-19. COVID-19 vaccination does not interfere with the JIA treatment and does not exacerbate the rheumatoid process. However, our results need to be confirmed in greater population. Our results may be helpful in everyday practice of pediatric rheumatologist who may refer to the findings of this study during conversation with patients or parents regarding the safety of COVID-19 vaccines. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
F. Peri 1 , S. Pastore 2 , A. Taddio 2 , F. Biscaro 3 , S. Martelossi 3 , A. Tommasini 4 1 Department of Medicine, Surgery, and Health Sciences, University of Trieste, Italy., 2 Institute of Maternal and Child Health, IRCCS Burlo Garofolo, Trieste, Italy., Trieste, 3 Pediatric Department, Presidio Ospedaliero di Treviso, Treviso, Italy., Treviso, 4 Institute of Maternal and Child Health, IRCCS Burlo Garofolo, Trieste, Italy, Trieste, Italy Correspondence: A. Taddio Introduction: Chilblain-like lesions has been a peculiar phenomenon described in children and adolescents during COVID-19 pandemic. Little is known about its recurrence and whether this condition may be prodromic to more complex rheumatological disorders. Objectives: To follow-up a cohort of children and adolescents that previously presented with chilblain-like lesions during early 2020 and 2021. Methods: We performed a telephonic survey of 31 patients diagnosed with chilblain-like lesions during the first two waves of COVID-19 at two north-eastern Italian hospitals. We collected data regarding new episodes, other signs and symptoms of systemic disease and need for treatment. Results: We reached 27/31 patients, most of them previously evaluated during early 2021. Seventeen patients (17/27, 63%) presented with new episodes of chilblain-like lesions. Relapse happened during winter months in all the patients; only two patients referred persistence of the condition also during the summer. Type I interferon signature tested positive (> 2) in 8/17 relapsed patients who underwent this examination. Most cases had a benign course with gradual resolution over several weeks with no need for specific treatment. Five adolescents received topical and systemic treatment due to painful lesions with functional impairment of hands and feet. Four patients had a minor relief from application of nifedipine cream and three patients had a progressive symptoms resolution after hydroxychloroquine treatment. None developed any signs or symptoms suggestive of a definite systemic rheumatologic disorder. Conclusion: Chilblain-like lesions presented a recurrent pattern triggered by cold environmental temperatures in our cohort of children and adolescents. None of them developed other worrisome symptoms and most cases did not need specific treatment. We noted a recurrence trend in patient with a positive type I interferon signature testA. K. Primero Nieto 1 , E. Faugier Fuentes 2 , A. Barba Aguilar 1 , E. Mercedes Pérez 2 , A. Guzmán Revilla 2 , H. Bermúdez Canales 2 , P. Ramos Tiñini 1 , H. Menchaca Aguayo 2 , S. Rodriguez Aguayo 2 1 Reumatología, Hospital Infantil de Mexico, 2 Reumatología, Hospital Infantil de México, Ciudad de México, Mexico Correspondence: A. K. Primero Nieto Introduction: The occurrence of autoimmune diseases can be generated by a variety of factors through hyperstimulation of the immune status. The factors can be classified as: genetic, environmental and hormonal. Viruses are an important component in the environmental factors affecting the immune system. Objectives: To describe a clinical case, who developed autoimmunity following SARS-CoV-2 infection. Methods: Clinical Case Results: Male adolescent aged 15 years. No personal pathologic history. Admitted to our unit with clinical manifestations of epistaxis of 6 days of evolution, asthenia, adynamia, headache, fever, jaundice, acholia and choluria. He was admitted to the ER with shock data requiring endotracheal intubation, with laboratory tests reporting autoimmune hemolytic anemia (AIHA) of warm antibodies; with positive nasal antigen test for SARS-COV-2. She received treatment with immunoglobulin and 5 pulses of methylprednisolone, with subsequent full dose of steroid and tapering schedule. During her follow-up, persistent thrombocytopenia was documented, so an approach for autoimmune diseases was initiated. She presented triple positive marker integrating probable Antiphospholipid Antibody Syndrome (APS); due to hematologic condition (thrombocytopenia) she was started on azathioprine, with adequate response at the time. Conclusion: This case describes the association of SARS-CoV-2 infection with autoimmune diseases such as APS and AHAI. Follow-up and subsequent treatment are described. The ongoing pandemic induced by SARS-CoV-2, which has spread through many countries, has generated curiosity in knowing the possible mechanisms of inducing hyperstimulation of the immune system (1). The receptors of this virus are angiotensin-converting enzyme-2 (ACE-2) and transmembrane serine protease-2 (TMPRSS2), which are used to penetrate cells. ACE-2 is also widely expressed in endothelial cells. The synthesis of multiple autoantibodies is known, with an effect of occurrence of autoimmune diseases, possibly pre-existing. (2) The association of severely ill SARS-CoV-2 infected patients with circulating B2-glycoprotein or anticardiolipin (aCL) autoantibodies has also been investigated. It was found in recent study that patients with severe COVID-19 had significantly higher aCL antibody levels than patients with moderate disease. These observations undoubtedly suggest the possible existence of molecular mimicry due to the similarity between the peptide regions identified in the b2GPI molecule (and recognized by anti-b2GPI) and the membrane proteins of various viruses and bacteria. (3) This is an example of antibody production secondary or parallel to SARS-CoV-2 infection, which triggers disease and may require treatment and long-term follow-up. It is therefore important to consider autoimmunity as a consequence of SARS-CoV-2 infection. Disclosure of Interest : None declared
M. T. Riccio, V. Aiello, F. Anselmi, B. Macri, M. Alessio, R. Naddei napoli, Italy Correspondence: M. T. Riccio Introduction: In December 2020, two anti-Sars-CoV2 vaccines were approved for pediatric population, which were initially intended for adult and frail population. The BNT162b2 and mRNA-1273 vaccines are chemically modified mRNA vaccines expressing the prefusion glycoprotein spike of Sars-CoV2 packed in lipid nanoparticles. This vaccine has been shown to be 94.1% efficacious to prevent symptomatic COVID-19 infection. In May 2021, these two vaccines were approved for teenagers aged 12 to 18 years, and in December 2021 for children aged 5 to 11 years. To date, many studies have been conducted on the efficacy of these vaccines in the adult population, while less is known in the pediatric population, particularly in children with chronic diseases being treated with background medication Objectives: To examine how children with chronic rheumatic diseases undergoing treatment with background medication respond to Sars-Cov2 vaccination. Methods: We administered a questionnaire to patients followed by the pediatric rheumatology unit of the maternal and child department of the AOU FEderico II in Naples. The questionnaire asked, how many doses were administered, if there were any side effects following the vaccination and if so which effects. After the second dose of vaccine had been given, we measured the anti-Sars-Cov2 antibodies, we assessed the presence or absence of antibodies. Once the data was collected, we divided the patients into two groups: a case group in which the therapy was not interrupted during the vaccination, and a control group that stopped the therapy. Results: We recruited 84 patients, of whom 83 were given the Pfizer vaccine and one modern. All received a single dose of vaccine, while 79/84 received a second dose. After the first vaccine dose, 54/84 reported collateral effects (4 fever, 31 injection site reactions, 2 menstrual cycle changes alterations, 3 headache, 8 muscle pain, 2 asthenia, 1 lymphadenomegaly). In 44 patients the symptoms resolved, 7 used symptomatic therapy. After the second dose the side effects occurred in 70/79 patients (12 fever, 3 headache, 1 cough, 2 chills, 5 pain, 4 asthenia, 24 injection site reactions, 1 lymphadenomegaly). In 36 patients the symptoms resolved spontaneously, in 15 patients symptomatic therapy was used. 11 patients discontinued immunosuppressive therapy. Conclusion: A great number of patients with chronic rheumatic diseases adhered to the vaccination campaign, no different or more severe collateral effects were reported in these than in the healthy population, and immunosuppressive drug administration and failure to discontinue did not lead to more severe collateral effects. Finally, we can state that anti-SarsCov2 vaccines can also be used in children with rheumatological diseases. The serological response of these patients is currently being evaluatedZ. Kolkhidova, S. Salugina, I. Nikishina, M. Kaleda, E. Fedorov, A. ShapovalenkoIt is known that the factors of severe COVID-19 are various comorbid conditions, including autoinflammatory diseases (AIDs) and concomitant immunosuppressive therapy. It is still unclear how COVID-19 affects the course and outcomes of AIDs. There is also data on the onset of AIDs after COVID-19. In the literature, information about the relationship between AIDs and COVID-19 is presented in some publications. Objectives: To study the frequency and features of the course of COVID-19 in children with various AIDs according to the Federal Rheumatology Center, to assess its impact on the course and outcomes of AIDs. Methods: The retrospective analysis included 36 pts (pts) with monogenic and multifactorial AIDs who had a COVID-19 confirmed by PCR test (12 pts), positive IgG (24) and/or IgM (2) antibodies. Results: For the period from 2020 to 2021, among 320 pts with rheumatic disease who underwent COVID-19, 36 (11.3%) had AIDs. Among them, Cryopyrin-associated periodic syndrome (CAPS) – 7 (20%), Familial Mediterranean Fever (FMF) -6 (17%), Blau-syndrome (BS) -2 (5%), most of them were pts with systemic juvenile idiopathic arthritis (sJIA) - 21 (58%). The number of boys and girls was the same. Median (Me) age was 13 years (y) [min 2; max 17]. The characteristics of pts are presented in Table 1. Me duration of AIDs at the time of COVID-19 was 8 y. All pts received stable therapy, which included DMARDs - 21 and Biologics (B) – 24 pts. The mean duration of therapy was 5.6 y. None of the pts received glucocorticoids. Half of pts (19) had mild clinical manifestations of COVID-19 (fever-11, rhinitis-5, anosmia-7, sore throat-1, cough-6, arthralgia-1, rash-1, dyspepsia-1), 17 (47%) children were asymptomatic. Pts with symptoms of COVID-19 compared with asymptomatic pts had a shorter duration of illness (5 vs 8 y) and received B for a longer period of time (5 vs 3 y). All pts were treated as outpatients except one girl, who required hospitalization due to initial signs of multisystem inflammatory syndrome with a good outcome. None of the pts demanded oxygenation support. At the time of, remission of AIDs was in 20 (56%), low activity in 10 (28%), high activity in 6 (16%) pts. After the COVID-19, a relapse and worsening of the disease developed in 11 (30%) pts, among them mainly pts with sJIA - 9 (82%). 3 pts required temporary withdrawal of DMARDs and B. Two girls after COVID-19 showed the development of sJIA 2 weeks and 3 months after infection. Conclusion: According to our study, pts with AIDs mostly had a mild or asymptomatic course of COVID-19, especially during remission. The therapy with DMARDs and B were continued in the majority of pts. Disease exacerbation after COVID-19 was noted in one-third of pts, more often with sJIA. We have noted 2 cases of sJIA after COVID-19 infection, thus not excluding the role of COVID-19 as a new triggering factor. Careful monitoring of the course of COVID-19 in children with AIDs, as well as long-term follow-up after COVID-19, is necessary. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
A. Schvartz 1 , R. Kechiche 1 , F. Bajolle 2 , S. Poignant 3 , R. Basmaci 4 , C. Pajot 5 , I. Melki 6 , L. Morin 7 , C. Adamsbaum 8 , N. Matsa 9 , M. Hofer 10 , I. Kone Paut 1,9 , C. Galeotti 1,9 on behalf of French Covid-19 Paediatric Inflammation Consortium 1 Service de Rhumatologie Pédiatrique, CHU Bicêtre, APHP, Le Kremlin Bicêtre, 2 Unité médico-chirurgicale de cardiologie pédiatrique, Hopital Necker Enfants Malades, APHP, Paris, 3 Service de Pédiatrie Générale, CHU Nantes, Nantes, 4 Service de Pédiatrie-Urgences, Hopital Louis Mourier, APHP, Colombes, 5 Service de Néphrologie, Médecine Interne et Hypertension pédiatrique, CHU Toulouse, Toulouse, 6 Service de Pédiatrie générale, Maladies infectieuses et Médecine Interne, CHU Robert Debré, APHP, Paris, 7 Service de Réanimation Pédiatrique et Médecine Néonatale, 8 Service de Radiologie Pédiatrique, 9 Centre de Référence des Maladies AutoInflammatoires et des Amyloses, Ceremaia, CHU Bicêtre, APHP, Le Kremlin Bicêtre, France, 10 Service de Rhumatologie Pédiatrique de Suisse Romande, Hôpital Universitaire de Lausanne, Lausanne, Switzerland Correspondence: A. Schvartz Introduction: Pediatric Inflammatory Multisystemic Syndrome associated to SARS-CoV2 (PIMS) or Multisystem inflammatory syndrome in children ( MIS - C ) happens 4 to 6 weeks after a SARS-CoV2 primary infection. PIMS has a wide clinical spectrum and this can lead to a late diagnosis responsible for poorer clinical outcome. Its early diagnostic recognition as well as its early management is important to avoid cardiac complications related to this pathology. Objectives: We intend to describe a symptom not described enough, associated with PIMS which can lead to a better understanding of the wide spectrum of the disease, and help for faster diagnosis. Methods: The JIR Cohort database, an international registry collecting data on patients with pediatric inflammatory diseases, was consulted to include patients between 03/15/20 and 12/31/2021. Results: Of the 140 patients in whom a diagnosis of PIMS was retained, we present a series of 38 patients (27%) who presented at diagnosis or during evolution, febrile torticollis or painful cervical involvement. These patients were on average 8.2 years old (0.6-15.2). The proportion of boys was 14 out of 38 (37%). Twenty-four patients out of 33 (73%) were hospitalized in intensive care. Ten patients out of 38 (26%) underwent cervical imaging, 5 (50%) had abnormalities such as collection or infiltration of the soft tissues. At the therapeutic level, 27/38 patients (71%) received corticosteroid therapy, 33/38 (87%) immunoglobulins, and 26/38 (68%) antibiotic therapy. Conclusion: PIMS is a pathology with significant clinical heterogeneity and severe consequences in case of delay in diagnosis and therapeutic management. In this epidemic context, it is important to consider PIMS in any patient with febrile torticollis, especially if he does not respond to antibiotics. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
C. Sembenini, G. Martini, D. Martino, A. Meneghel, F. Tirelli, M. Fastiggi, F. Zulian, M. E. Zannin Paediatric Rheumatology Unit, Department for Woman and Child Health, University of Padova, Italy , 2 Ophtalmology Unit University of Padua, Padua, Italy Correspondence: C. Sembenini Introduction: Juvenile idiopathic arthritis-related uveitis (JIA-AU) has been rarely reported as triggered by vaccines. Since the start of the vaccination campaign against SARS-CoV2, uveitis following the administration of the vaccine has been rarely reported. Objectives: To evaluate the possible association between anti-Covid19 vaccination and relapses of JIA-AU in paediatric patients Methods: A single-centre observational study was conducted in paediatric patients with JIA-AU followed at our Centre between April 2021 and March 2022. Results: Out of 77 patients affected by JIA-AU, who underwent anti-SARS-CoV2 vaccination, 66 (86%) did not have any complication while 11 (14%) developed a relapse of AU within 6 weeks (mean 24 days, range: 3-42 days) after 2 nd or 3 rd vaccine booster. The mean age of relapsed patients was 16.6 years (range 11-24); 10 presented unilateral relapse, one bilateral. All patients presenting JIA-UA relapse after vaccination, were on stable dose of treatment (all in methotrexate combined with adalimumab in 6 cases and abatacept in 2) and had been in AU remission for >12 months. In view of the vaccine, 10 patients had temporarily stopped systemic treatment: 7 patients for 2 weeks and 3 patients for > 2 weeks. The course of uveitis relapse was very benign as all patients went back into full clinical remission with just topical steroid therapy. No patients experienced arthritis relapse. Conclusion: Recurrence of JIA-AU after Sars-Cov2 vaccination was observed in a minority of patients, was mild, transient and regressed only with topical treatment. These findings underline the need for a close ophthalmological follow-up of all patients with JIA-related AU who underwent Sars-Cov2 vaccination. Further observations are needed to establish whether the vaccine itself or the combined temporary suspension of the systemic treatment are responsible of the AU relapses. Disclosure of Interest : None declared
S. Sener 1 , E. D. Batu 1 , U. Kaya Akca 1 , E. Atalay 1 , M. Kasap Cuceoglu 1 , Z. Balik 1 , Y. Bayindir 1 , E. Aliyev 1 , O. Basaran 1 , T. Karagoz 2 , Y. Ozsurekci 3 , Y. Bilginer 1 , S. Ozen 1 1 Pediatric Rheumatology, 2 Pediatric Cardiology, 3 Pediatric Infectious Diseases, Hacettepe University, Ankara, Turkey Correspondence: M. Kasap Cuceoglu Introduction: Multisystem inflammatory syndrome in children (MIS-C) is a cytokine storm syndrome that may mimic Kawasaki disease (KD) or may present as a shock-like syndrome. Objectives: In the presented study we describe the features that may help distinguish KD from MIS-C based on our clinical experience in pediatric patients. Methods: We retrospectively evaluated MIS-C patients with KD-like symptoms and those with KD, followed between July 2020 and October 2021. Results: Fifteen KD patients and 33 MIS-C patients with KD-like phenotype were included. The median age of MIS-C patients was older than KD patients at the time of diagnosis (7.6 vs. 3.4 years; p=0.004). Thirty MIS-C patients met the criteria for complete KD and the others for incomplete KD. Rash (p=0.044), oral mucosal changes (p=0.044), and cervical lymphadenopathy (p<0.001) were more frequent in KD. In contrast, MIS-C patients had more gastrointestinal symptoms (p<0.001), respiratory symptoms (p=0.044), and hypotension (p=0.002). MIS-C patients had lower lymphocyte and platelet counts than KD patients (p<0.001 for both). MIS-C patients often had high ferritin, D-dimer, troponin, and brain natriuretic peptide levels (p<0.001). MIS-C patients had significantly decreased left ventricular systolic functions (p=0.010). All patients received intravenous immunoglobulin as initial therapy. While most MIS-C patients were also given corticosteroid therapy, only five KD patients with refractory fever received corticosteroid therapy. Conclusion: As the COVID-19 epidemic continues to spread, there is an urgent need to improve our understanding of this new disease. Although both KD and MIS-C can be perceived as cytokine storms of children, they have distinctive featureT. Šinkovec Savšek 1 , M. Zajc Avramovič 1 , T. Avšič Županc 2 , T. Avčin 1,3 , N. Toplak 1,3 1 Dpt of Allergol., Rheumatol., Clin. Immunol., UCH, UMC Ljubljana, 2 Dpt of Microbiol., Immunol., Faculty of Medicine, Univ. Lj., 3 Faculty of Medicine, Univ. Lj., Ljubljana, Slovenia Correspondence: T. Šinkovec Savšek Introduction: Children with rheumatic diseases (RD) are often immunocompromised because of the disease and/or the therapy they receive. At the beginning of COVID-19 pandemic there was a great concern about the possibility of severe SARS-CoV-2 infection in these children. The best method of protection is vaccination, so as soon as vaccine was licenced, we aimed to vaccinate them. However, a lot of them already got the infection before vaccination. Objectives: Primary objective was to investigate the clinical presentation (CP) of SARS-CoV-2 infection in children with RD, the relapse rate (RR) of RD after infection and vaccination and to follow immunogenicity after infection and vaccination. Secondary objective was to follow safety and efficacy of vaccination against SARS-CoV-2 and to investigate if the immunogenicity and efficacy depended on the therapy. Adverse events (AE) after SARS-CoV-2 vaccination were studied and compared to healthy children. Methods: This was partially a retrospective study where we collected data about children who had SARS-CoV-2 infection at regular visits at rheumatology outpatient clinic in UMC Ljubljana. Children who were vaccinated against SARS-CoV-2 were followed prospectively. Serology was followed on a long term in both groups. For ach child we collected demographic data, diagnosis, therapy, time of infection or vaccination, CP of infection, AE after vaccination, the outcome of infection, serology (IgA, IgG) and possible relapse of RD after infection or vaccination. For serology anti-SARS-CoV-2 ELISA IgG and anti-SARS-CoV-2 ELISA IgA from manufacturer Euroimmun, Medizinishe Labordiagnostika AG, Lübeck, Germany was used. For statistical analysis Fisher’s exact test and T-test were used. Results: We collected data for 93 children with RD after infection (73% female) and for 43 after vaccination (63% female). In 93 children we registered 104 SARS-CoV-2 infections, 11 children got the infection twice. In 15% of events the infection was asymptomatic, 66% experienced mild CP, 18% moderate CP, 1% needed hospitalization. No statistically significant (SS) difference was detected on comparison of the CP between different therapy treatments for RD. 12% of children had a relapse after infection and 5% after vaccination, but the difference was not SS (p=0.23). No SS difference was detected in AE after vaccination between children with RD and a healthy control group which consisted of 92 children (59% female), p=0.48 (1. dose), p=0.86 (2. dose). After vaccination 44% of children got infected with SARS-CoV-2, on average 4.4±1.7 months after vaccination; 47% of those children were treated with TNFα inhibitors. A SS difference was detected in serology between children treated with TNFα inhibitors and those treated with other or no medication (IgG p=1,68*10 -3 , IgA p=1,49*10 -2 ). Conclusion: RR was 12% after infection and 5% after vaccination. There was a trend towards higher RR after infection. The CP of SARS-CoV-2 infection in children with RD was mostly mild, no child had respiratory distress, and no one needed oxygen. One child was hospitalized because of dehydration. AE after vaccination were comparable between children with RD and healthy control group. A SS difference was detected in serology between children after infection and vaccination. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
T. F. Spracklen 1 , S. Mendelsohn 1 , C. Butters 1 , H. Facey-Thomas 1 , R. Stander 1 , M. Erasmus 1 , J. Day 1 , C. Scott 1 , L. Zühlke 2 , T. Scriba 1 , K. Webb 1 1 University of Cape Town, 2 South African Medical Research Council, Cape Town, South Africa Correspondence: T. F. Spracklen Introduction: Multisystem inflammatory syndrome in children (MIS-C) is a severe acute inflammatory reaction to SARS-CoV-2 infection in children. Little is known about how MIS-C develops or its optimal treatment, particularly in Africa. Objectives: This study sought to characterise differential expression of immune-related genes in a cohort of South African MIS-C patients and controls. Methods: The cohort included 30 pre-treatment MIS-C cases and 54 healthy non-inflammatory paediatric controls. Febrile controls (n = 36) included patients with systemic lupus erythematosus (n = 7), Kawasaki disease (n = 8) and other inflammatory or infective conditions (n = 19). Longitudinal post-treatment MIS-C specimens were available for intravenous immunoglobulin (IVIG; n = 10) or IVIG and methylprednisolone (n = 30). Expression of 80Holms correction. Receiver operating characteristic (ROC) analysis was used to investigate discriminatory capacity of variables. Results: A total of 29 DEGs were identified in pre-treatment MIS-C compared to healthy controls. Only one gene, IL27 , was differentially up-regulated in MIS-C compared to other febrile conditions (p = 0.00056), and none were significantly down-regulated. IL27 expression could reasonably differentiate MIS-C from these febrile conditions (area under the ROC curve: 0.759; 95% CI: 0.632-0.886). Post-treatment follow-up data was available for 40 MIS-C patients for up to 1,200 hours after first treatment, and showed that IL27 and the majority of other DEGs resolved to normal levels in the timeframe with IVIG and/or methylprednisolone. All patients clinically recovered. Conclusion: These data indicate a role for IL-27 in development of MIS-C in South African children. IL-27 is a cytokine involved in differentiation of T helper 1 cells. Low serum levels of IL-27 have been described as a good prognosis indicator for adult COVID-19, and the up-regulation of this gene in our cohort suggests that IL-27 may be a key undescribed contributor to MIS-C phenotypes after SARS-CoV-2 exposure in children. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
A. Taddio 1,2 , S. Della Paolera 1 , F. Ricci 3 , M. Giangreco 2 , L. Abbagnato 4 , A. Meneghel 5 , A. Smarrazzo 6 , A. Mauro 7 , F. Biscaro 8 , A. Meini 3 , R. M. Dellepiane 9 , M. Fabi 10 , I. Floretta 11 , F. La Torre 12 , L. Mambelli 13 , L. Verdoni 14 , M. C. Maggio 15 , S. Stucchi 16 , M. Tardi 7 , R. Sottile 17 , F. Zunica 3 , G. Zuccotti 18 , D. Montin 19 , R. Caorsi 20 , A. Consolaro 20 , G. Simonini 21 , M. Gattorno 22 , M. Cattalini 3 on behalf of on behalf of Italian Pediatric Rheumatology Society - ReumaPED 1 University of Trieste, 2 Institute for Maternal and Child Health - IRCCS Burlo Garofolo, Trieste, 3 Pediatrics Clinic, ASST Spedali Civili di Brescia, University of Brescia, Brescia, 4 U.O.C. Pediatria ASST Lariana Sant’Anna, Como, 5 Department of Woman’s and Child’s Health, University of Padova, Padova, 6 UOC Pediatria Multispecialistica, Dipartimento di Emergenza, Accettazione e Pediatria Generale, IRCCS Ospedale Pediatrico Bambino Gesù, Roma, 7 Pediatric Emergency Unit, Santobono-Pausilipon Children’s Hospital, Napoli, 8 Division of Pediatrics, Presidio Ospedaliero di Treviso, Treviso, 9 Pediatric Intermediate Care Unit, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milano, 10 Department of Pediatrics, University of Bologna, IRCCS Sant’Orsola-Malpighi Hospital, Bologna, 11 Ospedale Santa Chiara, Trento, 12 Pediatric Rheumatology Center, Pediatric Unit, “Giovanni XXIII”, Pediatric Hospital, Bari, 13 UOC di Pediatria e Neonatologia, Ospedale di Ravenna, AUSL della Romagna, Ravenna, 14 Paediatric Department, Hospital Papa Giovanni XXIII, Bergamo, 15 Department of Health Promotion Sciences Maternal and Infantile Care, Internal Medicine and Medical Specialities “G. D’Alessandro”, University of Palermo, Palermo, 16 S.C. Pediatria, ASST Grande Ospedale Metropolitano Niguarda, Milano, 17 Department of Paediatrics, Pediatria 2, Santobono-Pausilipon Children’s Hospital, Napoli, 18 Department of Pediatrics, University of Milan, Children’s Hospital V Buzzi, Milano, 19 Department of Pediatrics and Public Health, University of Turin, Torino, 20 Clinica Pediatrica e Reumatologia, IRCCS Istituto Giannina Gaslini and DINOGMI, Università di Genova, Genova, 21 Pediatric Rheumatology Unit, AOU Meyer, University of Florence, Firenze, 22 Center for Autoinflammatory diseases and Immunodeficiencies, IRCCS Istituto Giannina Gaslini, , Genova, Italy Correspondence: A. Taddio Introduction: Multisystem Inflammatory Syndrome in children (MIS-C) is systemic mulkti-organ inflammation following SARS-CoV-2 infection in children. No clear data are available about the best treatment approach: while the use of immunoglobulin is widely accepted, the use of corticosteroids is controversial and the role of biologic treatment is not defined yet. Objectives: The main aim of this study was to define the better treatment option in MIS-C children and in particular to analyze the role of anakinra in this subgroup of patients. Methods: This is a multicenter retrospective cohort study. MIS-C patients were defined if they fulfilled the RCPCH clinical definitions. Patients were treated according to the attending physician’s decision. The patients were then divided in 4 groups on the basis of the treatment options that the patients underwent at time of admittance: immunoglobulins (IVIG), IVIG plus methylprednisolone (≤ 2 mg/kg/day), IVIG plus high dose methylprednisolone (> 2 mg/kg/day) and anakinra with or without other drugs (IVIG and/or methylprednisolone). Primary outcomes were defined as the presence of at least one of the following clinical characteristics: death, the failure of initial treatment needing additional treatment for clinical worsening based on treating physician decision and cardiac involvement at the end of follow-up. We then divided our population into 2 groups: patients with good outcome (absence of any of the primary outcomes) and patients with bad outcome (presence of at least 1 primary outcome). Results: At univariate analysis, persistent heart involvement at discharge was not associated with any clinical or laboratory test at time of diagnosis but but it was more frequent in those not receiving anakinra as initial treatment (3/20 vs 66/189; p=0.047). The need of vasoactive agents was associated with heart involvement at time of diagnosis as well as with the presence of hypotension/non-cardiogenic shock; similarly heart and pulmonary involvement were also associated with ventilatory support and intensive care admittance. Comparing the different treatment approaches adjusting for the propensity score we found that early treatment with anakinra was associated with higher probability to need intensive care admittance (p=0.0045; OR: 1,237-3,181), vaso-active agents use (p<0.0001; OR: 1,828-5,538) and initial treatment failure (p<0.0001; 8,003-30,112); however anakinra use was also associated to lower probability to have persistent heart disease at the end of follow-up (0,17-0,513) while no difference was found between high and low dose of corticosteroids at time of admission. Conclusion: The early treatment with anakinra seems to be safe and very useful in patient with MIS-C. However, anakinra could be considered the best treatment options for those patients with higher probability to develop a severe disease; unfortunately it is not clear which are the clinical or laboratory characteristics at onset which may predict a more severe course. Disclosure of Interest : None declared
C. Udaondo 1 , C. Cámara 2 , L. Miguel 2 , R. Alcobendas 1 , C. Muñoz - Gomez 1 , C. Millan - Longo 1 , B. Diaz - Delgado 1 , I. Falces - Romero 3 , M. Diaz - Almiron 4 , J. Cano Ochando 5 , A. Mendez - Echevarria 6 , A. Remesal 1 , C. Calvo 6 1 Paediatric Rheumatology, 2 Immunology, 3 Microbiology and Parasitology, 4 Biostatitics, Hospital La Paz, 5 National Microbiology Centre, Instituto de Salud Carlos III, 6 Paediatric and Infectious Diseases, Hospital La Paz, Madrid, Spain Correspondence: C. Udaondo Introduction: Immunossupressive treatments for rheumatic diseases (RD) could potentially interfere with vaccine response. There is a lack of information about efficacy of the mRNA SARS-CoV-2 vaccine in teenagers with rheumatic diseases (RD) including cellular response. Objectives: To evaluate safety and efficacy of the BNT162b2 vaccine, including humoral and cellular response, in teenagers with RD and immunosuppressive treatment compared with a control group. Methods: Teenagers from 12 to 18 years with RD followed at a Pediatric Rheumatology unit in Madrid and non-RD teenagers receiving BNT162b2 mRNA vaccination were assessed 3 weeks after complete vaccination. Humoral response was measured by total antiSpike antibodies, and cellular response by IFN-γ and IL-2 in blood stimulated with SARS-CoV-2 Spike and M proteins. Results: A total of 40 adolescents with RD and 24 healthy peers were recruited. The most frequent diagnosis was juvenile idiopathic arthritis (26/40, 65%) followed by Lupus (6/40, 15%). 60% of cases (23/40) received Tumour Necrosis Factor Inhibitors and 35% (14/40) methotrexate. No differences in spike-specific humoral or cellular response were found between groups (median IFN-γ response; 529 pg/ml in controls vs. 398 in RD patients, p 0.78, and median IL-2 in controls: 635 pg/ml vs. 497 in RD patients, p 0.22). 95% of RD patients and 96% of controls had maximum value of humoral response (IgG antibodies above 10 index). 40% of patients (26/64) had previous SARS-CoV-2 infection, 9 controls and 17 RD patients without differences. 70% of infections were asymptomatic. COVID-19 recovered individuals had higher IFN-γ than naive subjects in both groups (controls: median 859 pg/ml in recovered vs. 450 in naïve p 0.017, RD patients: 850 vs. 278 p 0.024). No serious adverse events or flares were reported following vaccination. Conclusion: Standard of care treatment for teenagers with RD did not affect the humoral and the cellular immunity to BNT162b2 mRNA vaccination compared to a control group, suggesting that no treatment discontinuation or additional dosing would be required compared to healthy peers. Previous SARS-CoV-2 infection was the most relevant factor in the immune responsV. Villarreal Treviño 1,2 on behalf of COLIBRI, V. Ramírez Nova 3 , I. Pelaez Ballestas 3 , S. G. Rosiles De la Garza 2 , F. Moctezuma Rios 3 , M.-G. Rodríguez Maldonado 4 , P. A. León 5 , M. De la O Cavazos 2 , N. Rubio Pérez 2 on behalf of COLIBRI, R. Hernández Martínez 5 , R. C. Calderón Zamora 2 , K. Ruiz Lopez 3 , J.-F. Heredia Gozález 3 , F. García Rodríguez 2 on behalf of COLIBRI on behalf of COLIBRI 1 Pediatrics, Hospital Materno Infantil SSA, Guadalupe, NL, 2 Pediatrics, Hospital Universitario “Dr. José E. González”, Monterrey, 3 Rheumatology, Hospital General de México, Eduardo Liceaga “Dr. Eduardo Liceaga”, 4 Rheumatology, 5 Pediatrics Intern Medicin, Cento Médico Nacional “La Raza”, Instituto Mexicano del Seguro Social, Mexico City, Mexico Correspondence: A. V. Villarreal Treviño Introduction: An emerging entity described during the Coronavirus disease 2019 pandemic was initially reported as cases of older school-aged children and adolescents presenting with prolonged fever, shock, abdominal pain, and cardiac dysfunction after SARS-CoV-2 infections, termed as Multisystem Inflammatory Syndrome in children (MIS-C). European and North American cohorts described this syndrome is more frequent in males, adolescents and from racial and ethnic minorities. Objectives: This study aims to describe epidemiological and clinical characteristics of Mexican patients with MIS-C at admission at four centers in Mexico. Methods: This is a cross-sectional retrospective study of four Hospitals of specialized-care in Mexico, 2 in the center of the country (Mexico City) and 2 in northeast (Monterrey). Patients classified with MIS-C according to the WHO definition, between April 2020 and April 2022 were included. Epidemiological and clinical characteristics at the diagnosis were collected and descriptive analysis using percentages, median, intercuartile ranges (IQR), and a Kaplan-Meier survival analysis was performed. Results: 106 participants, n=78 (73.5%) form the northeast and n=33.(31.1%) from center. n=67 (84.5%) were men, with a median age of 66.5 months (IQR 27-120). The median time from onset to care at the center was 6 days (IQR 4-8). MIS(-C) was characterized by fever n=106 (100%), mucocutaneus n=91 (85.8%), gastrointestinal n=62 (58%) and cardiocirculatory manifestations n=42 (39.6%) and increased inflammatory biomarkers n= 89 (90.8%) with a C reactive Protein median 17.3mg/dL (IQR 3-68.8), n=15.9 (15%)presented overweight according to the Body Mass Index percentile, schock n=29(27.4%), n=37 (35%) needed intensive care with a median length of stay at this unity of 7 (IQR 3-9) days. The laboratory data are shown in Table 1. The n=95 (90%( received Human Intravenous Immunoglobulin, n=81 anticoagulants. The median of hospitalization days were 8 (IQP 5-13), mortality higher than other cohorts n=6 (8%), Macrophage activation Syndrome reported in n=6 (8%) and n=5(5.7%) had evidence of thrombosis. Kaplan-Meier curve of time to discharge or death compared by the presence of hemodynamic alterations on admission, the curve shows higher day of hospital stay and death in patients with hemodynamic alterations at admission p< 0.01. Conclusion: This cohort showed more severe disease in MIS-C patients with hemodynamic alterations at admission. Mexican children showed similarities to other cohorts in the literature, sever cases at northeast population with increased mortality were observed. Tanslational studies and long-term follow-up of the patients are necessary to clarify the pathophysiology and predictive factors of severity to establish the long-term prognosis of this new entity. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
T. Marushko, O. Vovk, Y.-E. Kulchytska 1 Pediatrics-2, Shupyk National Healthcare University of Ukraine, Kyiv, Ukraine Correspondence: O. Vovk Introduction: The occurrence of infectious diseases in children with juvenile idiopathic arthritis on immunosuppressive treatment has its own peculiarities, so they may be at higher risk of severe coronavirus disease development following the infection with SARS-CoV-2. Objectives: To evaluate the COVID-19 infection incidence in children with juvenile idiopathic arthritis and to study the COVID-19 infection course in children with JIA who are being treated with disease-modifying antirheumatic drugs (DMARDs). Methods: We analyzed 63 clinical cases of Covid-19 infection in children with JIA who received DMARDs. Covid-19 infection in patients with JIA was diagnosed by a positive PCR test for SARS-CoV-2 and/or a positive serology test and presence of an infected family member. Results: We examined 63 children diagnosed with JIA, 73% of whom had SARS-CoV-2 infection. 32 patients received methotrexate, and 14 patients received biologic DMARDs (bDMARDs) (adalimumab) and methotrexate. There were no cases of severe Covid-19 disease course that required inpatient treatment. 12.4% of Covid-19 infected children on methotrexate treatment had no clinical symptoms of infectious disease. 1 patient from the group receiving adalimumab and methotrexate also had no clinical symptoms of infectious disease. 46.9% of patients on methotrexate had a mild course of coronavirus infection (rhinopharyngitis). 14.2% of children receiving bDMARDS and methotrexate had a mild course. 4 patients were diagnosed with viral pneumonia (1 child on methotrexate and 3 children on bDMARDS and methotrexate). 39.1% of patients, due to SARS-CoV-2 infection, were diagnosed with flare in the form of arthritis, which required intra-articular administration of glucocorticoids in 33.3% of cases and nonsteroidal anti-inflammatory drugs treatment in 44.4% of cases. Conclusion: JIA patients, compared with healthy children, are more vulnerable to SARS-CoV-2 infection. The clinical course of COVID-19 infection in children with JIA who have received immunosuppressive drugs such as conventional synthetic or biologic DMARDs may have a mild course of SARS-CoV-2 infection. However, it was found that SARS-CoV-2 infection had a significant impact on JIA activity, requiring the use of intra-articular glucocorticoid administration and prescription of nonsteroidal anti-inflammatory drugs. To better investigate the associations between JIA, pharmacological management, clinical manifestations, and possible consequences of SARS-CoV-2 infection in these patients, further studies with a larger number of cases are needed. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
, F. Haslak, A. Gunalp, A. Adrovic, S. Sahin, K. Barut, O. Kasapcopur errahpasa Medical School, İstanbul, Turkey Correspondence: M. Yıldız Introduction: The systems related to the internet and internet network have been in a great change and development since they were first used. While there was a one-sided flow of data in the first years of these systems, two-sided data transfer is possible today, which gives the opportunity to use internet systems for different purposes. One of the most important results of these changes has been the establishment of social media networks, the basis of which is personal data sharing. Although social media networks are quite new, they occupy a prominent place in people’s daily lives in today’s modern world. Objectives: The aim of this study is to assess the parental social media habits and its effect their willingness to allow their children to receive COVID-19 vaccination. Methods: A web-based questionnaire was circulated among the families of patients under 12 who have been following up at Istanbul University- Cerrahpasa pediatric rheumatology department. The families were asked about their social media habits and their intention to vaccinate to their children against COVID-19. Results: Parents of 138 children (75 females) with rheumatological diseases were completed the form. The families stated that the most common sources they used for getting information about COVID-19 were television/radio [99 (71.7%)], social media [71 (51.4%)] and health professionals [69 (50%)]. Instagram [123 (89.1%)], Facebook [96 (69.6)] and Twitter [45 (32.6)] was the most used social media platforms among the families. A total of 60.9% of the parents stated that they spend more than an hour a day on social media platforms. To stay informed was the first reason for using social media platforms, which is stated by 55 (39.9) of the parents . Other common reasons for using social media were to keep track of daily events (35.5%) and to keep touch with friends (17.4%). Majority of the parents (90.6%) were following a medical physician on social media and 89.1% of them stated that the statements about medical conditions by the physicians has a significant effect on their decisions if the statements are related to their field of expertise. When the parents were asked about their intention to vaccinate their children in the case the Ministry of Health recommends vaccinating children under the age of 12; 57 (41.3%) of parents answered “yes”, 14 (10.1%) “no” and the remaining “not decided yet”. There were no significant differences between the parents stated they would vaccinate their children and would not vaccinate their children according to social media accounts they have, the number of accounts, time spent on social media (p value for all > 0.05). Conclusion: The results of our study pointed out that there is no direct connection between the parental social media habits and willingness to their children. The fact that vast majority of families follow at least one doctor on social media and that declare the statements made by the doctors they follow on social media are important in their decision-making processes may be emphasizing the importance of the use of social media by physicians. Further studies are needed. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
A. Ziv 1,2 , M. Heshin-Bekenstein 2,3 , R. Haviv 4,5 , S. Kivity 5,6 , D. Netzer 7 , S. Yaron 7 , Y. Schur 7 , T. Egert 8 , Y. Egert 9 , Y. Sela 10 , P. J. Hashkes 11,12 , Y. Uziel 5,13 1 Pediatrics, MEIR HOSPITAL, Kfar Saba, 2 Sackler School of Medicine , Tel Aviv University, Tel Aviv , 3 Pediatric Rheumatology Service, Dana Children’s Hospital, Tel Aviv Medical Center, Tel Aviv, Israel, Tel Aviv, 4 Pediatric Rheumatology Unit, Meir Medical Center, Kfar Saba, 5 Sackler School of Medicine , Tel Aviv, 6 Rheumatology Unit, Meir Medical Center, Kfar Saba, 7 Community Medical Services Division, Clalit Health Services, Tel Aviv, 8 Inbar, NPO, Ramat Gan, 9 Inbar, NPO, Ramat Gan, 10 The Research Center for Internet Psychology, School of Communications, Reichman University, Herzliya, 11 Pediatric Rheumatology Unit, Shaare Zedek Medical Center, Jerusalem, 12 Faculty of Medicine, Hebrew University of Jerusalem, Jerusalem, 13 Pediatric Rheumatology Unit, Meir Medical Center, Kfar Saba, Israel Correspondence: A. Ziv Introduction: The effectiveness of the BNT162b2 mRNA COVID-19 vaccine for adolescents with juvenile-onset autoimmune inflammatory rheumatic diseases (AIIRD) is unknown. Several studies suggest attenuated immunogenicity in patients with AIIRD. Objectives: This study evaluated the effectiveness of the BNT162b2 mRNA COVID-19 vaccine in preventing COVID-19 infection in adolescents with juvenile-onset AIIRD compared to healthy controls. Methods: We used data from Clalit Health Services, the largest healthcare organization in Israel, to conduct an observational cohort study from June to December 2021, involving adolescents ages 12–18 years, diagnosed with AIIRD. Study outcomes included documented COVID-19 infection in relation to vaccination status and immunomodulatory therapy. We estimated vaccine effectiveness as one minus the risk ratio. Healthy adolescents in the same age group served as controls. Results: A total of 1,639 adolescents with AIIRD (juvenile idiopathic arthritis, systemic lupus erythematosus, or familial Mediterranean fever) were included and compared to 524,471 adolescents in the same age range. There was no difference in COVID-19 infection rates after the second dose of vaccine for those with AIIRD and healthy controls (2.1% vs. 2.1% respectively, p=0.99). The estimated vaccine effectiveness for adolescents with AIIRD was 76.3% after the first dose, 94.8% after the second and 99.2% after the third dose. Conclusion: We found that the BNT162b2 mRNA vaccine is highly effective against COVID-19 in adolescents with AIIRD, similar to healthy controls. Immunomodulatory therapy did not affect its effectiveness. These results can encourage adolescents with AIIRD to get vaccinated against COVID-19U01. Tuberculosis after Tocilizumab in SJIAPU02. Chronic non bacterial osteomyelitis in 11 Indian children - a single center experiencePU03. Clinical and laboratory presentation of Neonatal Lupus Erythematosus (NLE) - case report PU04. Juvenile systemic lupus erythematosus – experience of a tertiary hospital PU05. The rare cause of back pain – the crucial role of ultrasound as the first step to diagnose Takayasu arteritis Tuberculosis is a major cause of morbidity in low an middle income countries. Antitumour necrosis factor targeted agents (anti TNFs) are known to increase the risk of reactivation of latent tuberculosis infection (LTBI). However, the risk in non anti TNF biologics such as Interleukin 6 inhibitor Tocilizumab (TCZ) has been noted to be very low or absent. Sporadic cases have been reported. One may be question if it is primary TB infection or LTBI reactivation post TCZ Objectives: We present a 11 years old girl who was diagnosed with systemic onset juvenile idiopathic arthritis and given TCZ. However, she developed fever after the first dose which was subsequently diagnosed as tuberculosis. Methods: This 11 years old girl initially presented with a history of intermittent for 3 months which managed conservatively by antibiotics and antipyretics. On examination we found that there was left sided cervical lymphadenopathy. Other examination was within normal limits. The patient was admitted for almost a month however the fever was still persistent. Investigations revealed anemia, progressively increasing leucocytosis with a neutrophilic preponderance. Urine and blood cultures were negative. Bone marrow examination and cervical lymph node biopsy was normal. tuberculosis was ruled out. USG of abdomen and CT scan of thorax were within normal limits. As there was a maternal history of Carcinoma a PET scan was done which was also normal. The child received multiple antibiotics however the fever persisted. She was discharged later on as the fever spikes decreased. Two months after that the fever spikes increased however this time there was an evanescent rash, cervical lymphadenopathy and and left knee diagnosis. Finally a diagnosis of Systemic onset juvenile idiopathic arthritis was made and the child started on subcutaneous methotrexate. Due to relapses leflunomide was added an subsequently TCZ administered. The fever reappeared along with cough. Results: A chest Xray was done which showed mediastinal widening. Contrast enhanced CT scan of thorax showed Nodular opacity in RLL with few small pulmonary nodules and conglomerated Mediastinal necrotic lymph nodes. The tuberculin sensitivity test was also positive. All this pointed towards pulmonary tuberculosis and anti tubercular therapy was started. Conclusion: Tocilizumab is a humanized anti-IL-6 receptor antibody which inhibits the binding of IL-6 to its receptors, IL 6 has both pro- and anti- inflammatory action and is involved in Th17 and Th22 cell differentiation which is critical for anti-mycobacterial activity. It is produced early during mycobacterial infection. It has been note that administration of tocilizumab is associated with a very low or absent risk of tuberculosis reactivation. In our case the child was initially screened for tuberculosis before the diagnosis of sJIA. However, after administration of TCZ she still developed tuberculosis. If this reactivation of latent tuberculosis or primary infection remains debatable. Disclosure of Interest : None declared
Chronic non-bacterial osteomyelitis (CNO) is an autoinflammatory bone disorder usually affecting children and adolescents. CNO covers a wide clinical spectrum from rather mild, time-limited, monofocal bone inflammation to severe chronically active or recurrent multifocal bone inflammation. Clinical signs of bone inflammation include localized skin redness (rare), warmth and/or swelling, and pain. CNO is a diagnosis of exclusion. MRI is a choice of investigation in diagnosis , follow up and exclusion of other close mimics. Management usually involves non-steroidal anti-inflammatory drugs (NSAIDs), corticosteroids, disease-modifying anti-rheumatic drugs (DMARDs, usually methotrexate or sulfasalazine), anti-TNF agents or bisphosphonates and anti IL-1 agents especially in monogenic forms of CNO. Objectives: We aim to unveil characteristics of CNO in eleven Indian children at our center in India. Methods: This is a retrospective analysis of 11 children who visited our unit between Aug 2013 to March 2022 and were diagnosed with CNO after excluding close mimicshis Table Shows Characteristics of 11 Indian Children with CNO at our Unit Conclusion: Pre-adolescent girls are most commonly affected. Tibia is the most common bone involved and metaphysis is the most common site of involvement. Some of these patients presented with low grade fever and skin lesions. MRI and Bone Scan remains the first imaging modality. Most of the patients responded to adequate course of NSAIDs. Few patients required bisphosphonates and biologics to control the disease. Trial registration identifying number: 1. Curr Osteoporos Rep. 2017; 15(6): 542–554. Published online 2017 Oct 27. doi: 10.1007/s11914-017-0405-9 Chronic Recurrent Multifocal Osteomyelitis : Presentation , Pathogenesis & Treatment Sigrun R. Hofmann, 1 Franz Kapplusch, 1 Hermann J. Girschick, 2 Henner Morbach, 3 Jessica Pablik, 4 Polly J. Ferguson, 5 and Christian M. Hedrich 1,6,7ucic 3 , Z. Milosevic-Anđelkovic 3 , H. Stamenkovic 1,2 , T. Stankovic 1,2 , J. Vojinovic 1,2 1 Pediatrics, Faculty of Medicine, University of Nis, Nis, Serbia, 2 Pediatric Rheumatology and Immunology, 3 Neonatology Department, Clinic of Pediatrics, Clinical Center Nis, Nis, Serbia Correspondence: D. S. Lazarevic Introduction: Neonatal lupus erythematosus (NLE) is a rare acquired autoimmune condition that is present at birth with diverse clinical presentation. It is transient disorder with transplacental passage of mother’s antibodies. Often is misdiagnosed as intrauterine infection or sepsis Objectives: Clinical and laboratory presentation of newborn with NLE. Methods: We report a child with NLE in mother without positive history on autoimmune diseases. Results: We present premature male newborn, small for gestation age with clinical symptoms starting just after birth with leucopenia and thrombocytopenia, but without elevated inflammatory markers. Antibiotics were administrated despite all bacterial cultures were sterile. In the first weeks of life severe thrombocytopenia required administration of immunoglobulins and platelets transfusion, while erythematous rash on face has appeared. A detailed hematological and the most common neonatal virological diagnostic testing were performed. AIDS and syphilis were excluded. Serological tests have confirmed active herpes simplex infection and parenteral acyclovir were started until polymerase chain reaction (PCR) results were negative. Suspected immunodeficiencies and neonatal alloimmune thrombocytopenia were ruled out, as immunoglobulin levels and flowcytometry were normal. Within the first month skin rash have changed and extended on whole face and neck. Skin lesions becomes much reddish, annular and clearly demarked. Clinical spectrum with hematological disorders were enough to suspect on NLE. Anti-Ro, Anti-La and ANA antibodies in child have confirmed our diagnosis, while mother has and Anti dsDNA antibodies associated. ECG Holter monitoring have excluded congenital heart block. Only supportive treatment was advised. Conclusion: Neonatal lupus erythematosus should be considered in newborns with annular skin lesions, hematological disorders and without congenital heart block even in clinically healthy mothers. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
M. Bastos Gomes 1 , A. Costa Azevedo 1 , A. L. Carvalho 2 , I. Cardoso 3 , F. Aguiar 4,5 , M. Rodrigues 4,5 , S. Ganhão 4 , I. Brito 4,5 1 Pediatric Department, Unidade Local de Saúde do Alto Minho, Viana do Castelo, 2 Pediatric Department, Hospital de Braga, Braga, 3 Pediatric Department, Centro Hospitalar Vila Nova de Gaia/Espinho, Vila Nova de Gaia, 4 Pediatric and Young Adult Rheumatology Unit, Centro Hospitalar Universitário de São João, 5 Faculdade de Medicina da Universidade do Porto, Porto, Portugal Correspondence: M. Rodrigues Introduction: Juvenile systemic lupus erythematosus (jSLE) is a systemic autoimmune disease, which can affect several organs and systems, diagnosed in pediatric age. Despite representing 15-20% of SLE cases, it is characterized by a more severe course. Objectives: Characterize the population of patients with jSLE followed at a tertiary university hospital. Methods: Retrospective study, including the patients with jSLE followed at the Pediatric and Young Adult Rheumatology Unit, diagnosed between 1987 and 2021. Demographic, clinical and laboratory data were collected. Disease activity was evaluated using Systemic Lupus Erythematosus Disease Activity Index 2000 (SLEDAI-2K) and SLE Disease Activity Score (SLE-DAS) (2018), and the disease damage was evaluated using the Systemic Lupus International Collaborating Clinics (SLICC) / American College of Rheumatology (ACR) damage index. Results: A total of 44 patients were included, 93% female, with a median age at diagnosis of 15 years (IQR 12-16) and median follow-up of 8 years (IQR 5-13). There were 2 deaths (sudden death; both with pulmonary hypertension (HTP)). The clinical manifestations were: mucocutaneous (72%), hematological (70%), osteoarticular (59%), renal (41% - class IV: 63%, class V: 21%), constitutional symptoms (27%), vascular (22%), serositis (16%), gastrointestinal (14%) and pulmonary (2%) manifestations. All patients had positive anti-nuclear antibodies, 59% with high title (>1:1000). Ongoing treatment was: glucocorticoids (70%), hydroxychloroquine (78%), mycophenolate mofetil (41%) and azathioprine (20%). 13% received rituximab. SLEDAI-2K at diagnosis was significantly higher than at last patient assessment (10 vs 2, respectively, p<0.001). Regarding disease activity, 87% have SLEDAI≤4 and 42% SLEDAI=0; SLE-DAS ≤2.08 in 80%, SLE-DAS ]2.08-7.64] in 9% and SLE-DAS>7.64 in 11%. SLICC/ACR damage index was ≥ 1 in 13%. Conclusion: The most frequent manifestations in this cohort were mucocutaneous and hematological, which is in agreement with the literature. Major organs were commonly affected, including 41% with renal involvement, corroborating the greater morbidity associated with this age group. Deceased patients had risk factors: one received a delayed diagnosis and had shrinking lung syndrome and HTP, the other had an overlap with systemic sclerosis and had severe early-onset HTP. Most patients have low disease activity, which is possible in the face of early diagnosis and timely and effective immunomodulatory and/or immunosuppressive therapy, as well as regular follow-up in a specialized unit. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
Z. Pytelová 1 , J. Čivrný 2 , T. Klimas 2 , D. Klepárník 1 , K. Bouchalová 1 1 Paediatric Rheumatology, Department of Paediatrics, 2 Department of Imaging, Faculty of Medicine and Dentistry, Palacký University Olomouc and Olomouc Hospital, Olomouc, Czech Republic Correspondence: Z. Pytelová Introduction: Takayasu arteritis is a rare large-vessel vasculitis affecting the aorta, its major branches and the pulmonary artery, and common symptoms are non-specific myalgia, weight loss and fever or absence of peripheral pulse and claudication in later stages. Objectives: We aim to describe a case of a rare disease with early diagnosis and highlight the importance of thorough ultrasound imaging. Methods: We present a case report. Results: A 13-years-old girl was admitted for back pain lasting 1.5 months. The pain woke the girl up from sleep and there was no effect of NSAIDs. For a psoriatic lesion on her elbow, she was examined by a dermatologist once. On the day of admission, there were elevated inflammatory markers (CRP 172 mg/l, erythrocyte sedimentation rate, ESR 137 mm per hour), normocytic anemia and coagulopathy. During ultrasound imaging of the abdomen, the radiologist described signs of arteritis of the abdominal aorta. Immediately, CT angiography was performed with findings of involvement of abdominal aorta appropriate for inflammation process. The finding was assessed as Takayasu arteritis type 3 according to angiographic classification. MR angiography proved the diagnosis and will be used during follow-up. The patient received 5 pulses of corticosteroid therapy (methylprednisolone) followed by oral corticosteroid (prednisone). Infectious causes of inflammation were excluded. We observed a prompt improvement in clinical features. On the 12 th day, we started treatment with tocilizumab 162 mg administered subcutaneously once a week. After 4 weeks, ultrasound finding was normal and we observed a decrease in inflammatory markers. At the time of abstract submission, she is symptoms-free, on therapy with tocilizumab, and her CRP and ESR are normal. Conclusion: Takayasu arteritis is rare large-vessel vasculitis. Common symptoms are non-specific and thus, there could be a delay in diagnosis. In this case, our radiologist noticed inflammatory changes in the abdominal aorta during the standard abdominal ultrasound and it led to a diagnosis. This case highlights both the importance of doing thorough ultrasound imaging and the effect of therapy – high-dose corticosteroid followed by biologics led to clinical improvement. Patient Consent: Yes, I received consent Disclosure of Interest : None declared
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Trends and characteristics of fertility-sparing treatment for atypical endometrial hyperplasia and endometrial cancer in Japan: a survey by the Gynecologic Oncology Committee of the Japan Society of Obstetrics and Gynecology | 493da867-94e7-4fa0-9177-4efd34255356 | 10157339 | Gynaecology[mh] | Due to a tendency for delayed marriage, the age of pregnancy is delayed in Japan . This problem overlaps with the occurrence of gynecological cancer in the reproductive age group. Recently, the number of endometrial cancer (EC) patients younger than 40 years has been increasing . There are approximately 500 patients with EC younger than 40 years per year in Japan. Of them, 77% were stage IA . Using the National Cancer Database, Ruiz et al. reported that the proportion of endometrial cancer patients who were treated with progestin therapy increased from 2.4% in 2004 to 5.9% in 2014. The recommended fertility-sparing (FS) treatments in the National Comprehensive Cancer Network (NCCN) guidelines include hormone therapy (medroxyprogesterone [MPA] and megestrol acetate [MA]) and levonorgestrel-releasing intra-uterine devices (LNG-IUDs) . Japanese treatment guidelines for EC mention that FS treatment is a treatment option for young patients with atypical endometrial hyperplasia (AEH) and EC (endometrioid carcinoma grade 1 [ECG1] and lesion limited to the endometrium) . However, since a variety of FS treatment regimens have been widely adopted, the current trends in FS-treatment are relatively unknown. To elucidate current trends in FS treatment, a questionnaire-style survey regarding FS treatment was performed, in Japan Society of Obstetrics and Gynecology (JSOG) gynecological cancer registered institutions. In addition, this study was performed to identify factors correlated with the clinical response to FS treatment, disease recurrence, pregnancy outcome, and any deviations from the eligibility criteria by analyzing the detailed information of each patient, which were difficult to collect from meta-analysis. In our view, this is the largest-scale evaluation to date in a retrospective nationwide study of FS treatment for AEH and EC patients. 1. Study design and patients2. Statistical analysis The endpoint of this study was to examine the current trends in FS treatment for AEH and ECG1 patients in Japan. The secondary objective was to examine the associations of clinical characteristics with the pathological complete remission (CR) rate, recurrence-free survival (RFS), and pregnancy and live birth rates. RFS was measured from the end date of the initial FS treatment to the date recurrence was confirmed. Time to complete remission (TTCR) was measured from the day starting initial treatment to the day achieving CR. TTCR was classified into 2 groups (TTCR <6 and ≥6 months). Survival curves were calculated using the Kaplan-Meier method, and the curves were compared using the log-rank test. A Cox proportional hazards model and logistic regression analysis were used for multivariate analysis. Frequency distributions were compared using the χ 2 test, unless the expected frequency was <5, in which case, Fisher’s exact test was used. All statistical analyses were performed using JMP software (version 14; SAS Institute, Cary, NC, USA). A value of p<0.05 was considered significant. This study was conducted by the Committee on Gynecologic Oncology of JSOG in the 2017–2018 fiscal year. A nationwide, retrospective questionnaire style survey—as performed. The survey items included patient demographics (age, body mass index [BMI], complications, family history, desire to have children, etc.), examinations for diagnosis, pathological diagnosis, regimen of FS treatment, adverse events (AEs), presence of myometrial invasion [MI]), maintenance therapy (oral contraceptives/low dose estrogen progestin, estrogen+progestin, or progestin only), outcomes of initial and recurrent FS treatment, and pregnancy outcomes. AEs were assessed using the Common Terminology Criteria for Adverse Events (CTCAE; ver. 4.0; National Institutes of Health, Bethesda, MA, USA). The data of patients with AEH and EC receiving FS treatment between January 2009 and December 2013 were collected from JSOG gynecological cancer registered institutions. These institutions consisted of medical training institutions, cancer specialty hospitals, and local core hospitals. This study was approved by the Institutional Review Board (IRB) of Kurume University and JSOG (IRB registration No. 17310/ UMIN No. 000034254). The present study was conducted after obtaining approval from each IRB. We collected the data of 413 patients from JSOG gynecological cancer registered institutions, consisting of medical training institutions (n=262, 63%), cancer specialty hospitals (n=58, 14%), and local core hospitals (n=93, 22%). A total of 102 institutions had eligible patients. Finally, the clinical information (103 questions/patient) of 413 patients was collected. 1. Patients’ characteristics2. Initial treatment3. RFS in patients with AEH and ECG14. The pathological discrepancies of patients who did not achieve CR after initial treatment5. Cases of deviating from the eligibility criteria (patient with MI and ECG2)6. Pregnancy outcomes7. Treatments for patients with recurrence8. Occurrence of ovarian cancer9. Prognosis The prognosis of FS treatment was examined. The rates of patients with no evidence of disease (NED), alive with disease (AWD), and died of disease (DOD) were 95.6% (395/413), 2.7% (11/413), and 1.5% (6/413), respectively. The pathology before initial treatment of patients with DOD (n=6) was AEH in two cases and ECG1 in four cases. All of them were diagnosed by D&C before initial treatment. One patient (17%) was suspected of having MI before initial treatment. Two AEH patients (33.3%) achieved CR after initial treatment, although four ECG1 patients (66.7%) were PD. Five patients (83.3%) underwent surgery; all of their surgical pathology specimens showed high-grade carcinoma (carcinosarcoma: 2, ECG3: 1, clear cell carcinoma: 1, dedifferentiated carcinoma: 1) . The median follow-up time was 2,290 days. Patients’ median age and BMI were 35 years and 24.5 kg/m 2 , respectively. Most of the histological types were ECG1 (54.7%) and AEH (41.4%), although there were nine ECG2 patients. Major concomitant conditions were diabetes mellitus (DM) (9%), hypertension (8.7%), and polycystic ovarian syndrome (PCOS) (23.5%). We confirmed family history of cancer (Lynch syndrome suspected clinically) in 30 patients (7.3%). Twenty-six percent of patients had a history of infertility treatment . Thirty-six percent of patients had atypical genital bleeding at the first visit to the hospital. Fifty-two percent of patients had irregular menstrual cycles. The examinations for pre-initial pathological diagnosis included dilatation & curettage (D&C) (80.6%), endometrial biopsy by hysteroscopy (11.9%), blind endometrial biopsy (8.2%), and endometrial cytology (0.2%). MPA was used in 98.8% for initial treatment. In 408 patients treated by MPA, 360 patients (87.2%) used MPA alone, and 48 patients (11.6%) were combined with metformin. The main dosages of MPA were 600 (79.4%) to 400 mg (18.9%). The dosages of metformin were varied from 750 to 2,500 mg . AEs were observed in 4.4% of patients. Grade 3 massive genital bleeding was observed in 2 patients (0.49%). One of the patients underwent hysterectomy to control genital bleeding and discontinued FS treatment. A total of 253 patients (61.3%) took low-dose aspirin during FS treatment to prevent thrombosis. Grade 3 thrombosis was observed in only 1 patient (0.24%), even though she had been taking low-dose aspirin. Body weight gain (20% more) was observed in three patients (0.73%). There were no grade 4 AEs. CR after initial treatment was achieved in 78.2% (323/413) of patients. To accurately determine the response to FS treatment in eligible patients, 360 patients who matched common FS treatment criteria of several guidelines (Pathology: AEH or ECG1 without MI, Treatment: MPA 400 or 600 mg) were selected. Of these 360 selected patients, CR was achieved in 79.1% (285/360). Each treatment response (MPA 400 mg, 600 mg, and MPA + metformin) of initial treatments was higher in AEH patients (78.4%, 83%, and 95.5%) than in ECG1 patients (65.4%, 76.2%, and 83.3%) . We performed univariate and multivariate analysis to examine the relationship between clinicopathological factors (age, BMI, PCOS, family history of cancer, DM, histology, treatment, and treatment period) and the clinical response after initial treatment of AEH and ECG1 patients . On univariate analysis, there were significant differences in BMI (<25 vs ≥25 kg/m 2 : p=0.028), treatment (MPA 400 mg vs. MPA + metformin; p=0.003), and treatment period (<6 vs. ≥6 months; p=0.002). There were no significant differences, but some trends were seen in histology (AEH vs. ECG1; p=0.061) and treatment (MPA 600 mg vs. MPA + metformin; p=0.088 and MPA 400 or 600 mg vs. MPA + metformin; p=0.057, respectively). On multivariate analysis, BMI ≥25 kg/m 2 (hazard ratio [HR]=2.24), ECG1 (HR=2.28) and treatment period <6 months (HR=2.5) were related to a poor response to initial FS treatment. Univariate and multivariate analyses were performed with clinicopathological factors and RFS of AEH and ECG1 patients without MI treated with MPA 400 or 600 mg . On univariate analysis, there were significant differences in BMI (<25 vs. ≥25 kg/m 2 ; p=0.021), histology (AEH vs. ECG1; p=0.016), TTCR (<6 vs. ≥6 months; p=0.007), maintenance therapy (− vs. +; p<0.001), and pregnancy (− vs. +; p<0.001). On multivariate analysis, ECG1 (HR=1.73), TTCR ≥6 months (HR=1.51), maintenance therapy (−) (HR=2.1), and pregnancy (−) (HR=2.8) were associated with a significantly higher risk of recurrence on multivariate analysis in patients with AEH and ECG1 treated with MPA 400 or 600 mg. shows RFS curves by histology, TTCR, maintenance therapy, and pregnancy. The overall recurrence rate (ORR) was 39.5% in AEH, and 55.0% in ECG1. In terms of TTCR, ORR was 38.5% in TTCR <6 months and 55% in TTCR ≥6 months. In patients on maintenance therapy, ORR after achieving CR to initial treatment was lower (37.4%) than in patients not on maintenance therapy (57.6%). Furthermore, we found lower ORR in patients who could conceive than those who couldn’t. (29.3% vs. 57.3%). To examine the additional therapeutic effect of metformin, we reanalyzed the clinicopathological factors and RFS in patients treated with MPA + metformin or MPA (400 or 600 mg) . On multivariate analysis, there were significant differences in MPA 400 mg versus MPA + metformin (HR=0.33; p=0.026) and MPA 600 mg versus MPA + metformin (HR=0.36; p=0.021), in addition to histology, maintenance therapy, and pregnancy. However, there was no significant difference in TTCR (HR=1.38; p=0.093), which was significantly different in patients treated with MPA 400 or 600 mg. Among patients who did not achieve CR after initial treatment, it was suspected that there were some pathological discrepancies between before-and after-initial treatment. Therefore, we examined pathological discrepancies between before-initial treatment (diagnosed by D&C or not D&C) and after initial treatment (diagnosed by hysterectomy). Surprisingly, the pathological discrepancies was 81.3% (13/16) in AEH, while lower in ECG1 (19.6%, 9/46) . The rate of diagnostic discrepancies in AEH (n=16) differed by the method of pathological examination (D&C or not D&C). The rate of diagnostic discrepancies of AEH was 75.0% (9/12; AEH: 3, ECG1: 9) with D&C, and 100.0% (4/4; ECG1: 1, ECG2: 2, ECG3: 1) without D&C (endometrial biopsy). Of note, among patients (AEH + ECG1) who did not achieve CR after initial treatment, 8.1% (5/62) had high-grade carcinoma (endometrioid carcinoma grade3 [ECG3]: 2, clear cell carcinoma: 1, dedifferentiated carcinoma: 2). In this survey, there were 19 patients (4.6%, 19/413) who were suspected of having MI at pre-initial treatment by pelvic magnetic resonance imaging (MRI). CR rates were 42.1% (8/19) with MI and 74.6% (132/177) without MI. There was a significant difference in the CR rates between the 2 groups (relative risk=2.28; 95% confidence interval=1.4–3.6; p=0.005). The effectiveness of FS treatment in ECG2 was uncertain. In this survey, there were nine patients with ECG2. The CR rate after initial treatment was 88.9% (8/9), and the recurrence rate for those who achieved CR after initial treatment was 55.6% (5/9). A total of 217 patients desired children after achieving CR following initial treatment. 76% of these patients underwent infertility treatment, whereas 24% did not. In patients with infertility treatment, the pregnancy rate was 58.5%, and the live birth rate was 50.6%. On the other hand, in patients who did not have infertility treatment, the pregnancy and live birth rate were only 11.5%/7.7%. There were significant differences in the pregnancy and live birth rates between infertility and no infertility treatment groups (p<0.010) . Treatments for recurrent AEH and ECG1 patients without MI before initial treatment (n=126) who achieved CR after initial treatment were examined. Most recurrent sites were endometrium (98.4%, 124/126), with only 2 cases outside the uterus (1.6%, 2/126). The treatments for patients with recurrence were repeated FS treatment (42.9%, 54/126), surgery (40.5%, 51/126), repeated FS treatment to surgery (14.3%, 18/126), and unknown (2.4%, 3/126). Furthermore, we examined the efficacy of repeated FS treatment (MPA or MPA + metformin) for recurrent AEH and ECG1. The CR rates in recurrent AEH patients were 91.7% (22/24) with MPA, and 100% (6/6) with MPA + metformin. The CR rates of recurrent ECG1 patients were 90.9% (10/11) with MPA and 100% (1/1) with MPA + metformin. There were 15 cases of simultaneous ovarian cancer (3.6%) and one case of peritoneal cancer (0.24%). The timing of occurrence of ovarian cancer was before-FS treatment in 7.1%, during FS treatment in 20%, and after-FS treatment in 73.3%. Sixty-seven percent were diagnosed as primary ovarian cancer, whereas 13.3% were diagnosed as metastatic cancer from EMCA. Most pathology was endometrioid adenocarcinoma (85.7%) same as endometrial cancer . is study mainly focused on examining the factors that correlated with the response to initial treatment and recurrent risk factors who achieved CR to initial treatment. Furthermore, we specifically examined the pregnancy and live birth rate with and without the introduction of fertility treatment, the concurrent occurrence of ovarian cancer, the histological discrepancy before and after treatment in AEH, and deviations from the eligibility criteria. In terms of the response to initial treatment, we confirmed almost the same remission rate as the previous reports . Li et al. reported that the histology and BMI were significantly associated with a higher likelihood of achieving CR, although age, PCOS, and hormonal agents did not affect CR. In our study, the significant clinicopathological factors correlated to CR were histology (AEH), BMI (<25 kg/m 2 ), and treatment period (≥6 months). There is no consensus on the optimal duration of the treatment period to date . Based on a previous prospective study in Japan in which the treatment period was set at 6 months, it is possible that a certain number of the stable disease and progressive disease cases also completed treatment at six months . Wang et al. examined the treatment period in 68 patients (1 AH and 37 ECG1) to see whether a more extended treatment period affects oncologic results, causing disease progression or recurrence. They found cumulative complete response rates of 59% (≤6 months), 76% (6–9 months), and 95.5% (>9 months), respectively. In the present study, we also confirmed that a more extended treatment period (≥6 months) was correlated with CR. Further investigation of optimal treatment periods is warranted. About recurrent risk factors who achieved CR to initial treatment, we confirmed that ECG1, TTCR ≥6 months, maintenance therapy (−), and pregnancy (−) were associated with a significantly higher recurrent risk on multivariate analysis in the patients. Even in responders, the rate of recurrence was high in EC . The results of four meta-analyses showed 31%–41% of ECG1 patients develop recurrence after the initial response, and the potential risk factors associated with recurrence were BMI ≥25 kg/m 2 , PCOS, and EC . Furthermore, TTCR, family history of cancer, diabetes, pregnancy, progestin type, and maintenance therapy (−) have also been reported in other studies as independent risk factors for recurrence . From this study, we could confirm almost identical recurrent risk factors as previous studies. We confirmed the benefit of maintenance therapy for RFS; 55% of patients without maintenance therapy recurred within 5 years, whereas only 35% of patients on maintenance therapy recurred. About the importance of maintenance therapy to prevent a recurrence, KGOG study also emphasized this point . Maintenance therapy should be recommended for patients who do not express the desire to have children at the time of CR after initial FS treatment. In this study, 48 (11.6%) patients used metformin combined with MPA as initial treatment. Metformin increases insulin sensitivity and activates the AMPK pathway, counteracting the PI3K/ mTOR/Akt/FOXO1 signaling pathway, promoting endometrial proliferation . We found a better response rate to initial treatment and reduced recurrent risk as previous reports . When we analyzed RFS, including with MPA + metformin, TTCR ≥6 months was not a risk factor for RFS, although it was a significant risk factor in patients treated with MPA only. The addition of metformin to MPA might decrease the recurrent risk associated with longer TTCR. Because a small number of patients were treated with metformin, we could not conclude the response of the additional effect of metformin from this study. We would know this exciting answer from the ongoing prospective, randomized study of MPA + metformin versus MPA only (FELICIA trial) . Regarding the pregnancy and live birth rate in the present study, those with infertility treatment (58.5%/50.6%) were better than those for spontaneous pregnancy (11.5%/7.7%). Not only with assisted reproductive technology (ART) but there was also a relatively high pregnancy rate with timing treatment and intrauterine insemination. In previous meta-analysis, the live birth rate of patients who had ART was 39.4%, whereas it was 14.9% in patients who tried to conceive spontaneously . In the present study, the first- and second-year recurrence rates were 25% and 36%, respectively, in ECG1. Therefore, the recommended timing of pregnancy is early after achieving CR to initial treatment. The implementation of in vitro fertilization techniques increases the chance of conception and may also decrease the time to conception . The American Society of Clinical Oncology recommends that physicians “should refer patients who express an interest in fertility preservation to reproductive specialists” . The rate of ovarian cancer during follow-up was reported to be 3.6% in a previous meta-analysis . However, the detailed information of the patients was poorly reported (i.e., primary or metastatic, histology, and response to initial treatment) because the original primary reports did not include these. In the present survey, there were 15 cases of simultaneous ovarian cancer (3.6%). Of these, 67% were diagnosed with primary ovarian cancer, whereas 13% were diagnosed with metastatic cancer from EMCA. The developments of ovarian cancer were mostly detected post-FS treatment (73.3%). In addition, most patients (80%) achieved CR to initial treatment, and the histology of ovarian cancer was endometrioid carcinoma (85.7%), which is the same as endometrial cancer. Two cases of the patients diagnosed with primary ovarian cancer had suspected Lynch syndrome (20%, 2/10). The details of the etiology of ovarian cancer are unknown because genetic testing was not performed in this study. In the future, genetic testing of cases with concurrent ovarian cancer may help to elucidate these factors. Throughout the FS treatment period, gynecologists need to be very careful about the simultaneous occurrence of ovarian cancer. If ovarian tumors are detected during and post FS treatment, patients should be carefully examined by pelvic enhanced MRI and whole-body enhanced CT to rule out or confirm ovarian cancer and metastasis. In this nationwide survey, approximately 10% of patients (patients with ECG2 or MI) did not match the eligibility of standard FS treatment criteria. Through this research study, it was found that these originally off-label cases are being treated. We found initial FS treatment efficacy was equivalent to ECG1 for ECG2 patients but lower for MI cases than those without MI. Although, these are only the results of a few instances, and adhering to the standard indications for FS treatment is still essential. We found that patients with AEH who failed the initial FS treatment had more histologic discrepancies. In particular, the pathological discrepancies between pre- and post-treatment histology were more common in cases where the pre-treatment pathological diagnosis was made without D&C. This histological discrepancy due to differences in testing methods is also reported in the KGOG study . In our study, 8.1% of the patients with initial FS treatment failure had high-grade carcinoma. It is important to perform D&C for histological confirmation pre- and post-treatment. Especially in initial FS treatment failure cases, we need to thoroughly check for hidden high-grade carcinoma. The limitation of the present study is that it was a retrospective, questionnaire-style survey. In addition, this study was performed in a single country. The optimal progestin regimen remains to be elucidated, although MPA and MPA + metformin were mainly analyzed in the present study. The strength of the present study is that it was a nationwide, multi-center survey, and likely, the largest sample size compared with previous reports. Compared with a meta-analysis, although the number of patients was smaller, it was possible to accurately examine potential predictors affecting response to initial therapy, significant risk factors related to recurrence, ovarian cancer patients, and pregnancy data for each assisted reproduction treatment obtained for each patient, which was collected from the same questionnaire data, which covered the patients’ detailed background characteristics. In summary, the present study provides further insight into the current trends of FS treatment in AEH and ECG1 patients who want to have children. The patients who choose this FS treatment should be informed of the relatively low live birth rate, the high chance of recurrence, and the possibility of the occurrence of ovarian cancer, which could be life-threatening. Before FS treatment, detailed pathological examination is needed to rule out high-grade histology by D&C, MI, and concurrent ovarian cancer by MRI. If CR is achieved, gynecologists need to consult with reproductive specialists about infertility treatment, including assisted reproductive treatments, to maximize chances of a live birth. The trends of FS treatment shown in this study are clinically meaningful and may influence the future direction of investigation. |
Narrative medicine in clinical internship teaching practice | 40a03fec-76c0-4237-a7ef-433d790632c5 | 10512813 | Patient-Centered Care[mh] | Development of empathy and related humanistic skills is a key component of learning to become a physician. The field of obstetrics and gynecology has been a source of advocacy for patient-centered and empathic care. In the process of diagnosis and treatment, patients often undergo sensitive or invasive exams, which makes it necessary for obstetricians and gynecologists to have good humanistic quality and to exhibit empathy for patients’ situation . Medical students in the context of undergraduate education exhibit strong plasticity and these skills may be particularly necessary for medical students during their obstetrics and gynecology clinical rotations. Narrative medicine(NM) was first proposed by Professor Rita Charon of Columbia University in 2001, believing that narrative ability is the ability to recognize, absorb, explain, and be moved by the stories of diseases . NM emphasizes humanistic care and is a medicine practiced by clinical workers who have the ability. Its development is mainly through communication, understanding, and empathy with patients, helping medical practitioners understand the pain endured by patients in the disease . It is an inevitable product of the development of modern medicine . NM requires doctors and nurses to focus on patients’ real feelings of disease but not just data or image on the medical reports . NM improves the empathy and reflective abilities of medical workers through specific training methods such as fine reading and reflective writing . NM advocates that parallel medical record writing and reflective writing should be integrated into clinical teaching and practice to improve doctors’ attention to patients’ feelings of disease, as opposed to a situation in which doctors focus only on a number on their checklist . Growing research into the efficacy of integrating NM education into clinical education has suggested that NM has indeed improved the empathy of medical students . If the relevant theories and methods of NM can be integrated into medical education, this process may help medical students or clinical doctors provide better diagnoses and treatment experiences to female patients and increase patient cooperation . However, the heavy workload of medical courses and the difficulty of medical exams are of crucial importance for the career of medical students , leading them to be more willing to focus on learning professional knowledge and underestimate the cultivation of medical humanities literacy . The traditional clinical practice teaching mode mainly helps students consolidate their professional knowledge by combining theoretical knowledge with clinical practical cases. Owing to the long-term neglect of the cultivation of humanistic quality, medical students may not develop crucial qualities of empathy and patient-centeredness. The core purpose of NM is to cultivate an empathic understanding of patients’ experiences . According to the characteristics of a variety of theories and methods associated with NM, this approach is very suitable for clinical interns. First, during this time, medical students are still developing their clinical skills, and are likely to be open to learning. Second, the clinical internship is a process in which theory and clinical practice intersect. With the help of instructors, medical students can gradually cultivate their empathy for patients by coming into contact with patients while they master theories. To test the feasibility of this method, the Third Xiangya Hospital of Central South University took undergraduates who were engaged in practice in obstetrics and gynecology as its main research object to determine whether the integration of NM teaching into clinical practice in obstetrics and gynecology can help medical students improve their empathy regarding patients. In this study, the teaching method of NM is integrated into the undergraduate internship course and compared with the traditional teaching method to analyze the feasibility of integrating NM teaching into the development of humanistic quality in undergraduate medical education.
Participants Teaching method Implementation process Measures Data analysis Five-year clinical medicine senior undergraduate medical students were selected as the research subjects when they were engaged in practice in the Department of Obstetrics and Gynecology of the Third Xiangya Hospital of Central South University in 2019. In their pre-clinic and clerkship years, the development of humanistic skills was mainly taught by lectures based on ethics textbook in class. This study underwent institutional review board (IRB) review and received an exemption, and all students were provided with an information sheet detailing the purpose of the study and the ways in which they could opt out of participation. A total of 100 students were randomly divided into control group ( n = 50, 24 females) and experimental group ( n = 50, 28 females), which were taught by the same teacher.
Both the control group and the experimental group were taught by the same experienced teacher specializing in clinical education and humanistic quality education in the context of medicine. The control group received traditional teaching methods, which were guided by a combination of textbook professional knowledge with specific clinical cases, with the main purpose of teaching clinical professional knowledge. On the basis of teaching clinical professional knowledge, the experimental group integrated NM-related knowledge with practical methods, emphasizing the improvement of students’ humanistic quality in the process of engaging in medical activities. All training sessions, regardless of format, lasted for 2 hours. The teaching contents of the experimental group mainly included eight courses: introducing relevant theories and research progress related to NM; introduction to NM-related literary works; appreciation of film and television works related to NM; introduction to parallel medical record writing; narrative ward round: listening to the patient’s story and completing parallel medical record writing; parallel medical record sharing and discussion; reflective writing; reporting and sharing learning experiences. The teaching cases focused on the experiences of real patients as their main material. Students experienced the real lives of the patients behind the clinical data by listening to patients’ vivid stories. During their teaching rounds, the students were encouraged to listen to patients’ stories and to record their unique feelings using parallel medical records.
Before conducting the research, the investigators communicated with the lecturer fully regarding the research purpose, the specific implementation methods and the arrangement of theory and practice in the curriculum. Training and guidance regarding NM were provided to teachers to ensure uniform teaching methods and consistent classroom design. The Power Point course and task objectives were prepared one week prior to class. The class flow of the control group first featured an explanation of the relevant professional knowledge, which was followed by bringing the students to the ward to complete their teaching rounds; finally, 4–5 people were organized into a group to collect the patient’s medical history and write a complete medical record. The teaching process used for the experimental group first featured an explanation of the relevant professional knowledge, including NM-related knowledge, which was followed by the teaching ward round; finally, the students were encouraged to listen to the patient’s story and record it in the form of parallel medical records as part of the process of medical history collection. At the conclusion of the course, the students in the experimental group were surveyed using the course evaluation questionnaire.
The Davis empathy scale was first developed by Mark H. Davis in 1983 to evaluate empathy defined in terms of a multidimensional construct and is widely used for researching on the multidimensionality of the empathic process in the general population . This scale was used to examine the effect of NM on students’ empathy ability in this study. The Davis empathy scale includes 28 questions, each of which is scored on a scale ranging from 1–5: very unqualified, somewhat unqualified, uncertain, somewhat qualified, and very qualified. These 28 questions ranged across four dimensions: the fantasy scale, the perspective-taking scale, the empathic concern scale and the personal distress scale . According to the nature of the questions, they were associated with positive integers, i.e. plus points, and negative integers, i.e. minus points.
The empathy ability of all participants was measured before and after the course. All participants were included in the analysis. SPSS 20.0 software was used to conduct the statistical analysis. The measurement data were described by X ± s , and independent t tests and chi-square analyses were used to explore group differences in responses. The criterion of p < 0.05 was used to reject the null hypothesis.
Comparison of self-perceptions of empathy between the two groups of medical students Medical students’ understanding of NM and evaluation of the narrative course The role of the NM curriculum In the questionnaire concerning the role of NM in clinical medical work, medical students generally acknowledged the role of NM in medical education and medical practice. Fifty percent of the medical students thought that NM played a role in enhancing their doctor‒patient communication ability, 44% of the students thought that it was very useful, and only 6% of the students thought that it was not useful. Learning to listen is an important aspect of NM. Sixty-two percent of the medical students reported that listening was very important to the establishment of a relationship of harmonious communication, and an additional 36% of the medical students thought that listening was very important but that it may be difficult to implement in concrete situations. Sixty-two percent of the students noted that they often listened to patients’ stories during their internships, and 36% of the medical students listened occasionally. When asked whether NM could promote their reflection on their own behavior in medical practice, some students noted that they had taken action (24%) or were deeply touched (30%) or somewhat touched (38%) by NM, and only 8% of the students thought that it had little effect. Cultivating the empathy ability of medical students is a basic goal of NM. Ninety-six percent of the medical students thought that empathy ability could improve doctor‒patient communication, while 86% of the medical students thought that the NM teaching method was conducive to helping them develop empathy ability ( ).
According to the results of this survey, the differences between the two groups of medical students in terms of their self-perceptions of empathy scores were compared ( ). In the longitudinal comparison before and after the class, there were no significant differences between the control group and the experimental group ( p > 0.05). In the horizontal comparison between the control group and experimental group at the same time point, there were no significant differences between the scores on the empathy scale exhibited by the experimental group and those exhibited by the control group before the course ( p > 0.05), but after the course, the scores of the experimental group were significantly better than those of the control group ( p = 0.0146) ( and ).
After the course, the self-developed course evaluation questionnaire was distributed to students in the experimental group anonymously. Due to the integration of NM teaching into the internship course for obstetrics and gynecology, most medical students believed that they had a clear understanding (54%) or a deep understanding (30%) of NM. Regarding the necessity of continuing to provide NM-related courses in the future, 94% of the students believed that it was ‘necessary’ or ‘generally necessary, but more content and practical guidance are needed’. Seventy percent of the medical students reported that their overall impression of the course caused them to understand new medical concepts, and 24% of the students claimed they had made some gains. However, the teaching content of the course was too simple, and 6% of the students thought that the course was meaningless and a waste of time. In addition, 56% of the medical students thought that the NM course was rich in content and that it integrated various forms of teaching methods. Another 36% of the medical students thought that the curriculum arrangement was reasonable but that the content required further adjustment. Only 8% of the students thought that the curriculum arrangement was unreasonable and required large adjustments ( ).
The aim of the present study was to explore the effect of narrative medicine in clinical internship teaching practice, which may provide evidence for applying NM in clinical education. In this study, by comparing the scores of the two groups on the empathy scale, after completing the NM course, experimental group showed a higher level of empathy. Our results correspond to previous studies indicate that the integration of NM education into the clinical course has obvious advantages with respect to cultivating participants’ self-perceptions of empathy . Previous reports about the doctor-patient relationship showed that patients are non-adherent with treatment when they are not fully understood the purpose of medical treatment and provided some strategies to establish better understanding between patients and doctors . A recent study was conducted semi-structured interviews to explore the experience of doctors in communication with patients and further demonstrate that communication skills are absent during clinical practice . However, the content of doctor-patient communication includes not only disease-related information but also emotional communication. Thus, as Jacynthe et al. suggest, narrative medicine can help students reflect their professional identity, developing good professional identity abilities, promoting self-reflection in healthcare practice and having insight into patients’ suffering , so that to have better understanding experience of patients, thereby promoting good doctor-patient communication. The NM education is patient-oriented, while other medical curriculums are more technique-oriented . Some studies have shown that NM improved patients’ illness experience and compliance, which almost established a consensus . Although NM provide many methods to help improving doctor-patient relationship, it is still hard to play a role in clinical practice. For example, listening and reflection, as important skills emphasized by NM, play an important role in facilitating the doctor-patient relationship . However, our results are consistent with Abigail’s study that, although most participants understand the importance of listening and reflection, they worry that these methods are difficult to implement in their practical work due to the heavy pressure associated with clinical work and then influenced their work with patients . These evidences further proving that it is necessary to applying NM in medical education because of the difficulty in implementing in clinical work. Based on the results of the curriculum evaluation questionnaire, students’ perceptions of this new teaching model were objective and positive. Most students noted that they developed a comprehensive understanding of NM and a new understanding of the doctor-patient relationship as well as doctor-patient communication throughout the course. However, simultaneously, the students also objectively noted that the content of the teaching was too simple, indicating that we should add additional elements to the classroom teaching to attract students, which requires us to have a sufficient number of teachers and to enrich our teaching content and strengthen our teachers constantly. Integrating NM into the traditional classroom requires the lecturer to have a deep understanding of the relevant concepts and practical principles of NM. Therefore, it is necessary to improve the training of these teachers. There are still many limitations in this research. Firstly, the samples only include undergraduate students of one grade. In future research, graduate students, clinical residents, etc. should be included in the research scope. Secondly, in our study, there were only 8 courses of narrative medicine integrated into the teaching method, and the number of courses and the richness of teaching content should be increased. However, this study also highlights the benefits of integrating NM into internship classes to cultivate the empathy ability of medical students and reflects the feasibility of NM teaching in contemporary medical teaching. It is believed that this teaching mode will play an increasingly important role following a process of continuous exploration, practice, reform and innovation.
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Molecular glues of the regulatory ChREBP/14-3-3 complex protect beta cells from glucolipotoxicity | 80b40cb2-60f2-48b3-b87c-2076ba48dc1b | 11873037 | Digestive System[mh] | Type 2 diabetes (T2D) imposes an escalating burden on global health and economy. In 2021 537 million adults worldwide had diabetes, and this is expected to rise to 693 million by 2045 or even 1.31 billion by 2050 , making it one of the leading causes of global morbidity . Both major types of diabetes are characterized by insufficient functional β-cell mass to meet the increased demand for insulin. T2D develops due to decreased insulin response , triggering a vicious cycle of increased insulin demand and decreased peripheral tissue response. While lifestyle adjustments can alleviate insulin resistance , inadequate hyperglycemic control can lead to β-cell exhaustion, dedifferentiation, and eventual demise due to metabolic stress. This final stage is irreversible due to the restricted proliferative capacity of adult β-cells. Current T2D therapies mainly target insulin resistance and secretion, yet many T2D patients eventually become insulin dependent due to the loss of functional β-cells. Preventing the loss of functional β-cell mass remains one of the most important unmet needs in the armamentarium for the treatment of diabetes. An emerging potential target to treat T2D is the glucose-responsive transcription factor (TF) Carbohydrate Response Element Binding Protein (ChREBP) , . ChREBP is a key mediator in the response to glucose in pancreatic β-cells, controlling the expression of glycolytic and lipogenic genes . Small molecule modulation of ChREBP function may thus represent a promising approach to combat T2D; however, many TFs such as ChREBP are known as notoriously difficult to target with small molecules due to their lack of suitable ligand binding sites . However, several proteins interact with ChREBP to regulate its activation mechanism, including the 14-3-3 proteins , . The protein-protein interaction (PPI) between ChREBP and 14-3-3 potentially offers long-sought entries to address ChREBP via molecular glues , . The “hub” protein 14-3-3 is involved in numerous signaling pathways, as well as in the pathophysiologic state of diabetes . Molecular scaffold proteins belonging to the 14-3-3 protein family are widely conserved among eukaryotes , . In mammals, this family comprises seven isoforms, namely, β, γ, ζ, η, τ, σ, and ε . In the context of pancreatic β-cells, 14-3-3 proteins are integral to the regulation of insulin secretion, cell proliferation, and survival . They contribute to maintaining glucose homeostasis and influence key aspects of the cell cycle, impacting β-cell mass , . The diverse functions of 14-3-3 proteins encompass their involvement in mitochondrial activity , cell cycle progression , contribution to apoptosis, and cell survival pathways . ChREBP interacts with 14-3-3, via a PKA-responsive phosphorylation site S196 of ChREBP, and also via an alpha helix in its N-terminal domain (residues 117-137), which is one of the few known phosphorylation-independent 14-3-3 binding motifs , , . The molecular basis of this PPI has been studied by X-ray crystallography, revealing the presence of a free sulfate or phosphate ion binding in the 14-3-3 phospho-accepting pocket that interacts with both proteins (Supplementary Fig. ) . Adenosine monophosphate (AMP) has also been reported to bind to this phospho-accepting pocket, weakly stabilizing the protein complex and enhancing the 14-3-3-mediated cytoplasmic sequestration of ChREBP (Supplementary Fig. ) . Next to inorganic phosphate and AMP, ketone bodies regulate ChREBP activity by increasing its binding to 14-3-3 , . This may suggest that small-molecule stabilizers – molecular glues – of the ChREBP/14-3-3 PPI are potentially valuable tools to suppress glucolipotoxicity in T2D by inhibiting the transcriptional activity of ChREBP, thereby overcoming the current limitations of ‘direct’ targeting of TF functions. MLXIPL , the gene that expresses ChREBP, produces two major splice isoforms: ChREBPα and ChREBPβ. The ChREBPα isoform is the full length-isoform, containing the low glucose inhibitor domain (LID, including the nuclear export signal (NES)) at its N-terminal region. One of the mechanisms that retains ChREBPα in the cytosol is via its interaction with 14-3-3 (Fig. ) , . ChREBPβ lacks this N-terminal domain, resulting in a nuclear, constitutively active, hyper-potent TF , . In healthy conditions, glucose flux leads to dissociation of CHREBPα from 14-3-3 resulting in its translocation to the nucleus and induction of ChREBPβ transcription, ultimately leading to glucose-stimulated β-cell proliferation to meet the demand for insulin (Fig. ). However, under prolonged hyperglycemic conditions, a robust positive feedback loop is triggered in which the newly produced ChREBPβ binds to its own promoter sites leading to even more ChREBPβ synthesis eventually resulting in glucolipotoxicity and β-cell apoptosis (Fig. ) , , . These molecular mechanisms suggest that small molecule stabilization of the ChREBPα/14-3-3 PPI via a suitable molecular glue may prevent the nuclear import of ChREBPα under prolonged hyperglycemic conditions and thereby avert glucolipotoxicity (Fig. ). Indeed, our groups recently developed biochemically active stabilizers of the ChREBPα/14-3-3 PPI ; these compounds were only moderately active and more importantly lacked cellular activity, preventing cellular validation and downstream analyses of our hypothesis . In this work, we further improve these ChREBPα/14-3-3 PPI small-molecule stabilizers by using structure-activity relationship (SAR) analysis. This resulted in small-molecule stabilizers with a cooperativity factor (α) up to 220 for the ChREBPα/14-3-3 PPI. The introduction of a geminal -difluoro group into the pharmacophore not only significantly enhances their stabilization efficiency, most probably by slight bending of the pharmacophore arrangement, but is also crucial for obtaining cellular permeation and activity. X-ray crystallography confirms engagement of the molecular glues at the composite interface formed by the 14-3-3 binding peptide motif of ChREBPα and 14-3-3. Immunofluorescence shows retention of ChREBPα, by 14-3-3, in the cytoplasm of primary human β-cells upon addition of the stabilizers. Concomitantly, the transcriptional activity of ChREBPα is suppressed and thus prevents the upregulation of ChREBPβ in response to glucose and glucolipotoxicity. By exploiting this mechanism of action, β-cells are rescued from glucolipotoxicity, both in terms of viability and in terms of β-cell identity, including insulin production, by keeping ChREBP transcriptionally inactive at high glucose levels. This study provides the foundation for a potential new class of compounds for regulating ChREBP activity in T2D , .
Differential stability and regulatory roles of ChREBP isoforms under high glucose Focused library based on 1 established a crucial SAR Fluorination of compounds improves stabilization potency Molecular glues stabilizing the ChREBPα/14-3-3 interaction rescue human β-cells from glucolipotoxic death Compound 43 stabilizes the ChREBPα/14-3-3 interaction in situ preventing ChREBPα’s nuclear translocation in response to glucose or glucose + palmitate Effect of stabilizers on ChREBP downstream genes and preservation of β-cell identity Next, we assessed the effect of stabilizers on transcription of the ChREBP splice isoforms in human islets. While ChREBPα mRNA levels remained relatively stable in all conditions tested (Fig. ), ChREBPβ, which contains a well characterized carbohydrate response element (ChoRE) on its promoter , , was upregulated in response to high glucose, or to high glucose and high palmitate (glucolipotoxicity). Interestingly, compound 43 prevented upregulation of ChREBPβ both at mRNA level (Fig. ) and at protein level (Fig. c, ), indicating that inhibition of the nuclear translocation of ChREBPα by strengthening the ChREBPα/14-3-3 interaction with 43 is required for the blocking of the upregulation of glucose responsive genes such as ChREBPβ. TXNIP is another glucose responsive gene, which also contains a well characterized ChoRE and its upregulation is implicated in oxidative stress and β-cell death . Promoter luciferase assays demonstrated that 43 prevents the glucose response of the TXNIP gene (Fig. ). Importantly, β-cell identity marker genes, INS (Fig. ) and PDX1 (Fig. ), were both downregulated at the mRNA level in human islets exposed to glucolipotoxicity, consistent with the previously reported de-differentiation phenotype pertinent to β-cell demise , . Yet, in the presence of 43 this de-differentiation was prevented (Fig. f, ). Similarly, immunostaining for PDX1 showed a marked decrease in PDX1 protein levels in glucolipotoxicity, which was rescued by 43 (Fig. c, ). We also tested by qPCR in human islets other known 14-3-3 interactors, including USP8 , RELA , PIN1 , NFATC1 and BRAF (Supplementary Fig. ). Other β-cell maturity markers or glucose sensing and processing genes were unchanged by 43 (Supplementary Figs. ). However, the β-cell dedifferentiation marker- ALDH1A3 was significantly decreased by 43 in low glucose concentrations but unaffected in high glucose or glucolipotoxicity (Supplementary Fig. ). To test functional consequences, we measured the effects of 43 on Ca 2+ signaling in INS-1 cells, where 43 had no effect on calcium upregulation in response to glucose (Supplementary Fig. ). However, 43 enhanced mitochondrial activity in high glucose as evidenced by both active mitochondria staining (Supplementary Fig. ) and ATP/ADP ratio (Supplementary Fig. ). Together these data suggest that silencing ChREBPβ by 43 decreases de novo lipogenesis leading to less accumulation of toxic lipid species like ceramides, improving mitochondrial function.
ChREBPβ is a constitutively active transcription factor that promotes its own production by binding to its own promoter , . This raises questions of how ChREBPβ production gets turned off and what is its relationship to ChREBPα once it begins the positive-feedback mechanism. To address this, we analyzed the two isoforms’ half-lives. ChREBPβ, under high glucose concentrations, exhibits substantial instability, with a half-life as short as 12 min. In contrast, ChREBPα displays increased stability, with its half-life extending beyond 30 min (Supplementary Fig. ). These findings suggest that without continuous priming by ChREBPα activity, ChREBPβ production would be self-limiting, and provides a mechanistic explanation of how the positive-feedback production of ChREBPβ can be turned off when glucose levels decrease, or when ChREBPα is prevented from entering the nucleus when glucose levels are high.
To investigate the hypothesis that cytosolic sequestration of ChREBPα via molecular glue stabilization of the ChREBPα/14-3-3 PPI inhibits downstream glucotoxicity and β-cell apoptosis it was critical that our previously discovered stabilizer 1 (Fig. ) was first optimized to improve activity, but more importantly cellular efficacy. Stabilizer 1 was previously found to bind to the ChREBPα-peptide/14-3-3β interface (Supplementary Fig. ) . A challenge to optimization of 1 was the poor resolution of the ligand’s electron density in the X-ray co-crystal structure, particularly around the phenyl ring and linker of 1 . This poor electron density limited structure-guided optimization and indicated sub-optimal PPI stabilization (Supplementary Fig. ). To address this technical challenge our efforts were directed towards the development of an alternative crystallization approach. Gratifyingly, this co-soak crystallization approach resulted in a new co-crystal structure with an improved resolution of 1.6 Å. The improved resolution enabled a more reliable model building, including the atomic fitting of the phenyl ring and ethylamine linker of 1 . (Fig. , Supplementary Fig. ). In addition to the phosphonate of 1 interacting with the phosphate-accepting pocket of 14-3-3 (R56, R129, Y130), and R128 of ChREBPα (Fig. ), it was now observed that both the phenyl and phenyl phosphonate rings of 1 make hydrophobic contacts with side chains of both 14-3-3 and ChREBPα. Further, an intramolecular hydrogen bond between the amide moiety and the phosphonate group of 1 was resolved. Finally, the improved resolution of this crystal structure enabled detection of a water-mediated hydrogen bond between the carbonyl amide of 1 and the main chain amide of I120 of ChREBPα (Fig. ). Subsequent Fluorescence Anisotropy (FA) measurements revealed that compound 1 stabilized the ChREBPα/14-3-3 complex with a cooperativity factor (α) of 35 (Fig. ). The α and the intrinsic compound affinity to 14-3-3 (K D II ) were determined by fitting, using a thermodynamic equilibrium model, which allows for an objective comparison of different stabilizers (Supplementary Figs. , ) . With structural characterization of the ternary ChREBPα/14-3-3/ 1 complex in hand, attention was shifted to the optimization of compound activity. To gain a greater SAR understanding, a focused library was synthesized aiming to improve the stabilization efficiency and selectivity for this class of molecular glues, in which compounds were compared based on their EC 50 value derived from FA-based compound titrations (Fig. , Supplementary Figs. - , Supplementary Table ). To mimic the structure of the natural stabilizer AMP , the phenyl substituent of 1 was replaced by a purine ( 2, 3, 4 ). However, these modifications were not tolerated. Similarly, no stabilization was observed upon replacement of the phenyl moiety with either an indole ( 5 ) or a cyclohexane moiety ( 6 ), while naphthalene groups ( 7, 8 ) led to slightly more active compounds. Extension of the phenyl group with a methylpyridine functional group ( 9, 10, 11 ) increased the stabilization efficiency, with the N-2 position ( 9 , EC 50 = 7.6 ± 0.5 µM) eliciting the greatest response. Substitutions at the central acetamide were not tolerated ( 12, 13, 14 ). Furthermore, systematic shifting of the phosphonate group along the phenyl phosphonate ring showed that the ortho position is most favorable ( 1, 15, 16 ). Interestingly, replacement of the phosphonate with a phosphate group was tolerated ( 17, 18, 19 ) and the para positioned analog led to improved compound activity (EC 50 = 17.4 ± 3.2 µM). Conversion of the phenyl phosphonate into a benzyl phosphonate was however not tolerated ( 20, 21, 22 ). Phosphonate mimics, e.g., a boronic acid ( 23 ) or a sulfonamide ( 24 ) group were also inactive compounds. Lastly, we focused on modifications of the alkyl fragment of the phenyl ethyl amine moiety. Investigation of the SAR around the ethylene linker showed that replacement of the ethylene linker with a constrained (1 R , 2 S ) cyclopropyl ring ( 25 , EC 50 = 17.6 ± 1.7 µM) elicited similar activity to 1 , while stereoisomers 26 (1 S , 2 R ) and 27 (1 R , 2 R ) were not tolerated. Furthermore, ring expansion to the cyclopentyl ring ( 28 , 1 R , 2 R ) was inactive and installation of a cyclobutyl ( 29 ) or geminal -dimethyl groups ( 31 ) resulted in diminished activity relative to 1 . Interestingly, the introduction of a geminal -difluoro group however significantly enhanced (six-fold relative to 1 ) the stabilization of the ChREBPα/14-3-3 PPI, resulting in the best stabilization observed so far ( 30 , EC 50 = 5.8 ± 0.4 µM). Unfortunately, this analog was not amendable for phosphonate replacement with a sulfonamide or boronic acid, since this resulted in inactivity ( 58 , 68 Supplementary Table ). Merging the two best stabilizers ( 9 and 30 ) resulted in analog 73 with a similar EC 50 value compared to each individual substitution ( 73 , EC 50 = 11.0 ± 1.5 µM, Supplementary Table ). Co-crystallization of ChREBPα and 14-3-3 was successful for 30 , showing a clear density of both the ChREBPα peptide and 30 (Supplementary Fig. ). Overlaying the crystal structure of 30 with 1 revealed a high conformational similarity of 14-3-3σ and the ChREBPα peptide, with a comparable positioning of the phosphonate of 30 in the ChREBPα/14-3-3 phospho-accepting pocket (Fig. e, f). The geminal -difluoro group of 30 was, however, directed downwards into the 14-3-3 binding groove, causing an alternative bend of the phenyl ethyl amine moiety (Fig. ). Both fluorine atoms form interactions with 14-3-3σ; one directly contacts K49 and the other engages in a water-mediated polar interaction with N175 (Fig. ), thereby recapitulating other literature reports showing that fluorinated small molecules can form potent hydrogen bonds – . Additionally, due to the alternative bend of the linker, the amide of 30 was now positioned to interact with N123 and N124 of ChREBPα (Fig. ). These additional contacts of the molecular glue with both 14-3-3 and ChREBPα explain the increase in ternary complex formation by the introduction of the geminal -difluoro group.
Encouraged by the enhanced stabilizing activity of the fluorinated analog 30 , a focused library of fluorinated compounds was next synthesized and compared to their non-fluorinated analogs (Fig. , Supplementary Table ). Fluorination of analogs increased their stabilization potency consistently across all library members. Even the previously inactive p -Cl ( 32 ) and p -Br ( 33 ) substituted compounds now elicited a high activity when combined with the geminal -difluoro group substitution ( 40 : EC 50 = 6.6 ± 0.9 µM, 41 : EC 50 = 4.1 ± 0.4 µM, respectively). The fluorine functionality has a common place in medicinal chemistry as reflected by 20–25% of drugs in the pharmaceutical pipeline containing at least one fluorine atom , . Introduction of a fluoro group at the meta position of the phenyl ring even further improved compound activity ( 43 : EC 50 = 3.8 ± 0.2 µM). Additional to single fluoro-substitutions at the phenyl ring of 30 , double fluoro-substituted analogs were synthesized (panel IV in Fig. ). A 2,4-difluoro substitution resulted in the most potent stabilizer observed so far ( 53 , EC 50 = 3.1 ± 0.6 µM). Two-dimensional FA titrations were performed to investigate the cooperativity in ChREBPα/14-3-3/stabilizer complex formation. 14-3-3β was titrated to FITC-labeled ChREBPα peptide (10 nM) in the presence of different, but constant, concentrations of 30, 43 or 53 (0–500 µM) (Fig. b, , Supplementary Fig. ). The change in apparent K D of the ChREBPα/14-3-3 complex, over different concentrations of stabilizer (Supplementary Fig. ), showed that all three fluorinated compounds ( 30, 43 and 53 ) elicit a similar stabilizing profile, with a 2.4- to 6-fold increase in stabilization compared to the non-fluorinated parent analog 1 . Fitting the two-dimensional data using the thermodynamic equilibrium model . showed increased cooperativity for all three fluorinated compounds (α = 220 ( 30 ), 72 ( 43 ), 126 ( 53 )) compared to their defluorinated parent compound 1 (α = 35), while their intrinsic affinity for 14-3-3 was barely affected (K D II = 168 µM, 162 µM, 110 µM for 30, 43, 53 and 161 µM for 1 ) (Fig. b, , Supplementary Figs. , ). This indicates that the compound optimization has mainly improved the interaction with ChREBPα and not the binding affinity to 14-3-3. Of note, the mean squared error landscape of the two fitted parameters showed for some stabilizers that the α and K D II parameters are interconnected, meaning that a weaker intrinsic affinity, K D II , correlates to an increased cooperativity factor, α (Supplementary Fig. ). Phosphate- and phosphonate-based compounds may also act as inhibitors of other 14-3-3-client protein complexes, often acting as low micromolar (IC 50 ~ 1–20 µM) inhibitors , . To test the specificity of the ChREBPα/14-3-3 PPI stabilizers, we selected an array of eight representative 14-3-3 client-derived peptide motifs with differentiating binding sequences and included internal binding motifs BRAF , CRAF , p65 , USP8 , , one C-terminal binding motif ERα , one special binding mode Pin1 and another reported non-phosphorylated motif ExoS . Strikingly, all three compounds ( 30, 43, 53 ) displayed a high selectivity for stabilizing ChREBPα, without affecting any other client peptide up to 100 µM (Fig. , Supplementary Fig. ). Furthermore, titration of all seven 14-3-3 isoforms to ChREBPα in the presence of 43 showed PPI stabilization among all isoforms (Supplementary Fig. ). The ChREBPα stabilization selectivity of 43 was also preserved among different 14-3-3 isoforms (Supplementary Fig. ). These data demonstrate the highly selective nature of the molecular glue activity of these compounds by addressing a unique pocket only present in the ChREBPα/14-3-3 complex and via a stabilization mechanism, involving a large cooperativity effect. Crystallization of the new fluorinated compounds with the ChREBPα/14-3-3σ complex was also successful for 42 and 43 , resulting in a clear density for the entire molecule (Supplementary Figs. ). A crystallographic overlay with 30 revealed an additional conformational ‘clamping’ effect of helix-9 of 14-3-3σ in the presence of 43 (Fig. ), causing the hydrophobic residues of 14-3-3 (I219, L218) to be closer to the phenyl ring of 43 . This clamping effect of helix-9 is in line with previous observations for molecular glues with high cooperativity factors, for other 14-3-3 PPIs . A more detailed analysis showed that the meta -fluoro group of 43 is positioned at the rim of the hydrophobic interface of 14-3-3 and ChREBPα (Fig. ). The rest of stabilizer 43 has similar interactions with both ChREBPα and 14-3-3 as 30 (Fig. , Supplementary Fig. ). An ortho- fluoro substitution at the phenyl ring was less favorable ( 42 , EC 50 = 9.6 ± 1.1 µM) although it did result in a similar ‘clamping’ effect of helix-9 of 14-3-3 (Supplementary Fig. ). This ortho substituted fluoro was not positioned at the rim of the ChREBPα/14-3-3 interaction interface, instead it made a polar interaction with N175 of 14-3-3 (Supplementary Fig. ). While no crystal structure could be solved for the double fluoro-substituted analog 53 , the structure of 43 shows room for a fluoro-substitution at the para position of its phenyl ring, explaining the high potency of 53 (Fig. ). Likely, the electron withdrawing effects of the fluorine substitutions are enhancing hydrophobic contacts with the hydrophobic amino acids at the roof of the 14-3-3 groove. Concluding, fluorination of the scaffold enhanced stabilizing efficiency of the ChREBPα/14-3-3 complex, by strengthening the interactions at the rim of the PPI interface.
We tested six compounds ( 1, 12, 30, 43, 53, 66 ) for cytotoxicity and their ability to protect β-cells from glucolipotoxicity (summarized in Fig. ). Compound 1 , our previously reported parent compound that was moderately active in the biochemical assay, displayed cytotoxicity in INS-1 cells. In contrast, compounds 30, 43 and 53 , which were all active in biochemical assays, demonstrated no cytotoxicity in INS-1 cells and protected the β-cells from glucolipotoxicity, turning them into candidates for further cellular evaluation. Compounds 12 and 66 , which served as control compounds, were not active in the biochemical assay and were also neither generally cytotoxic nor rescued the cells from glucolipotoxicity. To test the efficacy of our compounds in mitigating β-cell death from glucolipotoxicity, we conducted dose-response experiments in rat insulinoma, β-cell-like INS-1 cells . We observed a significant attenuation of cell death in response to glucolipotoxic conditions at 5 and 10 μM of active compounds 30, 43 , and 53 , but not in the presence of the inactive compounds 12 or, 66 (Supplementary Fig. ). To investigate cadaveric human islets, we optimized the single-cell and population-level analyses using real-time kinetic labeling (SPARKL) assay , that measures the kinetics of overall proliferation (cell count, using NucBlue) or cell death rates (using YoYo3) specifically in β-cells using the rat insulin promoter (RIP)-ZSGreen adenovirus (Fig. , Supplementary Figs. ). The active compounds ( 30, 43, 53 ) all exhibited a remarkable decrease in β-cell death in glucolipotoxic conditions, as observed in Fig. c– while the ‘inactive’ control compounds showed no activity. Notably, for all five tested compounds, there was no discernible impact on β-cell number, indicating that over the course of 72 h, no meaningful proliferation occurred in treatment groups (Supplementary Figs. ). We also performed glucose-stimulated insulin secretion (GSIS) in primary human islets pre-treated for 24 h with 43 , in low or high glucose conditions. Remarkably, we found that 43 improved glucose-stimulated insulin secretion (GSIS) stimulation index (SI) in high glucose (Fig. ) with no effect on SI in response to KCl or on insulin content (Supplementary Figs. ). Our method was validated using TUNEL staining at 48 h using 10 µM 43 to show similar cell death patterns as the kinetic measurement (Supplementary Fig. ). Indeed, 43 , which we focus on for the rest of this study, also prevented glucose-stimulated proliferation, confirming our previous studies , , and glucolipotoxic cell death in INS-1 cells (Supplementary Fig. ). Together, these studies demonstrated that the three active compounds ( 30, 43, 53 ) prevent β-cell death while maintaining and even enhancing β-cell function in the context of glucolipotoxicity. Importantly. we also demonstrated that the effects of 43 were mediated by ChREBPβ as silencing of ChREBPβ had a similar effect on protection from glucolipotoxicity in human islet cells, while overexpression of ChREBPβ led to increased cell death under low, high, and glucolipotoxic conditions, and 43 was unable to protect human islet cells from apoptosis due to overexpression of ChREBPβ (Supplementary Fig. ).
To explore the mechanism by which 43 acts in cellulo as a molecular glue, we performed three sets of experiments. First, a proximity ligation assay (PLA) was employed in INS-1 cells using an antibody against ChREBPα and a pan 14-3-3 antibody. PLA assays show a fluorescent signal only when proteins are in close (<40 nm) and stable proximity to each other . In low glucose, we observed an interaction between 14-3-3 and ChREBPα. By contrast, following exposure to high glucose for 30 min, ChREBPα no longer interacted with 14-3-3, but the interaction was restored after 2 h (Fig. ). These observations are consistent with our previous study showing that ChREBPα transiently enters the nucleus to begin the feed-forward induction of ChREBPβ . Remarkably, in the presence of 43 , the interaction between 14-3-3 and ChREBPα remained unchanged at high glucose concentrations, thus confirming that 43 stabilized the interaction between 14-3-3 and ChREBPα in high glucose. In a second set of experiments, in which nuclear localization of ChREBPα was studied using a ChREBPα-specific antibody, we again found the same dynamics; ChREBPα entered the nucleus after 30 min of high glucose and exited the nucleus after 2 h. However, 43 blocked the transient translocation of ChREBPα in response to glucose (Fig. b, ). To confirm specificity of 14-3-3 binding to ChREBPα, we also studied the dissociation of 14-3-3 from NFATC1, previously demonstrated to translocate into the nucleus in response to glucose stimuli , . In the presence and absence of 43 , glucose promoted 14-3-3 dissociation from NFATC1, demonstrating the selectivity of our molecular glue to stabilize the interaction of ChREBPα and 14-3-3 (Supplementary Fig. ). Under glucolipotoxic conditions (20 mM glucose + 500 µM palmitate), we observed a similar translocation of ChREBPα to the nucleus after 30 min, however, the nuclear clearance of ChREBPα under glucolipotoxic conditions took longer compared to high glucose alone (Fig. d, ). AMPK activity cannot mediate 43 ’s effects since in the presence of 1 mM AICAR, a widely used AMPK activator, translocation of ChREBPα is not blocked (Fig. c, ). Because our nuclear translocation studies are based on immunodetection, this positive outcome could be a result of epitope masking, e.g., by a ChREBPα interaction with the transcription machinery or other heteropartners, which could block antibody binding. Therefore, a third approach used CRISPR/Cas9 engineered INS-1 cells wherein fluorescent tags to the 5′ and 3′ ends of ChREBP were added . An mCherry tags the N-terminal LID domain, which identifies ChREBPα exclusively, and an eGFP tag on the C-terminus represents both ChREBPα and ChREBPβ. In these cells, ChREBPα appears as yellow (red+green), and ChREBPβ appears green. Compound 43 clearly prevents the built up of nuclear ChREBPβ in both high glucose and glucolipotoxic conditions, thus confirming the immunostaining results (Fig. f, ). Together, these studies demonstrate that 43 stabilizes the interaction of 14-3-3 and ChREBPα under conditions of hyperglycemia and glucolipotoxicity.
The global epidemic of T2D necessitates the development of novel therapeutic and preventive strategies to arrest the progressive nature of this disease . ChREBP is an important regulator of glucose levels and is increasingly recognized as a potential target for T2D treatment . A small molecule regulation of its function is however challenging as TFs are notoriously difficult to target with ‘classical’ small molecules. Here, we sought to develop a molecular glue approach, based on the use of small molecule PPI stabilizers, molecular glues, for the ChREBPα/14-3-3 PPI. These compounds were then used to test if retention of ChREBPα in the cytoplasm may lead to a persistent, chemotherapeutically exploitable inactivation of its transcriptional activity. Through the development of cellularly active molecular glues, we were able to demonstrate that this unconventional strategy for targeting TF functions via TF retention in the cytoplasm indeed resulted in the desired, consistent, and efficacious glucolipotoxicity rescue phenotype. We also did not observe significant cytotoxicity on a cellular level. Besides its implications for T2D therapy, we therefore believe that 14-3-3 or other scaffold-mediated retention of TFs in the cytoplasm via customized molecular glues may represent a broader therapeutic approach that may also be extended to other, difficult to target TFs with medicinal relevance. This strategy also complements other non-conventional ‘molecular glue’ or PROTAC TF approaches such as TRAFTACs or other TF targeted degradation systems , . Sequence alignments of the interacting regions between 14-3-3 proteins and ChREBPα display the conserved sequences of the α2 and α3 helices of ChREBPα across multiple species, including human, chimpanzee, gorilla, rat, and mouse (Supplementary Fig. ). The alignment of specific 14-3-3 helices (helices 9, 7, and 3) that interact with ChREBPα’s α2 helix, highlighted in red within the crystal structure model demonstrates sequence conservation across the seven human 14-3-3 isoforms (Supplementary Fig. top). Cross-species alignment (human, chimpanzee, gorilla, rat, and mouse) for the 14-3-3 sigma isoform underscores the evolutionary conservation of interaction domains (Supplementary Fig. bottom), suggesting functional relevance in ChREBPα‘s regulation by 14-3-3 proteins. Our cellular active molecular glues simultaneously engage both protein partners in the composite ChREBPα/14-3-3 binding pocket, causing a cooperativity factor up to 220 for this PPI. The addition of geminal -difluoro groups increased the PPI stabilization efficiency significantly, overall leading to compounds with micromolar cellular potency. On a molecular level, this enhanced PPI stabilization by fluorination of the molecular glues was achieved by stimulating the clamping of helix-9 of 14-3-3 and contacting hydrophobic amino acids at the interaction surface of the ChREBPα/14-3-3 complex. This molecular mechanism also resulted in a very high selectivity of the compounds for the stabilization of the ChREBPα/14-3-3 PPI over other 14-3-3-based PPIs. Selective interfacing of these molecular glues with amino acid residues that only occur in the context of this specific PPI complex, thus result both in a large cooperative effect for PPI stabilization and high selectivity. Combined, this high cooperativity and selectivity for the ChREBPα/14-3-3 PPI, and probably increased membrane permeability, conferred cellular activity to our lead compound. Our studies also shed further light on the function and suitability of ChREBP as a target in metabolic disease. As T2D progresses, there is a progressive decline in β-cell mass due to the metabolic overload associated with a diabetic environment. ChREBP has long been identified as a potential mediator of this decline, in part by activating the pro-oxidative protuberant, TXNIP . Indeed, clinical trials have been launched with compounds that inhibit the induction of TXNIP in T1D . We recently described the destructive feed-forward production of ChREBPβ in β-cells in the context of prolonged hyperglycemia and diabetes , – . Unrestrained ChREBPβ leads to increased oxidative stress, which in turn leads to decreased expression of transcription factors necessary to maintain β-cell identity and function. Importantly, we found that deletion of the ChREBPβ isoform, using the Cre/Lox system, completely rescues β-cells from cell death and dedifferentiation due to glucolipotoxicity . Here, we found molecular glues, which prevented ChREBPα from dissociating from 14-3-3 and initiating the feed-forward production of ChREBPβ, that were also remarkably effective at preventing cell death and dedifferentiation from glucolipotoxicity. One additional consequence of the retention of ChREBPα was that the molecular glues attenuated cell proliferation (Supplementary Fig. ). This result was consistent with our previous findings that a modest induction of ChREBPβ is required for adaptive β-cell expansion in mice , , . Considering molecular glues as a potential therapeutic, it is likely that preservation of β-cell mass is more clinically relevant compared to the loss of an extremely low proliferation rate of human β-cells , . ChREBPα may be anchored in the cytoplasm by elements other than 14-3-3 including sorcin, a Ca 2+ -binding ER protein, and via C-terminal amphiphilic interactions with lipid droplets , . In addition, numerous post translational modifications , as well as interaction with importin α may play important roles in regulating ChREBP cellular location. Since 14-3-3 and importin α compete for the same ChREBP binding region, ChREBP/importin α-PPI inhibitors, as developed by Uyeda et al. could potentially work synergistically with ChREBPα/14-3-3 stabilizers in suppressing the progression of T2D. Importantly, upregulation of ChREBPβ and downstream genes(i.e., TXNIP and/or DNL genes) are implicated not only in β-cell demise but also in kidney failure , cardiac hypertrophy , ischemic and cardiovascular diseases , , liver steatosis , and NAFLD in particular in high glucose setting. Inhibiting ChREBPβ and downstream genes might prove to be valuable not only for preserving β-cell mass but may also be therapeutic in other tissues for the previously listed conditions. To summarize, we demonstrated the development of highly potent, selective, active stabilizers of the ChREBPα/14-3-3 PPI with a phosphonate chemotype that display activity in both INS-1 cell lines and primary human islets. This new compound class possibly presents the first foundations for the development of novel therapeutics against T2D. More broadly, this work delineates a conceptual entry to the forthcoming discoveries of molecular glues, and we envision that 14-3-3 PPI stabilization can not only be applied for the interaction with ChREBP but can be translated to other TFs as well and offers an orthogonal strategy for hard-to-drug proteins and pathways in general (Fig. ).
Ethics statement Protein expression and purification Peptide Sequences Fluorescence anisotropy measurements Compound titrations Cooperativity analysis X-ray crystallography data collection and refinement Synthesis of SAR library for 1 Cell lines Adenovirus Immunostaining Glucose stimulated insulin secretion (GSIS) analysis qPCR and PCR Proximity ligation assay (PLA) Proliferation and cell death Calcium imaging Active mitochondria staining ATP/ADP measurements Measurements were performed using a commercially available ADP/ATP bioluminescent kit (Abcam). Luciferase reporter Statistics research complies with all relevant ethical regulations. Non-diabetic islets were acquired from Prodo Laboratories and IIDP. Prodo Laboratories and IIDP obtains informed consent that covers all non-identifiable information, which can be found at ( https://prodolabs.com/humanislets-for-research ) and ( https://iidp.coh.org/ ) and compensation is not provided. These islets have been refused for transplantation, have been quality-controlled, and meet specific criteria for research purposes only. The research in this study was approved by the Icahn School of Medicine at Mount Sinai (IBC) Safety Committee (Approval number SPROTO202400000061).
The 14-3-3βFL and 14-3-3σΔC isoform (full length and truncated C-terminus after T231 (ΔC to enhance crystallization)) containing a N-terminal His6 tag were expressed and purified using standard methods as described previously .
The N-terminal FITC labeled ChREBP-derived peptide (residues 117–142; sequence: RDKIRLNNAIWRAWYIQYVKRRKSPV-CONH 2 ) was synthesized via Fmoc solid phase peptide synthesis as described previously . N-terminal acetylated ChREBP peptide used for crystallization was purchased from GenScript Biotech Corp. Peptides used for selectivity studies were purchased from GenScript Biotech Corp with the following sequences: BRAF: (5-FAM-RDRSS(pS)APNVH-CONH 2 ), CRAF: (5-FAM-QRST(pS)TPNVH-CONH 2 ), ERα: (5-FAM-AEGPFA(pT)V-COOH), EXO-S: (5-FAM-QGLLDALDLAS-CONH 2 ), P65: (5-FAM-EGRSAG(pS)IPGRRS-CONH 2 ), PIN1: (5-FAM-LVKHSQSRRPS(pS)WRQEK-CONH 2 ), USP8: (5-FAM-KLKRSY(pS)SPDITQ-CONH 2 ).
Fluorescein labeled peptides, 14-3-3βFL protein, the compounds (100 mM stock solution in DMSO) were diluted in buffer (10 mM HEPES, pH 7.5, 150 mM NaCl, 0.1% Tween20, and 1 mg/mL Bovine Serum Albumin (BSA; Sigma-Aldrich). Final DMSO in the assay was always 1%. Dilution series of 14-3-3 proteins or compounds were made in black, round-bottom 384-microwell plates (Corning) in a final sample volume of 10 µL in duplicates.
Compound titrations were made by titrating the compound in a 3-fold dilution series (starting at 500 µM) to a mix of FITC labeled peptide (10 nM) and 14-3-3β (concentration at EC 20 value of protein-peptide complex; 150 nM for ChREBP). For the selectivity studies concentration of 14-3-3β were: 300 nM for ERα, 3.65 µM for Exo-S, 10 µM for Pin1, 250 nM for USP8, 230 nM for B-RAF, 30 µM for P65 and 450 nM for C-RAF. Fluorescence anisotropy measurements were performed directly. Protein 2D titrations were made by titrating 14-3-3β in a 2-fold dilution series (starting at 300 µM) to a mix of FITC-labeled peptide (10 nM) against varying fixed concentrations of compound (2-fold dilution from 500 µM), or DMSO. Fluorescence anisotropy measurements were performed directly. Fluorescence anisotropy values were measured using a Tecan Infinite F500 plate reader (filter set lex: 485 ± nm, lem: 535 ± 25 nm; mirror: Dichroic 510; flashes:20; integration time: 50 ms; settle time: 0 ms; gain: 55; and Z-position: calculated from well). Wells containing only FITC-labeled peptide were used to set as G-factor at 35 mP. Data reported are at endpoint. EC 50 and K D values were obtained from fitting the data with a four-parameter logistic model (4PL) in GraphPad Prism 7 for Windows. Data was obtained and averaged based on two independent experiments.
The cooperativity parameters for compounds 1 , 30 , 43 , and 53 for 14-3-3 and ChREBP were determined by using the thermodynamic equilibrium system as described previously , and supported by references. The data from 2D-titrations was provided to the model including the K D I = 1.5 µM, P_tot = 10 nM, and the variable concentrations of 14-3-3 and stabilizer at each data point. Fit parameters were given the following initial guess values: K D II : 500 µM, α: 100.
A mixture of Ac-ChREBP peptide and compound (2 µL of 5 mM stock ChREBP peptide + 0.5 µL of 100 mM stock compound) was mixed in crystallization buffer (CB: 20 mM HEPES pH 7.5, 2 mM MgCl 2 , 2 mM βME) to a final volume of 4 µL. This was added to preformed 14-3-3σ/peptide 2d crystals at 4 °C that were produced as described previously . Briefly, single crystals were fished after 7 days of incubation at 4 °C and flash-cooled in liquid nitrogen. Diffraction data was collected at either the Deutsche Elektronen-Synchrotron (DESY Petra III beamline P11 proposal 11011126 and 11010888, Hamburg, Germany, wavelength 1.033220 Å, 04-11-2020 and 09-08-2024) (crystal structures of compound 1 and 30 ) or at the European Synchrotron Radiation Facility (ESRF Grenoble, France, beamline ID23-2 proposal MX2268, wavelength 0.873128 Å, 29-01-2021) (crystal structures of compound 43 and 42 ). Initial data processing was performed at DESY using XDS or at ESRF using DIALS after which pre-processed data was taken towards further scaling steps, molecular replacement, and refinement. Data was processed using the CCP4i2 suite (version 8.0.003). After indexing and integrating the data, scaling was done using AIMLESS . The data was phased with MolRep using 4JC3 as a template. Presence of soaked ligands and ChREBP-peptide was verified by visual inspection of the Fo-Fc and 2Fo-Fc electron density maps in COOT (version 0.9.6). If electron density corresponding to ligand and peptide was present, the ChREBP peptide was built in and the structure and restrains of the ligands were generated using AceDRG , followed by model rebuilding and refinement using REFMAC5 . The PDB REDO server (pdb-redo.edu) was used to complete the model building and refinement. Ramachandran statistics favored/outlier (%) were 98.77/0.00, 98.80/0.00, 96.40/0.00, and 96.80/0.00 for structures with compound 1, 30, 42 and 43 , respectively. The images were created using the PyMOL Molecular Graphics System (Schrödinger LLC, version 2.2.3). For refinement statistics see Supplementary Table . The structures were deposited in the protein data bank (PDB) with IDs: 8BTQ (compound 1 ), 8C1Y (compound 30 ), 8BWH (compound 42 ), and 8BWE (compound 43 ).
Detailed synthetic procedures and characterization of compounds marked with a star (*) in Table were described previously . The synthetic procedures and characterization of all remaining compounds are provided in the Supplemental Methods. Generally, all compounds obtained had been purified by reversed-phase high performance liquid chromatography (HPLC) and characterized by liquid chromatography mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR; 400 MHz for 1 H NMR and 100 MHz for 13 C NMR). Compounds were prepared as 100 mM stock solutions in DMSO before use in experiments, and stored at −20 °C.
INS-1–derived 832/13 rat insulinoma cells were maintained in RPMI 1640 medium with 10% FBS, 10 mM HEPES, 2 mM l -glutamine, 1 mM sodium pyruvate, and 50 mM β-mercaptoethanol 100 U/mL penicillin, 100 mg/mL streptomycin and further supplemented with 11 mM glucose, at 37 °C in a 5% CO 2 incubator. Human islets were isolated from human cadaveric islets donors provided by the NIH/NIDDK-supported Integrated Islet Distribution Program (IIDP) ( https://iidp.coh.org/overview.aspx ), and from Prodo Labs ( https://prodolabs.com/ ), the University of Miami, the University of Minnesota, the University of Wisconsin, the Southern California Islet Cell Resource enter, and the University of Edmonton, as summarized in Supplementary Table Informed consent was obtained by the Organ Procurement Organization (OPO), and all donor information was de-identified in accord with Institutional Review Board procedures at The Icahn School of Medicine at Mount Sinai (ISMMS). Human islets were cultured and dispersed as previously described .
Human islets were transduced with RIP-ZsGreen at MOI of 100. Overnight, the following day, islets were dispersed, and seeded in RPMI with 100 U/mL penicillin, 100 mg/mL streptomycin in the presence of an additional 100 MOI of the virus for 2–4 h. Following, FBS was added to a final concentration of 10%, compounds added and cells were continuously imaged for the course of 48–72 h.
INS-1 cells or dispersed human islet cells were plated on 12 mm laminin-coated glass coverslips. Cells were treated with the indicated conditions and times. Cells were washed with PBS and fixed in 4% paraformaldehyde. Immunolabeling was performed as previously described with primary antibodies directed against N-terminus ChREBP (1:250 ,), C-terminus ChREBP (Novus, cat # NB400-135, 1:250), Pdx1 (Abcam, cat # Ab47308, 1:500), and Insulin (Fisher, cat # MAB1417, 1:1000).
20 IEQ primary human were placed in 12 µm pore diameter inserts (Millipore) and incubated for 24 h with indicated treatment. Following, media was replaced with KREBS containing 2.8 mM glucose and 1% BSA, and GSIS analysis was performed as previously described . Insulin levels were measured using a commercial ELISA assay (Mercodia), following the manufacturer instructions.
mRNA was isolated using the Qiagen RNAeasy mini kit for INS-1 cells or for islets using the Qiagen RNAeasy mini kit. cDNA was produced using the Promega m-MLV reverse transcriptase. qPCR was performed on the QuantStudio5 using Syber-Green (BioRad) and analysis was performed using the ΔΔCt method. PCR for genotyping was performed using standard methods. Primer sequences are shown in Supplementary Table .
PLA was used to determine endogenous protein–protein interactions as previously described in ref. . Briefly, ChREBPα (by GenScript) or NFATC1 (cell signaling) and Pan-14-3-3 (Santa Cruz) antibodies were conjugated to Duolink oligonucleotides, PLUS and MINUS oligo arms, respectively, using Duolink® In Situ Probemaker Following a PBS wash, cells were fixed with 4% formaldehyde solution for 10 min at room temperature, and blocked with Duolink Blocking Solution for 1 h at 37 °C and then incubated with 4 μg/mL ChREBPα-Plus and 14-3-3-MINUS overnight at 4 °C. PLA was performed according to the manufacturer’s directions. No secondary antibodies were used because PLUS and MINUS oligo arms were directly conjugated to ChREBP and 14-3-3. Cells were imaged on a Zeiss 510 NLO/Meta system (Zeiss, Oberkochen, Germany), using a Plan-Apochromat 63×/1.40 oil differential interference contrast objective.
Cellular proliferation and cell death were quantified using Real-Time Kinetic Labeling (SPARKL) technique . Syto21 marked all cells, Yoyo3 marked all dead cells.
INS-1 cells seeded on glass bottom dish at low glucose. After 24 h media was replaced with KREBs buffer containing 5.5 mM glucose. Following 1 h incubation, Fluo-4 AM was added and incubated with cells for 45 min. The cells were then imaged at low glucose concentrations for 1 min, with images captured at 6 sec intervals. Glucose was supplemented to the cells to reach a final concentration of 20 mM and imaging was continued with the same settings for 5 additional min. The images were acquired using the confocal Cytation 10 system. Fluo-4 intensities were calculated using Gen5 Software.
INS-1 cells were incubated with 250 nM MitoTracker® Orange CM-H2TMRos (Life Technologies, Eugene, OR) for 30 min at 37 °C. Mitochondrial activity was analyzed based on fluorescence intensity using a confocal Cytation 10 system and Gen 5 software.
Measurements were performed using a commercially available ADP/ATP bioluminescent kit (Abcam).
The reporter assays were done as described previously . Briefly, INS-1-derived 832/13 cells that stably express the luciferase reporter gene under control of the human TXNIP promoter. Cells were harvested after 24 h and luciferase activity was measured using the Luciferase Reporter Assay System (Promega, Cat. #E1500) on Victor Nivo luminometer (PerkinElmer). Firefly luciferase activity was normalized to Renilla luciferase activity.
All studies were performed with a minimum of three independent replications. Data were represented in this study as means ± standard error of the mean (SEM). Statistical analysis was performed using two-way ANOVA on GraphPad (Prism) V9. |
Attitudes of medical workers in China toward artificial intelligence in ophthalmology: a comparative survey | 0fdf3f3b-6d60-4136-840c-90555e3ed9cb | 8501607 | Ophthalmology[mh] | In recent years, with the increase of computational speed, neural networks have regained prosperity after hitting its rock bottom. The deep convolutional neural network AlexNet winning the first place in the ImageNet competition has led to the rapid development of deep learning technology. After that, deep learning network models have emerged generation after generation, which has accelerated the development of artificial intelligence (AI) technology. Through AI, researchers can make the preliminary diagnosis of skin cancers, achieve rapid intraoperative diagnosis of brain tumors, diagnose 55 common diseases in pediatrics based on electronic medical records in Chinese, identify rare genetic diseases through facial photographs, and generate the findings that early and frequent patient movements can reduce the risk of post-intensive care syndrome and long-term dysfunction after analyzing patients’ movement activities in the intensive care units. Medical instruments are frequently used in clinical examinations in modern medicine, and imaging equipment is an important part. Imaging equipment is used for X-ray imaging, magnetic resonance imaging, ultrasound imaging, tomography imaging, and fundus photography, etc. The data in the ophthalmology field is diverse and huge, among which the most common types are fundus photographs and optical coherence tomography (OCT) images, making it the most extensively researched field with AI. Fundus photography and OCT are regular examinations used in ophthalmology, through which a vast amount of high-quality standard images can be obtained. These images are suitable for analysis and process by AI deep learning technology to further assist doctors in diagnosing ophthalmopathies. Using deep learning technology in AI, Google researchers have created an algorithm that can detect diabetic retinopathy and macular edema. By analyzing human retinal images, this algorithm can very accurately predict risk factors affecting cardiovascular health. The researchers from Sun Yat-sen University in China have developed a deep learning model called CC-Cruiser for recognizing congenital cataract, which is also able to diagnose blinding diseases such as age-related macular degeneration and diabetic macular edema after trained with deep learning algorithms based on OCT images . Besides, there are related studies that use AI technology for the segmentation of ophthalmic images , and the classification of ophthalmopathies , etc. Artificial intelligence technology has been studied so extensively in ophthalmology that some even have been on the commercializing stage , thus leading to some people believing that AI may be able to replace doctors. Some researchers investigated the attitudes of medical undergraduates to the application of AI in radiology and medicine , the attitudes of medical students in other regions to AI , the influence of artificial intelligence on radiology , as well as how to cope with the ethical challenges in medical AI , etc. However, few people know about medical workers’ familiarity with and their attitudes toward AI in ophthalmology. For this purpose, a questionnaire was designed to assess medical workers’ (health care workers or medical students) understanding level of and their attitudes toward AI. Meanwhile, the questionnaire also surveyed other professional technicians (engineers, teachers, technicians, experimenters, etc. in non-medical fields) using the same questions as a comparison. The survey of AI in ophthalmology-related questions cannot only evaluate the attitudes of medical workers (health care workers or medical students) and other professional technicians toward AI and clarify the dilemma facing the current technological development; it can also provide theoretical guidance for its future practice and application. At present, in the development of AI in ophthalmology, there are a lot of existing and foreseeable medical ethical problems. For example, the medical responsibilities are unclear, the problem of the security of patient privacy data and the risk of the weakening of the doctor’s professional status, etc. Analysis of the causes of these problems through this survey may help to put forward corresponding countermeasures so that we can draw on advantages and avoid disadvantages in the future development of AI in ophthalmology.
Using the Questionnaire Star APP (a professional questionnaire survey app in China, easy to edit and distribute survey questionnaires), we designed an electronic questionnaire that consisted of four parts. The first part was the respondent’s basic information, including the respondent’s sex, age, educational level, place of residence, work area and professional title; the second part was about the respondent’s basic understanding of AI, including whether the respondent understood AI, medical AI, and AI in ophthalmology, as well as the respondent’s evaluation of the current development of AI in ophthalmology; the third part was about the respondent’s attitude to AI, including whether he/she thought AI in ophthalmology would replace doctors, whether he/she had experience in AI in ophthalmology application, and his/her acceptance level of AI in ophthalmology; and the fourth part was about the respondent’s concerns about AI, which included the respondent’ specific concerns about AI in ophthalmology and whether he/she thought it was necessary to strengthen medical ethics research in the field. According to the pre-investigation experience and the principle of the rank order scale, the basis for the classification of the four ranks is determined. There were 4 grading used to reflect the basic understanding of AI in the second part of the electronic questionnaire. The grading “Completely” means the understanding level of AI ≥ 90 %; The grading “Almost” means the understanding level of AI between 50 % and 89 % (include 50 % and 89 %); The grading “A little” means the understanding level of AI between 10 % and 49 % (include 10 % and 49 %); The grading “Not understand” means the understanding level of AI ≤ 9 %. There were 4 grading used to reflect the respondent’s attitude to AI in the third part of the electronic questionnaire. The grading “Completely” means the acceptance level of ophthalmic AI ≥ 80 %; The grading “Partly” means the acceptance level of ophthalmic AI between 20 % and 79 % (include 20 % and 79 %); The grading “No” means the acceptance level of ophthalmic AI ≤ 19 %; The grading “Don’t understand” means the respondents didn’t understand the question. Respondents of the questionnaire were mainly members of the Zhejiang Society of Mathematical Medicine, with their locations covering various cities and counties mainly in Zhejiang Province. They worked as ophthalmologists, medical students, AI technicians, and professional technicians in other fields. Their educational levels were above junior high school and could understand the questionnaire well. The questions in the questionnaire have been investigated, sorted and summarized repeatedly in a broad and deep way. The survey is a targeted group survey. Questionnaires were sent to medical workers through professional ophthalmological or medical intelligence groups and other professionals through related professional groups. Before we collect the questionnaires for statistical analysis, each respondent was invited to fill out the questionnaire once based on a voluntary and anonymous principle and was informed that the results of the survey would be further used for statistical assessment and publication. This study is an epidemiological survey research design, containing 15 questions, and usually requires a sample size of at least 5–10 times the number of questions, usually about 15 times. Therefore, the sample we selected is between 15 and 20 times the number of questionnaires, with 291 medical workers and 271 other professional technicians. This study mainly uses statistical analysis, chi-square analysis and odds ratio analysis. The relevant indicators include statistical analysis results, chi-square value, odds ratio (OR) value, Probability (P) value and 95 % CI. A total of 562 respondents were counted, with 562 valid questionnaires returned. The results of the questionnaires are displayed in an Excel 2003 form. According to the Article 3 of the Measures for the Ethical Review of Biomedical Research Involving Humans issued by the National Health and Family Planning Commission in 2016, ethical review is unnecessary for the study.
Basic information of respondents Respondents’ basic understanding and attitudes towards AI Comparison of perceptions and attitudes towards AI between medical workers and other professional technicians Respondents’ concerns about artificial intelligence Of the 562 respondents, 291 were medical workers (51.8 %) with the rest being other professional technicians (48.2 %). As shown in Table , nearly half of them were from prefecture-level cities (47.7 %), about 1/4 (24.9 %) were from provincial capital cities, and the remaining were from other regions (27.4 %). 10.0 % had a doctor’s degree or higher education, 13.0 % had a master’s degree, and 77.1 % had a bachelor’s degree or lower education; 31.5 % had senior titles, 25.4 % were with intermediate titles, and 32.0 % were primary and ungraded professionals. The complex structure of these respondents had relatively good social representative value.
In Table , the percentage of respondents who completely understood and almost understood AI was 37.9 %, who understood a little was 52.1 %, and who didn’t understand AI was 10.0 %; the percentage of respondents who completely understood and almost understood medical AI was 31.7 %, who understood a little was 44.3 % and who didn’t understand medical AI was 24.0 %; the percentage of respondents who completely understood and almost understood AI in ophthalmology was 29.6 %, who understood a little was 34.5 %, and who didn’t understand AI in ophthalmology was 35.9 %. That is to say, the proportion of respondents whose understanding level of AI, medical AI, and AI in ophthalmology was “completely”, “almost” or “a little” was gradually decreasing in the mentioned order (AI, medical AI, and ophthalmological AI), while the proportion of people who did not know about AI, medical AI, and AI in ophthalmology was gradually increasing in the same order. Among the respondents in Table , there were 19.4 % of them thought that the current development of AI in ophthalmology was very good. About 42.7 % of respondents thought that its current development was good. There were 35.2 % of respondents thought that its current development was average. As shown in Table , on the question of whether AI in ophthalmology would replace doctors, there were 24.0 % of respondents said no. But 66.0 % of respondents thought it would partly replace doctors. There were 69.4 % of the respondents had no ophthalmic AI-related experience while the rest had applied or experienced AI in ophthalmology. There were 59.1 % of the respondents had a relatively high acceptance level of AI in ophthalmology, with only 2.1 % against it. Therefore, the popularity of AI in China is relatively high, and the respondents had a certain understanding of artificial intelligence (such as alpha go, etc.), but there were still deficiencies in professional fields (such as ophthalmology). The most of respondents had a high degree of acceptance of AI in ophthalmology, and held a positive attitude towards its current development, believing that AI can improve the quality of people’s lives. Although most respondents had no experience in ophthalmic AI, they still believed that ophthalmic AI would partially replace doctors. But doctors played a vital role in the diagnosis and treatment of diseases and would not be completely replaced.
Table ; Figs. and , and Fig. show that the proportion of medical workers whose understanding level was “completely understand” or “almost understand” was 47.4 %, 45.0 %, and 42.6 %, respectively, for AI, medical AI, and AI in ophthalmology. For other professional technicians, the proportion was 27.7 %, 17.4 %, and 15.5 %, respectively. The Pearson Chi-Square of work area and understanding level for AI, medical AI, and AI in ophthalmology was 43.207, 92.059, and 117.776 respectively. All the P was 0, which means the work area of the respondents was related to their understanding of AI, medical AI, and AI in ophthalmology. Table was a simple version of Table . In Table , option “completely” and “almost” were merged together, option “a little” and “not understand” were merged together. As shown in Table , all the P were equal 0, all the odds ratio (OR) were bigger than 1 and all the lower bound of the 95 %CI were bigger than 1. As shown in Figs. and , about 71 % of medical workers and 53 % of other professional technicians believed that the current development of AI in ophthalmology was good; while among those who completely understood and almost understood AI in ophthalmology, about 81 % of medical workers and 76 % of other professional technicians thought that the current development of AI in ophthalmology was good. For the two different groups by working fields, the respondents’ understanding level of AI, medical AI, and AI in ophthalmology tended to drop in the mentioned order. The proportion of respondents whose understanding level of these three AIs was “completely understand” or “almost understand” was greater among medical workers than among other professional technicians. The close combination of medicine and AI had enabled medical workers to understand more about AI. Therefore, AI is relatively well popularized in the medical field; meanwhile, there is a need to enhance the popularization of artificial intelligence-related knowledge among people in other fields. As shown in Figs. , and , about 77.0 % of medical workers and 57.9 % of other professional technicians believed that AI in ophthalmology would completely or partly replace doctors. There were 56.4 % of medical workers and 83.4 % of other professional technicians had no experience in the application of AI in ophthalmology, while among those with experience in the application of AI in ophthalmology, there were 84.3 % of medical workers and 73.3 % of other professional technicians would fully accept it. x In Table , option “completely” and “partly” were merged together, option “no” and “don’t understand” were merged together, option “no” and “strongly resist” were merged together; for question “Do you have an experience with an ophthalmic artificial intelligence application?”, option “having no related experience” was divided into one group, other options were combined into another group. As shown in Table , the P of the first and second questions were equal 0, the odds ratio (OR) of them were bigger than 1 and the lower bound of the 95 %CI of them were bigger than 1. So the work area of the respondents was related to the two questions. The P of the third question was 0.061, the OR was 2.357 but the 95 %CI was 0.883–12.312. So it wasn’t related to the work area of the respondents. For the two groups by working fields, most of people did not have experience in applying artificial intelligence to ophthalmology, but still believe that AI will partially replace doctors, which shows that the respondents have a certain understanding of AI, but AI had not been widely used in ophthalmology. Compared with other professional technicians, medical workers had more experience in AI. AI was developing rapidly in ophthalmology and had better prospects. There was only 1.8 % of the respondents believed that AI would completely replace ophthalmologists, indicating that people recognized the value of ophthalmologists and believed that even if AI developed better in the future, it is still only an aid to ophthalmologists rather than a replacement. At the same time, there was only 2.1 % of the respondents had an unacceptable attitude towards AI in ophthalmology, indicating that people recognized the combination of AI and the ophthalmology. This recognition had nothing to do with the working fields, but people believed that AI could improve people’s lives.
In Table , among the respondents, 56.4 % said that in the current ophthalmic AI practice, medical responsibilities are unclear; 49.3 % said that the quality of AI in ophthalmology services was difficult to guarantee; while the percentage of those who thought there existed problems such as extreme high prices, medical ethical risks and lack of political support was about 40 %. More than 90 % of the respondents thought there was a need to strengthen medical ethics research in the ophthalmic AI field. Among those who completely and almost understood AI in ophthalmology, 98.4 % of medical workers and 95.2 % of other professional technicians believed it was necessary to strengthen medical ethics research in the field, as shown in Fig. . This is enough to showcase the importance of addressing medical ethical issues in the ophthalmic AI field.
Among the medical workers, there was 42.6 % could understand ophthalmic AI, about 84.3 % fully accepted ophthalmic AI, there was 43.6 % had related applications experience in ophthalmic AI, there was 77 % believed that ophthalmic AI would completely or partially replace doctors, and there was 98.4 % believed that ophthalmic AI ethics research needs to be strengthened. These results help to make some targeted improvements in ophthalmic AI research, such as setting up more pilots so that people have more opportunities to experience ophthalmic AI and more accept ophthalmic AI, therefore, the application of ophthalmic AI can be promoted to the clinic. During diagnosis and treatment, the workload of medical workers is reduced. In the survey, the proportion of medical workers was roughly equal to the proportion of other professional technicians. The proportion of respondents who did not understand AI among other professional technicians was about three times that of medical workers. And the proportion of those who didn’t understand medical AI among other professional technicians was about 4 times that of medical workers. The proportion of people who did not understand AI in ophthalmology among other professional technicians was about four times that of medical workers. Hence one can see that the understanding level of AI, medical AI, and ophthalmic AI among the medical workers who participated in the survey is higher than that among other professional technicians, suggesting that in China, AI is relatively more widely popularized among medical workers. In recent years, AI has been more and more widely introduced in the medical field . Due to work or academic exchanges, medical workers have more opportunities to learn about medical AI than other professional technicians. The other professional technicians in this survey were technical personnel whose research areas had no connection to medicine or AI. As a result, they had relatively less knowledge about medical AI and fewer opportunities to apply or experience medical AI, especially in the more specialized clinical ophthalmology field. Naturally, those in the survey who had no medical background would have a difficult time imagining the application of AI in medicine. Therefore, the real-life application of AI in other areas should be further popularized in our society to gain more of people’s trust before AI can be used in medical care. Concerning the attitudes toward AI, both medical workers and other professional technicians were relatively confident in human doctors, with only a very small proportion of people thinking that AI in ophthalmology would completely replace ophthalmologists. Most respondents were relatively rational, believing that ophthalmic AI would only partly replace ophthalmologists. As people become more rational about AI in ophthalmology, the condition has been more and more favorable for the healthy development of AI in the medical field. As suggested by Turing for the healthy development of AI, “Instead of trying to produce a program to simulate the adult mind, why not rather try to produce one which simulates the child’s? If this were then subjected to an appropriate course of education one would obtain the adult brain.” This conception has functioned as guidance in the research of medical AI. From JAMA’s publication of AI used for DR diagnosis in 2016 to the U.S. Food and Drug Administration’s approval of IDx-DR in 2018 , there has been no real diagnostic systems that can fully diagnose and identify all the 4 grades of DR, which means there is still a long way for ophthalmic AI to go from laboratory research to clinical application. But undeniably, in a country such as China where ophthalmologists are in much dire need (the over 1.4 billion Chinese people only share about 44,800 ophthalmologists) , the application of AI can help to diagnose and treat many more patients, which would reduce the burden on ophthalmologists, thus probably having led to the higher acceptance level of ophthalmic AI among medical workers. Meanwhile, those other professional technicians, despite a relatively lower understanding level, also basically held a positive attitude to acknowledging AI in ophthalmology. As far as the application of ophthalmic AI goes, there are currently some pilot centers in China that provide opportunities to experience its real-life application. However, due to the rarity of such centers, only close to half of the medical workers involved in the survey had related experience while 4/5 of the other professional technicians had no such experience. This indicates that the application of AI in ophthalmology is not yet popularized. It has very few real-life applications, mainly due to technical and ethical issues. On the technical side, the systems that had relatively good diagnosing abilities turned out to be not well performed in the complex real-life scenarios, requiring further improvement. And on the ethics side, the medical responsibilities are not clearly defined for the artificial intelligence diagnostic systems and there are no related policies to follow or to regulate it with. Therefore, although the research of AI in ophthalmology is going well, more pilot centers are needed to explore the problems that may be encountered before actual application; it still takes prudence in its real-life application. In recent years, the application of AI in ophthalmology is very deeply researched, but there are not so many studies on related policies and ethics. The survey found that unclear medical responsibilities and difficulty in guaranteeing service quality respectively ranked as the No. 1 and No. 2 concerns about the use of AI in ophthalmology, with 60 % of the respondents worrying about the “unclear medical responsibilities.” These concerns are sufficient to show that the country needs to improve its regulation system of AI and strengthen the exploration of relevant medical ethics issues. Only when the relevant regulation system and ethics issues have been addressed can we guarantee the real-life practice of medical AI, and establish people’s confidence in medical AI so that they can truly accept its relevant application. Therefore, the next step is to research on AI technology that can eliminate ethical risks and non-technical countermeasures, etc. The survey mainly analyzed the respondents’ understanding and acceptance level of AI in ophthalmology, as well as the respondents’ concerns about AI in ophthalmology. The respondents in this survey are mainly from Zhejiang, China, an important part of the Yangtze River Delta, which is one of china’s relatively developed regions. With almost no respondents from remote areas, the survey results do not represent those of medical workers and other professional technicians nationwide. Besides, although the respondents are relatively evenly distributed among the different groups, the total number is relatively small. The subjects of the survey are mainly divided into medical workers and other professional technicians. This survey mainly studies the understanding and attitudes of ophthalmology AI in different working fields, and the research angle is relatively single. The follow-up surveys should try to broaden the survey scope and increase the angle of research angle (doctors, patients, etc.), making the findings more credible and broadly representative.
The survey results revealed that the medical workers had a higher understanding level of AI in ophthalmology than other professional technicians, making it necessary to popularize ophthalmic AI education among other professional technicians. Most of the respondents did not have any experience in ophthalmic AI, but generally had a relatively high acceptance level of AI in ophthalmology, and there was a need to strengthen research into the medical ethics issues of the field. The next step of this research is to expand the scope of survey, increase the angle of survey, and carry out research on AI technology that can eliminate ethical risks and research on non-technical countermeasures,promoting the clinical application of AI.
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Europium Nanoparticle-Based Lateral Flow Strip Biosensors Combined with Recombinase Polymerase Amplification for Simultaneous Detection of Five Zoonotic Foodborne Pathogens | 672ef5e3-ef07-4f69-a862-a2951eade50f | 10295953 | Microbiology[mh] | A zoonosis is an infectious disease that has jumped from non-human animals to humans, which is caused by viruses, bacteria, parasites and fungi . The rapid and widespread distribution of zoonotic strains poses a major threat to livestock and human health on a global scale . The risk of zoonotic diseases in humans increases with the consumption of animal products . According to the World Health Organization (WHO) , 600 million cases of illness caused by contaminated food are reported worldwide each year, resulting in 420,000 deaths and 33 million people at risk. Since 2000, the global economic cost of zoonotic outbreaks has exceeded USD 10 billion . Zoonotic strains have caused a huge economic burden worldwide, examples include Listeria monocytogenes ( L. monocytogenes ) , Streptococcus suis ( S. suis ) , Staphylococcus aureus ( S. aureus ) , Salmonella enterica ( S. enterica ) and Escherichia coli O157:H7 ( E. coli O157:H7 ) . In 2022, the Ministry of Agriculture of the People’s Republic of China (PRC) confirmed that salmonellosis, swine streptococcosis and listeriosis are important zoonotic diseases . The European Union (EU) observed that S. aureus and E. coli O157:H7 were major foodborne zoonotic strains in the 2019–2020 period . The five pathogenic bacteria mentioned above are commonly found in undercooked foods, contaminated animal products and food processing environments. The consumption of contaminated products (such as meat and milk products) or exposure to contaminated environments can cause various human diseases ( ) [ , , , , , , , , , , , , , , , , ]. Therefore, rapid detection of these pathogens is required for effective disease diagnosis and biomonitoring. Traditional detection methods for zoonotic pathogens based on bacteriological, morphological and polymerase chain reaction (PCR) techniques have drawbacks, such as their time-consuming nature, complex preprocessing steps and strict experimental conditions . Biosensor technology is becoming increasingly popular in pathogen detection as an alternative to traditional methods due to its higher specificity, sensitivity and economic feasibility . Lateral flow strip biosensors (LFSBs) are highly efficient biosensors with the advantages of chromatographic separation and immunological recognition, which allow the visualization and quantification of target products . LFSB detection performance largely depends on the performance of the signal nanomaterial . Traditional LFSBs used gold nanoparticles (AuNPs) as labels, but this method was limited by low signal strength and poor quantitative detection . In recent years, to compensate for the disadvantages of AuNPs and improve the sensitivity of LFSBs, several new signaling nanomaterials have been developed, such as up-converting phosphor (UCP), quantum dots (QDs) and europium nanoparticles (EuNPs) . Among these, lanthanide chelates are complexes of rare earth lanthanide ion-chelating ligands with unique fluorescence characteristics. The chelates possess unique fluorescence characteristics, which make them preferable to other fluorescent markers. Specifically, they have a longer fluorescence decay time, narrower emission spectrum and larger Stokes shift . In recent studies, it was found that the use of EuNPs considerably enhanced the sensitivity and signal-to-noise ratio of lateral flow immunoassay strips . EuNP-LFSBs have been widely used in clinical diagnosis , veterinary drug residues , antibiotic detection and zoonotic strain assays . To further improve the sensor sensitivity, the strategy of nucleic acid signal amplification is extensively used in sensors. Recombinant polymerase amplification (RPA), developed by Piepenburgin in 2006 , is an established method for isothermal nucleic acid amplification. The RPA system relies on three enzymes for nucleic acid amplification, recombinase (UvsX and UvsY), single-strand binding protein (gp32) and strand replacement DNA polymerase (Bsu). The recombinase enzyme directs short oligonucleotide primers, forming filaments that recognize a homologous target sequence. Once the specific site is found, the enzyme opens the double strands to allow for the hybridization of the primer and target sequence. This process is aided by the single-stranded DNA binding protein, which prevents dissociation of the primers. The strand-displacing polymerase copies the DNA by adding bases onto the 3′ end of the primer. The process can be performed at temperatures between 37 °C and 42 °C within 15–20 min , reducing the requirement for high-precision technologies. Compared to other isothermal amplification methods ( ) [ , , , , , , , , , ], RPA technology has high sensitivity, specificity and low-instrument dependency. In the present study, we developed quintuple RPA-EuNP-LFSBs for the rapid detection of L. monocytogenes , S. suis , S. aureus , S. enterica and E. coli O157:H7 from complex samples. The use of a mini automatic nucleic acid extractor (Auto-Pure Mini) for pre-sample processing can significantly reduce the preparation time and improve the nucleic acid purity. In addition, a fluorescent strip reader can be employed to accurately quantify the detection results. To enable the simultaneous detection of five zoonotic strains, we designed five pairs of specific primers that targeted conserved genes of the bacteria. Key parameters were optimized, such as the RPA primer concentration, reaction time and temperature, magnesium ion (Mg 2+ ) concentration and selection of the nitrocellulose (NC) membrane material. The proposed method can efficiently detect objects within 20 min (including strip detection) at 37 °C. By constructing five antibody-loaded test lines, the quintuple RPA-EuNP-LFSB method facilitated the simultaneous field detection of five target pathogens with a low detection limit (10 1 CFU/mL). This method overcomes the limitations of single detection objects and low sensitivity in rapid detection. It effectively enables joint inspection of multiple zoonotic strains in the field and has promising market prospects.
2.1. Bacterial Culture Preparation and DNA Extraction 2.2. Reagents and Apparatus 2.3. Preparation of EuNP-mAb 2.4. Primer Design and Assembly of Quintuple RPA-EuNP-LFSBs 2.5. Multiplex Reaction Protocols for RPA 2.6. Optimization of Quintuple RPA-EuNP-LFSBs 2.7. Sensitivity and Specificity 2.8. Artificially Contaminated Food Samples 2.9. Analysis of Quintuple RPA-EuNP-LFSBs in Field Samples 2.10. Statistical Analysis A total of 40 bacterial strains were utilized, including 20 strains of the five target pathogens and 20 strains of non-target pathogens, as presented in . All strains were sourced from the American Type Culture Collection (ATCC) and the China Medical Culture Collection (CMCC), except for CJ 10102 and CJ 10217, which were obtained from laboratory stock. The standard strains of S. suis , S. aureus and L. monocytogenes were streaked and cultured on tryptic soy agar plates (Hopebio, Qingdao, China). After 16 h of incubation at 37 °C, a single colony was extracted in brain–heart infusion broth (BHI, Thermo Fisher Scientific Inc., Waltham, MA, USA), and cultured at 37 °C for 18 h with shaking (200 rpm). Under the same conditions, the remaining strains were plated on the nutrient agar plate (Hopebio, Qingdao, China) and single colonies were cultured in Luria–Bertani broth (LB, Sangon, Shanghai, China). After incubation at 37 °C for 6 h, bacterial culture suspensions were colonized to solid culture plates for colony counting. The number of colonies counted was multiplied by the dilution factor and divided by the volume of the culture plate to obtain the colony-forming units per milliliter (CFU/mL). Use of the mini automated nucleic acid extractor (Auto-Pure Mini) as a sample preparation tool simplifies the operational steps while providing efficient and rapid extraction techniques ( in ). The operating mechanism was selective adsorption of the target extract through magnetic beads modified with specific chemical groups, enabling efficient high-throughput DNA extraction. The operation process of the Auto-Pure Mini was based on previous experiments . The extracted DNA was quantified using a spectrophotometer and stored at −20 °C.
We prepared carboxytetramethylrhodamine (TAMRA) monoclonal antibody (mAb), carboxy fluorescein (FAM) mAb, tetrachlorofluorescein (TET) mAb, cyanine 5 (Cy5) mAb, and biotin mAb in the laboratory. 2-(N-morpholino) ethanesulfonic acid (MES) was obtained from Yuchun Biological Technology Co., Ltd. (Shanghai, China). Goat anti-mouse polyclonal antibody (pAb), bovine serum albumin (BSA), 1-(3-di-methylaminopropy1)-3-ethylcarbodimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), Tween-20 and glycerol were obtained from Merck & Co., Inc. (Rahway, NJ, USA). Carboxylate-modified EuNPs with a diameter of 200 nm were procured from Shanghai Uni Biotech Ltd. (Shanghai, China). Sample pads, conjugate pads, adsorption pads and backing cards were obtained from Dean Biotechnology Co., Ltd. (Hangzhou, China). The NC membranes, including Millipore 90 (M90), Millipore 180 (M180), Sartorius CN95 and Sartorius CN140, were purchased from Microdetection Biotechnology Co., Ltd. (Nanjing, China). A mini automatic nucleic acid extractor (Auto-Pure Mini, Allsheng Instruments Co., Ltd., Hangzhou, China) was used for the pre-treatment of food samples. An HPY001 row film integrated machine (Wilfen Automation Equipment Co., Ltd., Haining, China) and a CM2000 guillotine cutter (BioDot, Irbine, CA, USA) were used to prepare test strips. A fluorescent strip reader (Suzhou Helmen Precise Instruments, Suzhou, China) was used to quantify the fluorescent band intensity, and a ML-49 Portable Ultraviolet 365 nm flashlight (Moweal Biotechnology Co., Ltd., Shanghai, China) was used for visual inspection. A homogenizer (Bioprep-24, Allsheng Instruments Co., Ltd., Hangzhou, China) was used for food homogenization.
To prepare the anti-digoxin mAb conjugated with EuNPs, we proceeded as follows. Firstly, 2 mg of carboxyl EuNPs (10 mg/mL), 30 µL of EDC (10 mg/mL) and 90 µL of NHS (10 mg/mL) were dissolved in 800 µL of MES (0.05 M, pH 8.2). Then, the solution was activated by slow shaking and left to incubate at room temperature for 30 min. After activation, the excess EDC/NHS was removed by centrifugation at 12,000 rpm for 25 min. The precipitate was dissolved in 1 mL of borate buffer saline (BBS, 0.05 M, pH 8.2), followed by the addition of 2 mL of anti-digoxin mAb (10 µg/mL). The mixture was gently shaken for 2 h at room temperature. On completion of protein coupling, 110 µL of blocking solution (15% BSA) was added and the solution was then rotated at room temperature for 1 h. To separate any unreacted polyclonal antibody and BSA, the EuNP-mAb conjugate was centrifuged twice at 13,000 rpm for 20 min. Finally, the sediment was suspended in 1 mL of a storage solution containing 0.1% BSA ( w / v ) and kept at 4 °C.
Before designing the RPA primers, MegAlign software (LaserGene, DNASTAR Inc, Madison, WI, USA) was used to analyze the conservation of the following five genes: hlyA from L. monocytogenes (GenBank: HM58959), nuc from S. aureus (GenBank: EF529607.1), gdh from S. suis (GenBank: AF229683), fimY from S. enterica (GenBank: JQ665438.1) and rfbE from E. coli O157:H7 (GenBank: HM58959). The five genes were highly conserved and there was no homologous sequence among them. According to the TwistDx instruction manual, the specific primers of S. suis were designed using Primer Premier 6.0 software (Premier Biosoft, San Francisco, CA, USA), while the remaining primers were referenced from previous experiments ( ) . All primers were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). The quintuple lateral flow strip consisted of a sample pad, a conjugate pad, NC membranes, an adsorption pad and a backing card. The sample pad and conjugate pad needed to undergo pre-treatment which required soaking them in PBS buffer solution (0.05 M, pH 7.4, containing 1% BSA and 0.05% Tween-20) for 30 min, followed by drying them at a constant temperature of 37 °C for at least 16 h. The prepared EuNP-mAb was evenly distributed onto the conjugate pad (1% BSA, pH 7.4) and the sample pad was wetted with 0.05 M PBS for 30 min. Samples were then dried overnight in a drying oven at 37 °C. As shown in a, 2.0 mg/mL goat anti-mouse pAb was immobilized as the C line. Anti-Cy5 mAb, anti-FAM mAb, anti-TET mAb, anti-TAMRA mAb and anti-biotin mAb were immobilized as the T1, T2, T3, T4 and T5 lines, respectively. The distance between the test line (T line) and control line (C line) was 2 mm. Then, the prepared materials were assembled and cut into 2.5 mm wide strips using a strip cutter. Finally, the strips were stored at room temperature below 20% humidity.
The TwistAmp Basic Kit (TwistDX, Cambridge, UK) was used for RPA amplification. The target DNA was prepared as per the above scheme, and sterile water was used as a negative template control (NTC). The final reaction system for the quintuple RPA-EuNP-LFSBs experiment was 50 μL containing 25 μL of 2× reaction buffer, 2 μL of each of the forward primers and reverse primers (10 μM) for the five target pathogenic bacteria and 0.5 μL of each of the templates. The mixture was added to the lyophilized enzyme precipitate and mixed well. Then, 2.5 μL of 14 mM Mg 2+ was added to the cap of the tube. The RPA reaction was incubated at 37 °C for 25 min. After the reaction was completed, the amplification products were promptly transferred into ice to stop the reaction.
To achieve optimal performance of the quintuple RPA-EuNP-LFSBs, it was necessary to optimize several key parameters: the primer concentration of RPA, reaction time, reaction temperature, Mg 2+ concentration and selection of NC membranes. To determine the optimal primer concentration, different concentration gradients ranging from 150 to 450 nM were set. Then, the effectiveness of these concentrations was compared at eight different temperature gradients (33, 34, 35, 36, 37, 38, 39 and 40 °C) and ten different time points (0, 2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20 and 22.5 min) to identify the optimal conditions. After optimizing the initial three conditions, seven different magnesium concentrations (0, 2.8, 5.6, 8.4, 1.2, 14 and 16.8 mM) were evaluated. Four types of membranes with pore sizes and capillary ascent rates were used, with manufacturer-provided rates of fluid flow: CN-140 (134.3 s/40 mm capillary speed down web), CN-95 (96.9 s/40 mm), M-90 (80–100 s/40 mm) and M-180 (175–185 s/40 mm). The material showing the highest fluorescence intensity was selected as the final condition for the test strip.
The sensitivity of quintuple RPA-EuNP-LFSBs was determined using five pathogenic bacteria in the mid-exponential growth phase. The bacterial strains were diluted to 10 0 CFU/mL, 10 1 CFU/mL, 10 2 CFU/mL, 10 3 CFU/mL, 10 4 CFU/mL, 10 5 CFU/mL, 10 6 CFU/mL, 10 7 CFU/mL and 10 8 CFU/mL. The five reference pathogens were mixed at an equal volume concentration level. In the negative control, the DNA template was substituted with an equal volume of double-distilled water. In the specificity experiment, the quintuple RPA-EuNP-LFSBs was assessed with the DNA extracted from 40 bacterial strains ( ). The reaction was performed under the optimal reaction conditions of quintuple RPA-EuNP-LFSBs. Each experiment was performed independently three times, and each test strip was scanned three times.
The samples tested in this study were sourced from local supermarkets in Hangzhou, China, including chicken, pork, beef, lamb, duck and milk. According to the bacteriological analytical manual (BAM) for L. monocytogenes , S. aureus , S. enterica and E. coli O157:H7 , all food samples were certified as negative for four pathogens. Then, 225 mL of buffered protein water (BPW) was added to each sample (25.0 g ± 0.1 g or 25.0 mL ± 0.1 mL) under sterile conditions to culture L. monocytogenes , S. aureus , S. enterica and E. coli O157:H7 . According to the national standard for S. suis detection , the test sample was determined to be negative. As mentioned above, 225 mL of Todd Hewitt broth (THB) was added to the sample under sterile conditions to culture S. suis . All samples were homogenized using a homogenizer at 9000 rpm for 2 min. Then, reference bacteria (the IDs of strains: ATCC 19115 for L. monocytogenes , ATCC 25923 for S. aureus , ATCC 700794 for S. suis , ATCC 13076 for S. enterica and ATCC 35150 for E. coli O157:H7 ) with concentrations of 10 4 CFU/mL, 10 3 CFU/mL, 10 2 CFU/mL and 10 1 CFU/mL were added to each sample homogenate. The food samples from each group were extracted using an Auto-Pure Mini, and the obtained DNA was used as the template for RPA. The experiment was divided into two groups. One group was only inoculated with one target strain. Another group was inoculated with five target strains simultaneously. The quintuple RPA-EuNP-LFSB testing was performed under optimal reaction conditions. The fluorescence reader read the fluorescence intensity, and the sample recovery rate was calculated.
The utility of using quintuple RPA-EuNP-LFSBs as a surveillance tool for detecting L. monocytogenes , S. suis , S. aureus , S. enterica and E. coli O157:H7 in food was assessed. Six types of food samples were randomly purchased from local markets (Hangzhou, China), including chicken, pork, beef, lamb, duck and milk. All food samples were verified as being free of the target pathogenic bacteria. Then, all samples were weighed to 25.0 g ± 0.1 g or 25 mL ± 0.1 mL. Homogenate was prepared in 225 mL BPW or 225 mL THB. The mixture was oscillated at 37 °C and 200 rpm. After incubation for 16 h, 1 mL of enrichment mixture was extracted. The pathogens were determined using quintuple RPA-EuNP-LFSBs and BAM methods.
All measurements were performed in triplicate for each experiment, and all strips were read three times with a fluorescent strip reader. Data were exported through the fluorescent strip reader software (Suzhou Helmen Precise Instruments, Suzhou, China). The T/C value, which is the ratio between the T line and the C line, was calculated using Microsoft Excel software (Microsoft Inc., Redmond, WA, USA).
3.1. Assay Principle 3.2. Optimization of the Quintuple RPA-EuNP-LFSBs 3.3. Sensitivity and Specificity 3.4. Detection of Quintuple RPA-EuNP-LFSBs in Artificially Contaminated Food 3.5. Detection of Quintuple RPA-EuNP-LF/SBs in Actual Samples The quintuple RPA-EuNP-LFSBs is a membrane-based sensor for detecting L. monocytogenes , S. suis , S. aureus , S. enterica , and E. coli O157:H7 in contaminated food. The principle involved the RPA amplification of target fragments followed by visualization on LFSBs ( a). The hlyA gene of L. monocytogenes , nuc gene of S. aureus , gdh gene of S. suis , fimY gene of S. enterica and rfbE gene of E. coli O157:H7 have been used as target genes of the five zoonotic foodborne pathogens in previously reported assays . In this study, the five forward primers were labeled with Cy5, FAM, TET, TAMRA and biotin at the 5′ end. All reverse primers were tagged with digoxin ( ). After multiplex RPA amplification by using a TwistAmp Basic Kit, five specific products were generated in a single tube: Cy5-digoxin-, FAM-digoxin-, TET-digoxin-, TAMRA-digoxin- and biotin-digoxin-tagged double-stranded DNA. As shown in b, EuNPs were functional microspheres with chemical stability and a high lanthanide-specific fluorescence ratio . The EuNPs had the advantages of good stability, high labeling efficiency and sensitivity . The bindings of EuNPs and anti-digoxin monoclonal antibody ( c) were combined with the labeled duplex DNA on the conjugate pad. Then, the conjugates were transported to the NC membrane by capillary force. The different products were captured by anti-Cy5 monoclonal antibody (for detection of L. monocytogenes in T1), anti-FAM monoclonal antibody (for detection of S. aureus in T2), anti-TET monoclonal antibody (for detection of S. suis in T3), anti-TAMRA monoclonal antibody (for detection of S. enterica in T4) and anti-biotin monoclonal antibody (for detection of E. coli O157:H7 in T5), in the five test lines. The remaining EuNP-mAb was immobilized by the anti-mouse polyclonal antibody (pAb) on the control line. For positive samples, the visible test line formed on the NC membrane. Additionally, when there were no amplification products, the C line was always visible. As shown in d, the strips can be qualitatively evaluated by the naked eye under a handheld UV lamp (365 nm). Furthermore, a fluorescent strip reader can be used for quantitative measurement. The fluorescence signals of the T and C lines were collected and analyzed using the portable instrumentation. These signals were converted into standard curves based on the T/C value and the contents of the substance.
RPA primer concentration, reaction time and temperature, Mg 2+ concentration and the selection of NC membrane material were systematically optimized through a series of experiments, resulting in the achievement of better detection efficiency and sensitivity of the quintuple RPA-EuNP-LFSBs. The concentration of primers was found to be a critical factor affecting the efficiency and specificity of RPA reactions in previous studies . An inadequate primer concentration was shown to reduce the speed and yield of the RPA reaction. Conversely, an excessively high primer concentration resulted in non-specific amplification and primer dimer formation . With reference to previous experiments , a single RPA-EuNP-LFSBs of 150 nM was used as the initial primers concentration. The quintuple RPA-EuNP-LFSBs were optimized at 150 nM–450 nM. As shown in a, the primer concentration was divided into seven groups, and the T/C values gradually increased as the primer concentration increased. The amplification efficiency reached consistency when the primer concentration was 450 nM for L. monocytogenes , 400 nM for S. aureus , 450 nM for S. suis , 400 nM for S. enterica and 400 nM for E. coli O157:H7 . In addition, the optimum temperature for the RPA reaction was generally 37–42 °C, and the optimum reaction time was generally between 10–25 min . By optimizing parameters such as temperature and time, the sensitivity and specificity of the RPA technology could be improved to achieve efficient and accurate environmental analysis . As shown in b, the best T/C value was found in the temperature range 37–39 °C, and 37 °C, with a lower energy consumption, was selected for subsequent detection, in the eight groups of reaction temperature optimization. Additionally, the reaction temperature could be easily maintained through various methods such as heating, using a water bath, or relying on human body temperature. As shown in c, the RPA reaction times of the 10 groups (0–22.5 min) were determined, and there was no significant difference between 15 min and 22.5 min. In order to provide maximum sensitivity while minimizing the measurement time, 15 min was used in the subsequent experiments. The amplification products were detected after incubating at 37 °C for 15 min. To corroborate the optimized reaction conditions, including RPA primer concentration, reaction temperature and time, the results of electrophoresis gel are shown in . When conventional PCR is selected to identify pathogenic bacteria, the reactions must be completed in less than 1 h through a specific thermal cycler . The quintuple RPA-EuNP-LFSBs involve an isothermal reaction and do not rely on instrumentation, thereby reducing their operation time compared with PCR-based detection. The concentration of Mg 2+ in RPA is believed to affect the amplification efficiency, as suggested by previous studies . Therefore, optimizing the Mg 2+ concentration was essential for multiple RPA reactions to occur efficiently. As shown in d, five pathogenic bacteria showed superior results in the range 14 mM–16.8 mM, and 14 mM was identified as the optimal concentration of Mg 2+ for RPA. Research has indicated that the adsorption capacity of surface antibodies varies depending on the type of NC membrane material used . Different types of NC membranes have different porosity and flow rates, which significantly affected the results of the quintuple RPA-EuNP-LFSBs. Four groups (M90, M180, CN95, CN140) were selected as the NC membranes with different materials to compare the fluorescence intensity. The results are shown in e; CN140 has a higher sensitivity and higher fluorescence signal intensity than the other materials.
The optimized parameters were used to evaluate the sensitivity of quintuple RPA-EuNP-LFSBs. Five pathogenic bacteria were 10-fold serially diluted from 10 8 to 10 0 CFU/mL. The same concentration levels of the five bacterial solutions were mixed together in equal volume. The fluorescence reader was used to quantitate the digital signals of the C and T lines, and the standard curves were established. The experiments were repeated three times. As shown in , the T/C value, the ratio of fluorescence signal intensity, increases with a high concentration of template DNA. There were no distinct detection lines when the concentration was below 10 1 CFU/mL. The visual detection limits were 1.5 × 10 1 CFU/mL for L. monocytogenes , 3.2 × 10 1 CFU/mL for S. aureus , 2.2 × 10 1 CFU/mL for S. suis , 1.9 × 10 1 CFU/mL for S. enterica and 1.7 × 10 1 CFU/mL for E. coli O157:H7 . Therefore, the average sensitivity of quintuple RPA-EuNP-LFSBs was 10 1 CFU/mL. The correlation coefficients (R 2 ) for each variable are as follows: R 2 = 0.9852 for L. monocytogenes , R 2 = 0.9678 for S. aureus , R 2 = 0.9708 for S. suis , R 2 = 0.9719 for S. enterica , and R 2 = 0.9611 for E. coli O157:H7 . Biosensors based on nucleic acid amplification ( ) [ , , , , , , , , ] have been applied for the detection of pathogenic bacteria, such as pNC-based strip biosensors, electrochemical biosensors and SERS-based LF strip biosensors. Compared with traditional methods, these biosensors have the advantages of simplicity, sensitivity and specificity. However, they are insufficient when faced with the requirement of multiple targets in the field. In this paper, the quintuple RPA-EuNP-LFSBs provide a multi-objective, highly sensitive, synchronous and rapid detection tool for testing zoonotic foodborne pathogens. The important indexes with which to evaluate the efficiency of the detection methods are specificity and accuracy . A total of 20 bacterial target strains ( ), including L. monocytogenes (n = 4), S. aureus (n = 4), S. suis (n = 4), S. enterica (n = 4), E. coli O157:H7 (n = 4) and 20 other non-target pathogens, were used to verify the specificity of quintuple RPA-EuNP-LFSBs. The quantitative results obtained with a fluorescence reader for the five target strains are presented in a. The lateral flow strip photograph captured under UV light is shown in b. The results indicate that only the first 20 target pathogens showed positive signals in the detection, while the 20 non-target pathogens showed no signal. This indicated that quintuple RPA-EuNP-LFSBs were specific to their corresponding targets.
The quintuple RPA-EuNP-LFSBs successfully identified six types of spiked food samples, i.e., chicken, pork, beef, lamb, duck and milk. To simulate multiple strains in food samples, we prepared a concentration of 1.9 × 10 4 CFU/mL for L. monocytogenes , 3.8 × 10 4 CFU/mL for S. aureus , 2.4 × 10 4 CFU/mL for S. suis , 2.2 × 10 4 CFU/mL for S. enterica and 1.9 × 10 4 CFU/mL for E. coli O157:H7 , and diluted them from 10 4 to 10 1 CFU/mL. Equal volumes of the same concentration of target bacteria were added into the samples for artificial contamination. The experiments were divided into two groups: single contamination and contamination with five strains. The artificial contamination of the individual strains in the samples, with recovery rates of 91.6 to 101.1% for the spiked samples, is shown in . The co-existence of the five target bacteria in food samples and their recovery rates in spiked samples of 90.6 to 101.6% are presented in . Compared to other biosensor detection methods ( ), the quintuple RPA-EuNP-LFSBs were able to detect the lowest detection limit (10 1 CFU/mL) of five bacteria in various food sample matrices. These results demonstrate that the method was able to accurately and consistently differentiate the spiked samples.
To further verify the capability and accuracy of quintuple RPA-EuNP-LFSBs, we evaluated 15 food samples including chicken, pork, beef, lamb, duck and milk. All food samples were extracted for genomic DNA and subjected to quintuple RPA-LFIA, BAM or national standard detection, and the results were consistent as shown in . The positive detection rate of S. aureus was 6.7%, the positive detection rate of E. coli O157:H7 was 13.3%, and the remaining three strains were negative. The quintuple RPA-EuNP-LFSBs demonstrated good performance in actual samples detection, which made them more suitable for field detection or detection in areas with resource shortages. In addition, the quintuple RPA-EuNP-LFSBs were more cost effective, with the estimated cost per reaction of around 12 USD, than the test price of real-time PCR detection kit on the market.
In this paper, we presented the development of quintuple RPA-EuNP-LFSBs, which utilized europium nanoparticles, recombinant polymerase amplification and a lateral flow strip biosensor. This innovative approach offers rapid DNA extraction, amplification of target genes and the capability to detect five pathogenic bacteria simultaneously. The rapid synchronous amplification and visual judgment of multi-objective results for five zoonotic foodborne pathogens ( Listeria monocytogenes , Staphylococcus aureus , Streptococcus suis , Salmonella enterica and Escherichia coli O157:H7 ) were successfully conducted via one-tube RPA and demonstrated the capability of detecting multiple targets. The entire process was completed within 20 min (including 5 min for test strip display) at 37 °C using optimized key parameters. The average sensitivity of the quintuple RPA-EuNP-LFSBs reached 10 1 CFU/mL. The recoveries of the five pathogens ranged from 90.6 to 101.6% in the spiked sample experiments. Furthermore, the actual sample detection results were consistent with those of culture assay. In summary, the proposed quintuple RPA-EuNP-LFSB method was designed for its ease of use and excellent fluorescence performance, enhancing its practicality and availability. It can not only achieve the purpose of simple, sensitive and specific detection, but also provides an effective technical means for the field inspection of multiple zoonotic diseases with good market promotion prospects.
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The preclinical study of biocompatibility of tyrosine polycarbonate bioresorbable scaffold in small caliber porcine peripheral arteries | fc239446-e1d0-44f0-951e-4deb36a5b1ce | 11950639 | Cardiovascular System[mh] | Peripheral arterial disease (PAD) remains a major challenge for healthcare systems across the globe with over 200 million people affected worldwide . The number is set to grow further due to the aging population. PAD’s most severe manifestation, chronic limb-threatening ischemia (CLTI), will potentially occur in up to 11% of patients requiring urgent intervention to prevent imminent limb loss . The advances in techniques and technology facilitated the increased use of endovascular methods over bypass surgery . The drug-eluting stents (DESs) proved to be superior to balloon angioplasty and bare-metal stents in preserving patency for up to 12 months in infra-popliteal lesions , . Although the acute results have substantially improved, the restenosis rate remains high in the long term, particularly in the below-the-knee (BTK) patients’ subset . Furthermore, the presence of a permanent metallic scaffold implicates several significant limitations. The rigid implant affects the vessel by impairing the vasomotor tone, inducing endothelial dysfunction, chronic inflammation, and consequently excessive late lumen loss , . Furthermore, the distinctive anatomy of the infra-popliteal region creates concern over excessive external force leading to stent compression and infrequently fracture . Metallic stent placement in the BTK artery may preclude future bypass surgery underscoring the need for a well-weighted decision . The drug-eluting resorbable scaffolds (DRS) were created to overcome the limitations of traditional devices. The intriguing “leave nothing behind” concept sparked an interest in the initial generation of PLLA-made devices in the coronary territory. The rapid rise of DRS in coronary application was suddenly halted by reports of increased rates of stent thrombosis with the leading PLLA-based scaffold as compared with contemporary DES , . As a result, all DRS in development at the time have been prematurely stigmatized with a class-failure label, even though many were technologically different, and some used entirely different materials such as metallic alloys or tyrosine polycarbonate (Tyrocore) . Although the concerns about negative long-term safety outcomes proved largely unfounded with these non-PLLA based coronary scaffolds , , the use of DRS in coronary applications remains very limited. However, the favorable potential of DRS in BTK arterial disease has been recognized – . The recently presented results of LIFE BTK confirmed that DRS are not just an interesting theoretical concept, but a viable treatment option in peripheral arteries, showcasing their superiority in terms of efficacy, while retaining a similar safety profile to that of balloon angioplasty . The favorable outcomes translated into the recent FDA approval that was granted to Espirit BTK (Abbott) for the treatment of below-the-knee lesions. To that end, we present preclinical results of novel MOTIV bioresorbable scaffolds for the treatment of BTK lesions.
The study hypothesized that the Tyrocore-based scaffold validated in coronary clinical use retains its early integrity and biocompatibility in a more biomechanically demanding peripheral vasculature, even at longer lengths, previously not evaluated. Study device Experimental model Accordance statement The study is reported in accordance with ARRIVE guidelines. Experimental procedure The MOTIV scaffold (REVA Medical, Inc. San Diego, CA) is a balloon-expandable, sirolimus-eluting, open-cell scaffold design (Fig. ) made from Tyrocore, a novel polycarbonate polymer comprised of analogs of the natural amino acid tyrosine (desaminotyrosine) and biocompatible hydroxy- esters (e.g., lactic acid). In comparison to previously published properties of poly-L-lactic acid (PLLA), Tyrocore features higher ultimate tensile strength (100–110 MPa vs. 50–70 MPa for PLLA) and elongation (ductility, 150–200% vs. 2–10% for PLLA) . The radiopacity of the scaffold structure is equivalent to currently marketed metallic stents. After implantation, the scaffold elutes a substantial portion of sirolimus within 30 days. The coating used for the drug elution is the same polymer that is used to make the scaffold platform, applied abluminally to elute sirolimus at 1.97 µg/mm 2 dose density. Tyrocore polymer features a steady resorption profile. One year after implantation, the vessel becomes uncaged, and the material fully resorbs within 4 years. The following MOTIV stent sizes (strut thickness) and lengths were used in the study to allow for testing of both short and long scaffolds: 2.5 mm (95 μm) x 24 mm; 3.0 mm (105 μm) x 12, 18, 24, 36 and 48 mm; 3.5 mm (115 μm) x12, 18, 24, 36, 48 and 60 mm.
The study was approved by the Institutional Animal Care and Use Committee and conducted in accordance with the Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals (National Research Council, National Institutes of Health publication no. 85 − 23, revised 1996) at U.S. Department of Agriculture-licensed, Association for the Assessment and Accreditation of Laboratory Animal Care International-accredited animal research facility.
The study is reported in accordance with ARRIVE guidelines.
A total of nine (9) Yorkshire swine were enrolled in the study. All animals were sourced from an accredited vendor (Manthei Hog Farm; 23130 112th St NW, Elk River, MN 55330). Three (3) animals were implanted with the test article in naïve peripheral arteries and survived to 30 days (Group 1), and six (6) animals survived to 90 days (Group 2). All procedures were performed under general anesthesia maintained with the use of isoflurane in 100% O 2 (inhalation; 0–5%) and propofol (IV.; 2–8 mg/kg). The exact distribution of animals and scaffolds among animals and time points is presented in Table . Scaffolds were implanted with a targeted balloon-to-artery ratio of 1.1:1 into the iliac arteries using online Quantitative Vascular Angiography (QVA). Optical Coherence Tomography (OCT) imaging was performed post-implantation to confirm good apposition to the arterial wall. In absence of endovascular imaging-guided sizing, 9/20 scaffolds appeared undersized in post-implant OCT and were post-dilated to optimize apposition. Group 1 and 2 animals underwent OCT imaging on all implanted vessels at post-implant and termination, excluding the ones designated for Scanning Electron Microscopy (SEM) (5 vessels in total, two (2) from Group 1 and three (3) from Group 2). Following termination (euthanasia was performed under general anesthesia with the use of pentobarbital sodium and phenytoin sodium solution) vessels containing the scaffolds were subjected to histology analysis.
OCT pullbacks were performed at baseline (post-implant) and pre-termination at 30 and 90 days using Abbott’s ILUMIEN OPTIS system and the Dragonfly™ Duo catheter. Images were analyzed by an in-house core laboratory (CRF Skirball Center for Innovation, Orangeburg, NY) using commercial software (ILUMIEN OPTIS, Abbott, Santa Clara, CA). Lumen area and the inner and outer scaffold areas, and corresponding diameters were measured planimetrically after calibration. Percentage area of stenosis (% AS), neointimal area (NA) and neointimal thickness (NIT) were derived from these measurements. The %AS was calculated according to the formula: %AS = (neointimal area/inner scaffold area) x100%. Neointimal area was calculated as inner scaffold area – lumen area. Neointimal thickness was calculated as inner scaffold diameter – lumen diameter. The ratio of uncovered struts (ROUS) refers to the ratio of uncovered struts to the total number of struts. Covered struts were defined as having ≥ 20 μm of neointima over the strut. A ratio of covered struts ≥ 95% was considered acceptable for all devices. Late structural strut discontinuity was assigned if either of these two conditions existed: (1) two struts overhung each other in the same angular sector of the lumen perimeter, with or without malapposition, covered or uncovered; or (2) there was an isolated, covered or uncovered strut(s) located more or less at the center of the vessel, without obvious connection with other surrounding struts in OCT. Scaffold fracture was assumed in the case of overriding contiguous struts, disconnection from the expected device circularity, and isolated struts lying in the lumen. Malapposition refers to a strut that is not in contact with the artery wall. Malapposition was considered when the axial distance between the strut’s surface and the luminal surface exceeded the strut thickness (95 μm for 2.5 mm; 105 μm for 3.0 mm; 115 μm for 3.5 mm scaffold). A strut that appeared non-apposed in the side branch regions, which often have geometry distortion, was excluded from the analysis. Malapposition distance, location, the total number of malapposed struts for the device, and averages for each group were reported. Of note, the OCT properties of the Tyrocore polymer are remarkably different from those of PLLA scaffolds; the abluminal surface of the strut is strongly reflective of light, resulting in a single line resembling a metallic strut reflection. As such, the visualization of the entire strut (and scaffold) footprint in the “box” form previously characterized for the PLLA scaffolds is not possible. Histological preparation All peripheral arteries implanted with the scaffolds were collected and perfusion-fixed with 10% NBF. The scaffolded arterial segments, as well as proximal and distal reference regions were processed and embedded in SPURR media, sectioned at three levels: proximal, middle, and distal, and stained with hematoxylin and eosin (H&E) and elastin trichrome (ET).
The scaffold-containing coronary arteries designated for SEM were fixed en-bloc and later trimmed into two longitudinal sections. Each long section was processed and mounted onto an SEM stub for imaging. Digital SEM imaging was performed using a JEOL JSM-6610 microscope.
Photomicrographs of histological ET-stained sections were measured with computer-assist software (Image-Pro® Plus 7). Measurements included the cross-sectional areas for external elastic lamina (EEL), internal elastic lamina (IEL), percent area stenosis (%), average neointima thickness (µm), neointimal area (NA, mm 2 ), and medial area (MA, mm 2 ). The derivative parameters were calculated as follows: percent area stenosis (based on internal elastic lamina area - IELA) = (1 − LA/IELA) × 100; average neointima thickness (µm) = (IELD-LD)/2 × 1000; neointimal area (NA, mm 2 ) = IELA-LA; medial area (MA, mm 2 ) = EELA – IELA. Histological analysis SEM analysis Statistical analysis Blinded analysis of all slides was performed via light microscopy. Slides were evaluated for standard healing parameters, such as injury, endothelialization, inflammation, multinucleated giant cells, residual red blood cell distribution, fibrin, neovascular buds, medial necrosis/hemorrhage/fibrosis, adventitial/perivascular inflammation (severity and distribution) and assessed semi-quantitatively. Other factors analyzed included neointimal maturity, strut-associated calcification, adventitial fibrosis and presence of granulomas. All parameters were interpreted from the H&E and ET-stained slides. Per the consensus document for Drug-Eluting Stents in Preclinical Studies , it is recognized that granulomata occur regularly (typically less than 10% of vessels treated in a study) and vessels affected may be excluded from the analysis.
All luminal regions of each treated vessel were evaluated for endothelial coverage and maturity, thrombus formation, and the presence of inflammatory cells and platelets. Endothelialization coverage and maturity were evaluated over and between struts if an outline of the struts underlying the neointima was visible. Proximal and distal hosts (non-treated segments) were also examined. The evaluation of healing was assessed from images at 50x, 100x, and 250x magnifications. Electron micrographs taken at other magnifications were evaluated if provided. Non-parametric SEM features were scored between 0 and 4 (endothelialization, thrombus, leukocytes, platelets) with scores at or near zero denoting within the normal limits state.
For parametric variables, means ± SD were calculated for each group and assessed with Student t test, or analysis of variance and correction for multiple comparisons. Dichotomous variables such as injury score, endothelialization, and inflammation cannot be averaged but were presented categorically as mean ± SD. Numerical scores from the grading scale were converted to a percentage ranging from 0 to 100%.
All animals (9) were successfully implanted and survived to 30 or 90 days. There were no adverse events reported on treatment day, nor during the observation period. OCT SEM Histopathology Vessel healing Biological response to the scaffold Quantitative histomorphometric evaluation All vessels remain patent at all examined levels at both time points. The average percent area stenosis for the stented segments was 31% (20–36) and 35% (20–57) for 30- and 90-day respectively. One animal from Group 2 demonstrated the highest EEL area and the highest neointimal area, and thus increased percent area stenosis by 57% (Table ).
Following deployment of the devices, OCT was performed on 14 MOTIV scaffolds in 9 animals (no OCT analysis was performed on the SEM cases). An average overstretch ratio of 1.05 ± 0.07 was achieved for Group 1 animals, and 1.02 ± 0.06 for Group 2 animals based on OCT calculations post-implant. Following post-dilation where it was deemed necessary as described in Methods, no evidence of inadequate deployment nor malapposition was observed in the final post-deployment OCT. At termination, all treated segments featured satisfactory patency (Figs. and ). The average neointimal thickness was 0.21 ± 0.05 mm in Group 1 (30 days) and 0.18 ± 0.10 mm in Group 2 (90 days)( p = 0.39); the percent area stenosis (PAS) was 28 ± 7% in Group 1 and 24 ± 11% in Group 2 ( p = 0.35); the neointimal area was 1.7 ± 0.35 and 1.42 ± 0.73 ( p = 0.33) respectively. The neointimal area, thickness, and PAS between the two groups were comparable ( p > 0.050). The outer scaffold area did not differ statistically among time points (Group 1 vs. Group 2–7.82 ± 0.51 vs. 7.52 ± 0.97, p = 0.51 at baseline; 7.23 ± 0.46 vs. 7.05 ± 0.99, p = 0.69 at termination). All struts examined were fully covered by neointima in both Groups. An average total of 56 ± 8 struts were counted in Group 1 and the ratio of uncovered struts (ROUS) was 0%; no malapposition, stent fracture, or late strut discontinuity was observed. The satisfactory structural integrity was also confirmed by post-mortem Faxitron imaging (Fig. ). An average total of 52 ± 5 struts were counted in Group 2 and the ROUS was 0%; no malapposition, stent fracture, or late strut discontinuity was observed. The detailed OCT results are summarized in Table .
SEM analysis of two (2) MOTIV-treated vessels after 30 days demonstrated complete endothelialization (score 4) with no noteworthy platelet, red blood cell, or leukocyte adhesion, no fibrin deposition, and no unresorbed surface thrombus (all score 0). Minor surface artifacts (e.g., dehydration cracking along a strut), as well as endothelial erosion, were occasionally observed. SEM analysis of three (3) MOTIV-treated vessels after 90 days demonstrated complete endothelialization (score 4) with no noteworthy platelet (mostly 0) or leukocyte adhesion, no fibrin deposition, and no thrombosis (all score 0) (Fig. ). One animal consistently demonstrated what was interpreted to be terminal endothelial erosion/denudation artifact and red blood cell pooling. Minor surface artifacts (e.g., surface cracking or peeling) were also occasionally observed. The polymer degradation/resorption was not observed in SEM analysis.
Vessel injury t 30 days the average in-stent injury score was low, indicating a good apposition of the struts to the internal elastic lamina. There were only focal struts that penetrated IEL and were contacting the media, typically within a single quadrant. The 90-day cohort expressed similar results confirming a proper scaffold apposition in all but one vessel that demonstrated severe injury (stent strut penetrating the IEL, media, and EEL) at multiple struts/within multiple quadrants at every stented level. This injury was associated with extensive eosinophilic-rich granulomatous inflammation. The distal stented level had the least overall injury of the examined levels and demonstrated severe injury and accompanying inflammation around ~ 50% of the vessel circumference, with the remaining struts showing no to minimal injury, as well as minimal inflammation (Table ).
Adequate vessel wall healing at both time points was characterized by a typically fully mature neointima and complete reendothelialization at all sites. At 30 days, minimal to moderate fibrin deposition was observed adjacent to the struts and was overall decreased at 90 days with minimal fibrin deposition occasionally observed. No unresorbed luminal thrombus was observed in any vessel (Table ).
Overall, the inflammation scores were low for all vessels (characterized by minimal lymphocytes, macrophages, and multinucleated giant cells) on both time points except for one animal. The average inflammation (excluding multinucleated giant cells [MNGCs]) was 0.6 for the stented vessel segments at 30 days and was characterized by minimal scattered lymphocytes and macrophages (average MNGCs score was 0.9). As for the 90 days cohort, the inflammation scores were generally low with an average inflammation (excluding MNGCs) of 0.8 for the stented segments, characterized by minimal scattered lymphocytes and macrophages (average MNGCs score was 1.0) (Fig. ; Table ). In one animal from 90-day cohort the inflammation was defined by typically confluent or circumferential granulomatous formation, characterized by many granulocytes (predominately eosinophils but with many neutrophils) and macrophages, with fewer multinucleated giant cells and lymphocytes. These eosinophil-rich granulomas were observed adjacent to all struts at the proximal and mid-stented levels and were adjacent to half of the struts within the distal level. At every level, this inflammation was associated with moderate to severe injury in which the strut penetrated the IEL and media and contacted or penetrated the EEL. Extension of inflammation into the adventitia was also observed at every level and was moderate in severity, affecting up to 25% (distal level) or up to 50% (proximal and mid-levels) of the adventitial circumference .
The MOTIV DRS is a novel scaffold designed for the treatment of below-the-knee lesions, characterized by its thin struts, rapid and broad expansion capability, and most uniquely, its radiopacity. The study aimed to evaluate the safety and performance of the MOTIV DRS in the porcine peripheral arteries model. Intravascular imaging confirmed that all scaffolds maintained their device integrity by showing no notable malapposition, fracture, or late strut discontinuity at 30- or 90-days post-termination in the stented internal iliac arteries. All struts were fully covered/embedded at both time points and the vessels remain patent. The measured percent area stenosis was comparable between groups. The average values of 28 ± 6% (30 days) and 24 ± 11% (90 days) respectively are acceptable and comparable with numbers presented for the drug-eluting stents tested in porcine peripheral arteries , . These findings were corroborated by histomorphometry revealing comparable percent area stenosis values except for one vessel that demonstrated the greatest EEL area and greatest neointimal area. This indicates that the intense inflammatory response noted in that vessel was associated with both positive vessel wall remodeling with expansion of the EEL area, as well as neointimal hyperplasia (and thus increased percent area stenosis – 57%). Overall, almost all tested devices showed a very good biocompatibility that was indicated by the low inflammation scores, reflecting minimal infiltration of lymphocytes, macrophages, and multinucleated giant cells. Nevertheless, in one animal mentioned above, an excessive reaction in the form of granulomatous inflammation was described. Similar findings were previously reported and are known to occur regularly in swine and are considered to represent a type IV hypersensitivity reaction . It is also recognized that in humans, late stent thrombosis in sirolimus-eluting stents has been attributed to a similar hypersensitivity reaction . Recent studies suggest biodegradable/bioresorbable-coated polymers may result in reduced late inflammatory reactions and overall better outcomes compared to permanent polymers or bare metal stents but note that different polymer coatings and any significance to clinical use in humans remain to be elucidated . This animal may therefore represent an individual ‘reactor’ demonstrating pig-to-pig variability in immune responsiveness. Microscopic evaluation confirmed a low injury caused by implanted scaffolds. The struts were well apposed and rarely penetrated through the intima to more external layers. At both time points the stents were fully covered with typically fully mature neointima indicating a good vessel wall healing. The described fibrin deposits were minimal to moderate at 30 days’ time point and decreased over time being occasionally observed at longer follow-up. Fibrin is considered a surrogate biomarker for the presence of an anti-proliferative drug since delayed healing is an inevitable consequence of pharmacologic containment of neointimal proliferation with the agent eluted from the scaffold surface. Fibrin deposits have been shown to peak at 1-month post-implant for the Absorb scaffolds in the porcine coronary artery model . MOTIV scaffolds exhibited rather scarce fibrin deposits. This would be a desirable finding as long as it does not occur at the price of decreased anti-restenotic efficacy. However, it does not seem to be the case for the scaffold in the present study, as it demonstrated adequate containment of neointimal response, comparable to the ones reported in other preclinical studies, even with drug-eluting stents , . SEM data provide the most reliable assessment of endothelial coverage of the stented segment of a peripheral artery. SEM analysis relies upon en-face views with suitable magnifications of the entire luminal surface of a bisected vessel, which is more comprehensive than light microscopic evaluations commonly performed on limited numbers of transverse sections ( n = 3) per treated vessel. At 1 and 3 months, the MOTIV-treated vessels exhibited complete endothelial coverage over and between struts with, in general, no noteworthy findings observed (such as thrombosis, fibrin deposition, platelet adhesion, etc.). Minor surface artifacts and endothelial erosion were occasionally described. They can be attributed to the device preparation (dehydration cracking along a strut) and/or terminal angiography performed and are a common finding when utilizing so accurate and high-resolution modality. However, oThese findings can be attributed to the excessive and abnormal inflammatory reaction reported in this animal as described earlier. The positive preclinical findings of the tested scaffold may not be directly translated to the clinical setting owing to the lack of atherosclerotic disease in animals and thus should be interpreted with caution. Nevertheless, they constitute a valuable source of information helping to understand the associated healing process and uncover any potential threats, knowing the shortcomings of the first generation PLLA scaffolds. The described good safety and biocompatibility profile of MOTIV stents is in line with recently presented results of a multi-center study in BTK lesions demonstrating improvement in Rutherford Classification and wound healing (58 patients, 60 study limbs, and 76 Motiv scaffolds) at 24 months (81.7% of stents were patent with a limb salvage rate amounting to 95%) . The Tyrocore-made scaffolds (MOTIV, Fantom) were designed to overcome the limitations of first-generation PLLA DRS. For instance, the literature suggests an unfavorable peristrut rheology for first-generation thick-strut DRS, as compared with metallic stents , . Low shear stress regions and altered flow patterns in between Absorb struts were identified by three-dimensional angiographic reconstruction techniques and computational fluid dynamic data . Low endothelial shear stress has been long recognized as a powerful local stimulus for atherogenesis, formation, and progression of an early atherosclerotic plaque to high-risk plaque . Lastly, an ex-vivo porcine arteriovenous shunt model and a rabbit model of iliofemoral stent implantation demonstrated increased thrombogenicity quantified by platelet aggregation and inflammatory cell adhesion in Absorb when compared to thin strut biodegradable polymer coated DES . The evidence summarized above suggests the key role of lowering strut thickness without loss of radial strength to further improve DRS technology . The struts of MOTIV scaffolds are around 27–39% (dependent on the scaffold size) thinner than the ones of Absorb (95–115 μm vs. 157 μm). Interestingly, despite the disappointing long-term results of first-generation PLLA scaffolds in coronary arteries, the reported long-term outcomes of ABSORB BTK showed primary patency (defined as freedom from peak systolic velocity ratio > 2.0 or target vessel occlusion) and freedom from clinically driven target lesion revascularization rates of 89.2%/80.3%/72.9% and 97.2%/90.7%/90.7% at 12, 36, and 60 months, respectively. The authors reported no late or very late scaffold thrombosis . Even more important, the recently published results of multicenter Life BTK RCT (261 patients with CLTI) comparing the Esprit scaffold (thin strutted Absorb) against angioplasty (randomized in a 2:1 ratio) showed that the DRS constitutes a viable treatment alternative . The accumulating preclinical and clinical data suggest that the BTK arteries might be an appropriate target for bioresorbable technologies. Combined with the proper implantation and vessel preparation technique, such as PSP (predilation, sizing, and post-dilation) achieving positive long-term results was possible even with the discontinued first-generation scaffolds. On this background, gradual improvement on the shortcomings of the early technologies has allowed progress with current generation of devices for this very challenging arterial territory. Limitations Some study limitations must be acknowledged. The relatively small number of animals and devices evaluated should be considered when interpreting the results. The 30- and 90-day interim data do provide a snapshot of the healing profile and have been previously accepted as sufficient preliminary preclinical evidence for novel DRS , , but the follow-up of 3 months is now considered quite short for animal studies of DRS given the multi-year degradation process and the delayed nature of adverse clinical sequelae identified for Absorb. However, it needs to be emphasized that an earlier generation coronary device - the Fantom scaffold, made of a similar tyrosine-based polymer, was previously studied preclinically out to 4 years revealing no concerns . Additionally, recently presented 5-year clinical outcomes of the FANTOM II study corroborated the long-term device safety with MACE rate, target lesion failure, and scaffold thrombosis reported in 6.3%, 5.8%, and 1.3% patients respectively . Furthermore, as described previously, MOTIV scaffolds identical to the one studied in the present study demonstrated a favorable 24-month clinical safety profile and angiographic results . Secondly, although the porcine peripheral arteries model is a well-established and desired one , the implantation of scaffolds in native vessels of young animals without atherosclerotic disease may limit the ability to directly translate the preclinical results to clinical settings. However, the previously mentioned clinical data do support our findings.
Implantation of the MOTIV DRS up to 60 mm long in small-caliber peripheral arteries of swine resulted in 100% patency rate and adequate vascular healing at 30-day and 90-day timepoints. The Tyrocore-based DRS retained their integrity and radial strength throughout the course of the study and confirmed their favorable biocompatibility in small-caliber porcine peripheral arteries thus confirming the study hypothesis. The scaffolds retained their necessary structural integrity (and thus also, presumably, sufficient radial strength) throughout the course of the study and confirmed their safety in the preclinical setting. Combined with positive clinical results, the MOTIV DRS has a chance to constitute an alternative for the treatment of below-the-knee lesions, adding to currently limited interventional armamentarium.
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Exploring the incidence and influencing factors of postoperative nausea and vomiting after laparoscopic bariatric surgery: a protocol for a retrospective observational study | 1fc09742-5f6a-4177-aaee-0ae5ebbf773e | 11748921 | Laparoscopy[mh] | Obesity, an escalating global health epidemic, poses a profound threat to the well-being of individuals worldwide. As reported by the WHO in 2022, 2.5 billion adults aged 18 years and older were overweight, with 890 million individuals classified as obese. Exceeding 390 million children and adolescents aged 5–19 years were overweight, with 160 million of these individuals being obese. Obesity is a principal contributor to mortality and leads to a myriad of complications, including, but not limited to cancer, sleep apnoea, respiratory distress, type II diabetes, hypertension and cardiovascular diseases. Traditional interventions, such as lifestyle modifications through exercise and dietary restrictions, have been demonstrated as insufficient in managing severe obesity. Bariatric surgery is widely acknowledged as the most effective treatment for obesity and its associated complications, with an increasing number of patients opting for this procedure. Nevertheless, postoperative nausea and vomiting (PONV) is a prevalent issue among patients undergoing bariatric surgery, with an incidence rate ranging from 60% to 90%. Despite the implementation of prophylactic measures, a substantial proportion of patients, approximately 50–70%, still experience PONV. The occurrence of PONV poses a risk for a range of serious complications, including aspiration, suture line disruption, anastomotic leaks, malnutrition, postoperative haemorrhage, subcutaneous emphysema, pneumothorax, increased intragastric pressure, venous hypertension, dehydration and electrolyte imbalances. These complications can prolong hospital stays and increase healthcare expenditures due to extended recovery periods. Moreover, PONV significantly affects patients' comfort and satisfaction, emerging as a leading cause of readmission following bariatric surgery. Therefore, identifying the risk and protective factors for PONV following bariatric surgery is of paramount importance to facilitate early and effective prevention strategies for PONV. Previous studies have identified high-risk factors for PONV following bariatric surgery, which primarily include patient factors (such as female gender, younger age, non-smoking status, history of alcohol abuse, history of motion sickness or PONV, preoperative reflux symptoms, body mass index and complications such as diabetes and sleep apnoea), anaesthesia techniques (including opioids, volatile anaesthetics, neostigmine and prolonged anaesthesia duration), surgical factors (such as surgery duration, induced pneumoperitoneum, antrectomy, increased intragastric pressure, proximal gastric pouch expansion and gastropexy), intraoperative hypotension, postoperative recovery status and pain severity. In recent years, studies have identified several potential measures to reduce the incidence of PONV following bariatric surgery. These measures encompass the use of pharmacological agents (such as dexamethasone, sugammadex, propofol, aprepitant and dexmedetomidine), opioid-free analgesia and multimodal analgesia, total intravenous anaesthesia in lieu of volatile anaesthetics, multimodal antiemetic therapy and goal-directed fluid therapy. The majority of previous studies on PONV in the context of bariatric surgery have focused on patients who have undergone laparoscopic sleeve gastrectomy. However, no study has yet examined the incidence and influencing factors of PONV across all types of laparoscopic bariatric surgeries. Consequently, this study aims to explore the occurrence and associated factors of PONV following all types of laparoscopic bariatric surgeries.
Study design and setting Inclusion and exclusion criteria Primary outcome Planned secondary outcomes Outcome measure Potential related factors Data collection methods Data management Statistical analyses Statistical method Missing data Sensitivity analysis is is a retrospective, single-centre observational study that will be conducted at the Third People’s Hospital of Chengdu. This study will retrospectively include patients who underwent all types of laparoscopic bariatric surgeries at our centre from December 2019 to April 2024. At our centre, all types of bariatric surgeries are performed with laparoscopy. Refer to for an overview of data sources and study processes.
The study will include adult patients who underwent elective laparoscopic bariatric surgery with general anaesthesia at the Third People’s Hospital of Chengdu from December 2019 to April 2024. The exclusion criteria will be as follows: patients under the age of 18 years or over 60 years, patients with a history of drug addiction, patients with inflammatory bowel disease and patients lacking data on PONV.
The primary outcome is the factors associated with PONV in patients who underwent laparoscopic bariatric surgery under general anaesthesia.
The planned secondary outcomes include the correlation between PONV and postoperative recovery parameters, such as postoperative fever, postoperative ICU (Intensive Care Unit) admission, duration of resumption of feeding and length of hospital stay after surgery in patients who underwent laparoscopic bariatric surgery under general anaesthesia.
Patients will be considered to have PONV if they develop symptoms of nausea and/or vomiting and receive rescue antiemetic measures within 24 hours postsurgery. The rescue antiemetic measures are detailed in .
Based on the literature review, clinical practice and the available data, we plan to include the following potential factors associated with PONV in our analysis. The planned potential factors are detailed in . Box 1 Planned potential factors associated with PONV Factors Age (years) Sex Body mass index (kg/m 2 ) ASA (American Society of Anesthesiologists) classification Prohibition period (hours) Fasting time (hours) Apfel risk score Reflux oesophagitis Hiatal hernia of oesophagus Helicobacter pylori infection Arrhythmia Hypertension Cardio dysfunction Diabetes Impaired glucose tolerance Apnoea Bronchitis Anaemia Liver dysfunction Renal dysfunction Nerve block Types of surgery Midazolam Penehyclidine Lidocaine Dexmedetomidine Venous general analgesia drug Total intravenous anaesthesia Opioids NSAIDs (Non-Steroidal Anti-Inflammatory Drugs) Positive inotropic drugs Vasoconstrictor drugs Neostigmine Intraoperative prophylactic medication Crystalloid-to-colloid ratio Average liquid (mL/kg/hour) Blood loss (mL) Operative time (min) Anaesthesia time (min) Analgesic pump usage Postoperative moderate-to-severe pain Postoperative prophylactic medication Note: Tthe Apfel risk score includes female gender, a history of motion sickness or PONV, non-smoking status, and postoperative opioid use. The types of surgery include laparoscopic sleeve gastrectomy (LSG), laparoscopic sleeve gastrectomyLSG plus duodenojejunal bypass (LSG-DJB), laparoscopic sleeve gastrectomyLSG plus jejunojejunal bypass (LSG-JJB), laparoscopic Roux-en-Y gastric bypass (LRYGB), laparoscopic one-anastomosis gastric bypass (LOAGB).
Data on patients undergoing laparoscopic bariatric surgery from December 2019 to April 2024 will be extracted from the DoCare Anesthesia Clinical Information Management System (Suzhou Medical System Technology, V.5.0), the iPainfree pain management information system software (Jiangsu Apon Medical Technology, V.1.0) and the case information system (Union Digital Medical Record browser, V.2012.4).
Data from all participants will be available in the electronic medical record database of the Third People’s Hospital of Chengdu and will be simultaneously cross-validated and transcribed into Microsoft Excel by two researchers. These records will be securely stored on a password-protected computer.
Sample size Based on the annual volume of bariatric surgeries performed at the Third People’s Hospital of Chengdu, we conservatively estimate that the final data will include over 2500 patients. This cohort will represent the largest exploration to date of factors contributing to PONV following laparoscopic bariatric surgery.
Categorical covariates will be summarised by frequency and percentage, while continuous covariates will be described by mean and SD, or median (25th–75th percentile) for those not adhering to a normal distribution. Univariate logistic regression will be used to identify potential risk or protective factors associated with PONV. Variables with p values from the univariate logistic regression no more than 0.10, along with those deemed clinically significant, will be incorporated into a multivariate logistic regression model. Factors with p values <0.05 in the multivariate logistic regression analysis will be considered as independent risk or protective factors for PONV. The outcomes of the regression analyses will be expressed as OR and 95% CIs. The variance inflation factor will be computed to evaluate the multicollinearity. All analyses were performed using R (V.4.3.3; R Development Core Team, Vienna, Austria).
We will initially attempt to manually fill in the missing data. If data remain absent, variables with a missing rate exceeding 20% will be excluded from the analysis; those with a missing rate of 20% or less will be addressed through five imputations. Continuous variables were imputed using predictive mean matching, whereas binary variables were imputed by using logistic regression models. The multiple imputation procedure will be performed by mice package in R (V.4.3.3; R Development Core Team).
Sensitivity analysis will be conducted by performing the primary analysis—univariate and multivariate logistic regression as mentioned above—on a complete dataset, which was created by excluding patients with missing data. This approach will assess the robustness of our findings and mitigate potential biases that may arise from missing data.
The incidence of PONV following bariatric surgery, which ranges from 60% to 90%, is markedly higher than that observed after general surgery, with rates fluctuating between 10% and 40%. Despite this disparity, there are no evidence-based guidelines tailored specifically for the prevention of PONV in patients undergoing bariatric surgery. The identification of risk factors for PONV is crucial, as it empowers anaesthetists to implement targeted preventive strategies. Consequently, we intend to conduct a retrospective observational clinical study to investigate these risk factors and to evaluate the efficacy of various preventive interventions. This study, which will be the largest of its kind, will undertake a comprehensive analysis of potential risk factors as reported in the previous literature, thereby enhancing the statistical power of our findings. Furthermore, this research will be the first investigation to encompass all types of laparoscopic bariatric surgeries, facilitating a more nuanced understanding of the risks and preventive factors associated with PONV in the laparoscopic bariatric surgery setting. This study will further validate the controversial factors associated with PONV in previous research, including gender, age, obesity, smoking history, opioid medications, surgery duration and anaesthesia duration. Additionally, this research will investigate new potential factors related to PONV, including preoperative comorbidities across various systems, types of bariatric surgeries, neuromuscular blocking agents, vasoactive drugs, and the crystalloid-to-colloid fluid ratio. The findings of this study could be instrumental in the design of prospective studies and may provide evidence-based support for the development of future PONV guidelines for this patient population. This retrospective study has several limitations. First, missing data are inevitable and will interfere with the results of such retrospective research. Sensitivity analysis will be conducted to verify the robustness of the results. Second, the observational nature of the study may have potential unmeasured confounding factors, which necessitates a cautious interpretation of the observed associations. Despite these limitations, retrospective studies can provide valuable insights and generate hypotheses for further exploration in prospective studies employing more rigorous methodologies.
The institutional review board of the Third People’s Hospital of Chengdu approved this study (approval number: 2024 S-252). Informed consent was waived by the ethics committee, and all data were anonymised prior to the study authors’ visit. Registration has already been completed in the Chinese Clinical Trial Register (ChiCTR) under registration number ChiCTR2400088738. The results will be presented at conferences and published in a peer-reviewed journal. Trial status Data collection commenced on manuscript submission. The analyses are planned to be completed by the end of 2024.
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A silent pandemic of violence against providers in obstetrics and gynecology: A mixed‐methods study based on a global survey | 50c2c835-4803-4a2e-9688-c865d525f74c | 11649870 | Gynaecology[mh] | INTRODUCTION Workplace violence (WPV) directed against healthcare providers is a major public health concern, of consequence not only to providers but to the healthcare system as a whole. According to WHO, WPV is an incident where staff are abused, threatened, or assaulted in circumstances related to their work, involving an explicit or implicit challenge to their safety, well‐being, or health. WPV against healthcare providers is common and seems to be increasing. A recent meta‐analysis of 253 studies including 331 544 participants found a mean 1‐year incidence of WPV of 61.8%. A 2018 survey conducted by the American College of Emergency Physicians demonstrated that out of more than 3500 emergency department (ED) doctors, 47% had been physically assaulted, in 97% of cases by a patient. In the USA, injuries caused by violent attacks against medical professionals grew by 67% between 2011 and 2018—with healthcare workers five times more likely to experience WPV than workers in all other industries. Data on violence against providers in the field of obstetrics and gynecology (OBGYN) is limited. A survey from 2018 among obstetricians and gynecologists in China found that two‐thirds had experienced violence while on duty in the previous year alone. Recent systematic reviews of the phenomenon of WPV against physicians indicate that its incidence varies between countries and contexts. , To our knowledge, no previous original study has had a global scope and collected qualitative data on healthcare providers' experiences of violence and the context within which it occurs. The aim of the present study was to both quantify and qualify the life‐time experience of WPV in a global sample of providers in OBGYN. MATERIALS AND METHODS We performed a mixed‐methods study based on responses to an online survey sent out in October 2023. The survey content was developed through several iterations within the FIGO Committee on Women Facing Crises and directed primarily towards doctors in OBGYN. The survey was translated into French, Spanish, and Arabic, and was available to attendants of the FIGO World Congress in Paris on October 9–11, 2023, through a QR code displayed at the welcome counter. The survey was subsequently emailed to all national societies of obstetrics and gynecology for dissemination. The survey was constructed around four mixed research questions on the experience and perceptions of WPV: What character of WPV do providers experience while on duty? What are the physical and psychological consequences of experiencing WPV? What contextual contributing factors for WPV do providers identify? What support structures exist for providers that experience or are at risk of WPV and how are they perceived? The survey contained 18 categorical questions on the type of violence experienced, impact on physical and psychological well‐being, impact on job satisfaction, situation triggers, and support and education structures within the healthcare system. These questions were paired with open‐ or close‐ended questions nuancing the categorical responses. For example, the question “Did the violence affect your emotional and psychological well‐being?” was followed by the question “How did the violence affect your emotional and psychological well‐being.” Here we used an integration strategy of matching to be able to integrate quantitative and qualitative findings. , Baseline characteristics were presented as absolute numbers and rates, and as relative rates according to whether the respondent had or had not experienced WPV. Associations between baseline characteristics age, gender, profession, length of professional experience, and region of residence were calculated using unconditional binomial logistic regression. Background variables with a significant association to having experienced violence were included in an adjusted model, unless they correlated too strongly with each other ( r >0.6). Apart from region of work, potential confounders were suggested by previous studies. Text data were interpreted at the manifest level. The whole text was read through for familiarization. Within the limitations of using text data, we used a mix of deductive and inductive coding with survey questions/themes used as overarching codes and subthemes extracted within each theme. Three researchers (ME, RS, and AR) reviewed the text data. We used mixed methodology to enhance the understanding of the experience of WPV. Quantitative and qualitative data were interpreted separately in parallel and then integrated using joint display in a convergent design where subthemes were assessed in relation to quantitative data for confirmation, complementarity, and contradiction. Approval for the study was obtained from the Swedish Ethical Review Authority (reference no. 2020–04629). RESULTS Between October 9, 2023, and January 31, 2024, we collected 1251 responses from 77 individual countries. The total number invited to scan the QR code or who were sent the study through their national societies is not known, meaning that the response rate could not be calculated. We excluded respondents who reported a non‐medical profession ( n = 17), who did not specify their profession ( n = 84), or who did not respond to whether they had experienced violence ( n = 204). Categories were not mutually exclusive and a total of 235 respondents were excluded. Our analyzed sample consisted of 1016 participants. We retained respondents who identified as “Other doctor,” as many were assumed to be under specialization in OBGYN or work closely aligned with OBGYN. Inclusion into the study is shown in Figure . Among our respondents, 804 (75.1%) identified as female, 204 (20.1%) as male, and 1 (0.1%) as non‐binary. Specialists in OBGYN represented 781 (76.9%), while 235 (23.1%) were either nurse/midwives, doctors under specialization in OBGYN, or with an affiliated specialization, such as surgery or anesthesia. There was an even distribution among respondents in age and length of professional experience. Eight regions of the world were represented as follows: Africa ( n = 149, 14.7%), Australasia ( n = 5, 0.5%), Central Asia ( n = 503, 49.5%), East Asia ( n = 57, 5.6%), Europe ( n = 88, 8.7%), the Middle East ( n = 84, 8.3%), North America ( n = 12, 1.2%), and South and Central America ( n = 107, 10.5%). Overall, out of 1016 respondents, 764 (75.2%) reported having experienced either verbal, physical, or another type of WPV. Gender and profession were not associated with an increased risk of violence, although men were more likely to have experienced physical violence (Tables and ). There was a linear increase in adjusted risk of having experienced violence with decreasing age and decreasing length of professional experience. Working in Central Asia, South or East Asia, Europe, or the Middle East was associated with a lower adjusted risk of having experienced violence. Baseline characteristics as well as associated risk of experiencing violence are presented in Table . The most common perpetrator according to our respondents was a relative of a patient (Figure ). Among all 1016 respondents, 669 (68.8%) had experienced verbal violence. Characterizations of verbal violence included shouting, insults, threats of physical violence including homicide, racism, and forceful accusations of incompetence, negligence, and responsibility for adverse outcomes, perceived to be outside the control of the provider. Many respondents reported frequent, sometimes daily, verbal abuse throughout their careers and abuse by large groups of people at a time. Frequently, respondents described disregard and lack of understanding for the context within which doctors had to operate: “If she dies, it's your fault!! You don't want to take care of me! I'm going to kill you and your entire family!!” [OBGYN, Venezuela, translated from Spanish]. A smaller group ( n = 123/1016, 12.1%) reported that they had experienced physical violence. Many respondents described being spit at, scratched, or slapped; however several described more severe assaults, such as being punched, held by the neck, kicks to the body or face, fractures, and threats at gunpoint. “I have experience being slapped, hair pulled, scratched, kicked, punched, grabbed in a sexual manner, spit at, human waste thrown at me, and have been verbally assaulted and threatened.” (Midwife, USA). A minority ( n = 11/1016, 1.1%) reported having experienced what we characterized as another type of violence. Two respondents reported sexual violence, including rape. For most of these respondents, however, the violence consisted of threats of actions involving parties outside the patient–doctor relationship, such as the legal system, the media, or the police. The threat of social media was especially prominent. “A patient was seriously ill and his relatives kept shouting at me even threat(e)ned to take my video and make it viral.” (Doctor, Pakistan). A joint display of quantitative and qualitative data for the occurrence and characterization of violence is shown in Table 2 Among the 764 providers who had experienced violence, 106 (13.9%) described a transient or long‐lasting physical impact. In the text data, some respondents described wounds or fractures needing to heal; however, more often, the violence was associated with chronic pain, migraine or tension headache, or the advent of chronic disorders, such as high blood pressure or autoimmune disease. “Chronic pain is exhausting, I never hurt like I do now 3 years later.” (OBGYN, Colombia). More respondents ( n = 362, 47.4%) reported the violence impacting their psychological health and well‐being. Characterizations of the psychological impact of violence in the text data confirmed and complemented these findings with many respondents describing anxiety, depression, and insomnia. Many respondents further described fear of facing similar clinical scenarios, permanently losing empathy for patients, and becoming despondent and withdrawn from friends and family. Some respondents described severe psychiatric illness: deep depression requiring hospitalization, medication or therapy; acute stress disorders; symptoms suggesting post‐traumatic stress disorder; suicidal ideation or attempt; and self‐harm. “I lost sympathy with patients and (their) relatives. Lost interest in dealing patients. Felt disgusting. Cannot focus on professional work. Depression for the whole family.” (Doctor, Pakistan). Close to one‐third of the 764 respondents ( n = 222, 29.1%), having experienced violence, reported that the violence had affected their job satisfaction. Qualitative data complemented this finding. While some described transient discomfort or difficulty meeting patients after the event, many described more profound effects, such as loss of empathy with patients. Several respondents described that the incident of violence had a radical impact on their subsequent career choices. “Maybe I am not brave enough to change my career but I definitely won't let any of my kids be a doctor…” (Doctor, Bangladesh). A joint display of quantitative and qualitative data for the impact of violence on physical and psychological health and job satisfaction is shown in Table . A little over half of the 764 providers ( n = 438, 57.3%) who had experienced violence reported the incident; however, only 216 (28.3%) reported having received any kind of support. Qualitative analysis indicated that many respondents did not report the incident due to a feeling of resignation in knowing that nothing would be done, or a lack of recognition on their part that the incident deserved attention. Many respondents understood violence as “the risks of the job.” “Work with what pleases God. Trust in God. Do not ask about anything else.” (OBGYN, Jordan, translated from Arabic). Confirming our quantitative results, formal support structures were described as poor or lacking, with mainly informal support offered from colleagues. “I was abandoned by my director, only a support from my colleagues on the psychological and administrative level.” (Doctor, Democratic Republic of the Congo, translated from French). Only a minority of total respondents ( n = 88/1016, 8.7%) had received training on WPV. Institutional policies on WPV were reported as effective or somewhat effective by 412 (40%) of respondents and not effective by 322 (31.7%). Qualitative data largely confirmed the absence of effective policies, with respondents often expressing powerlessness faced with their experience. “There is no law, no mechanism to handle any sort of abuse towards doctors. Once the dust settles you are supposed to chin up and return to your work like nothing happened.” (OBGYN, Pakistan). Two‐thirds of respondents ( n = 654/1016, 64.4%) believed there should be stricter legal consequences for violence against medical providers. Many respondents cited the need for zero tolerance for abuse, including verbal abuse, against healthcare providers. “The ‘zero tolerance’ policy for violence against [healthcare providers] in the NHS is helpful and I believe does sometimes prevent violence, particularly physical violence.” (Midwife, UK). A joint display of data on support structures and training surrounding WPV is shown in Table . The presence of triggers or contributing factors for the violence was asked of all 1016 respondents, whether or not they had personally experienced violence, and they answered as follows in order of frequency: staff shortages ( n = 394, 38.8%); lack of security personnel ( n = 381, 37.5%); long waiting times ( n = 379, 37.3%); staff attitudes ( n = 294, 28.9%); perpetrator mental health issues ( n = 262, 25.9%); genuine medical negligence ( n = 194, 19.1%); and substance abuse on the part of the perpetrator ( n = 108, 10.6%). Confirming the categorical data, many respondents described that insufficient infrastructure, staffing, and resources contributed to abuse against providers. Complementary text data showed that respondents recognized that fear, pain, and desperation on the part of patients or relatives were catalysts to violence and resulted from lack of knowledge or unrealistic expectations about what providers could achieve. Several respondents also described a pervasive distrust of medical providers, exacerbated by social media, that led to a climate of fear and antagonism between patients and providers. “Bad reception upon arrival. Husband's fatigue: patient having been referred from another health facility. Prejudices about the staff.” (OBGYN, Côte D'Ivoire, translated from French). A joint display of findings on triggers for violence against providers is shown in Table . DISCUSSION 4.14.2Main findings We found that WPV against OBGYN providers is pervasive across settings, with young and junior providers being most at riskseems to be triggered by a complex interplay of factors, and insufficient structures and policies exist to prevent violence and support those affected. All previous studies indicate that WPV against healthcare workers as a group is common and likely underreported, with 1‐year incidence rates of verbal and physical violence in the range of 33.8%–78% and 8.5%–31%, respectively, across five systematic reviews. , , , , Several of these studies further confirm our finding that young or less experienced providers are more exposed to violence, and that being male and a nurse is associated with a higher risk of physical violence, which our study did not find. , , , That our senior providers reported lower rates of lifetime WPV experience may seem counterintuitive as older doctors have more years in which to experience violence. It is possible that older respondents have forgotten the violence they experienced when they were new on the job. It is also possible that violence towards doctors is increasing and junior doctors are at the frontline of an environment where violence has become more prevalent. Several studies and systematic reviews indicate that this is the case, with WPV further exacerbated during the recent COVID pandemic. , , , What our study adds to the current evidence is a characterization of WPV both in terms of what the experience consisted of and its repercussions. Our results indicate that even verbal abuse can lead to complex, long‐lasting, and severe mental distress. Previous research on violence suggests a distinction between affective and predatory violence, with the latter being more detrimental for the victim. Affective violence arises from of a “fight or flight” response, which, in the context of medical care, is often fear or pain. In contrast, predatory violence is an intentional and deliberate attack motivated by a grievance. Consistent with this, many respondents recognized that threats to their person and accusations of mismanagement and incompetence had particularly painful and had long‐lasting effects. A type of violence that emerged in our data, which has not been previously reported, was the threat of leveraging social media to discredit providers. The Internet is a source of legitimate information about the quality of services, which makes this type of abuse more difficult to categorize. Nevertheless, workplace safety policies must recognize the illegitimate use of social media as another form of violence against providers, be prepared to counter personal abuse that emanates from social media platforms, and redirect criticisms of services to legitimate channels. We also found indications that, although respondents expressed understanding for factors that triggered the violence, their empathy and engagement with patients and similar clinical scenarios sometimes changed permanently for the worse. This study further highlights the complex setting in which WPV in healthcare settings occurs, in that both the aggressor and the victim are under duress. The stress of an untreated emergency, the unrecognized pain of a loved one, and navigating labyrinthine and unaccountable healthcare systems are easily understood to heighten feelings of desperation and anger. At the same time, healthcare providers may be operating under the extreme pressure of insufficient time and resources to meet patient needs. Our data support a prevalent atmosphere of distrust towards providers fueled by unrealistic or unmet expectations. In both the USA and India, providers increasingly report fearing for their safety and the use of weapons or private security staff has become more prevalent. , Almost two out of five providers in our study believed their hospital's policies were, to some degree, effective in preventing and managing WPV. However, a recent Cochrane review concludes that although a wealth of interventions exist to mitigate WPV, their focus is primarily on de‐escalation and there is no evidence that they work to reduce the occurrence, or severity, of WPV. As previous authors have noted, there is a lack of evidence‐based research on successful measures to prevent or mitigate WPV. Most studies indicate that support structures are not only ineffective, but lacking. A recent systematic review of 17 studies on the prevention of WPV found that training improved providers' perceived ability to handle violent events but no study measured the interventions' effect on the incidence of WPV or its repercussion for the provider. A recent study found a strong association between a hospital having solid policies on workplace safety, and having solid policies on patient safety. This mirrors our finding that WPV often occurs in a context that is perceived as unsafe for both patients and providers. Tolerance for long waiting times or poor outcomes are likely higher in a clinical setting in which the patient and their relatives trust the institution providing care. The FIGO Committee has developed a tool kit to prevent and mitigate WPV against OBGYN, the development process of which will be described in another article. It is likely that addressing violence against healthcare providers requires a comprehensive approach involving healthcare institutions, policymakers, law enforcement agencies, healthcare professionals themselves, and the broader community. Strengths and limitations To our knowledge, this is the first global survey on WPV among OBGYN providers and the only study examining the prevalence and character of WPV also through a qualitative lens. Using a retrospective survey as data measurement risks both selection bias, in that people affected by violence are more likely to respond, and recall bias, in asking people to remember events far in the past. The collection of qualitative data through a survey, unlike in an interview setting, did not allow for individualized follow‐up questions, which could have nuanced the responses and the quality of the data. Nevertheless, the quantity, detail, and depth of our text responses suggest that the experience of WPV and its repercussions are not easily forgotten. CONCLUSION Our findings suggest that WPV against OBGYN providers is frequent across settings, and that victims experience wide‐ranging and long‐lasting physical and psychological repercussions that affect performance, satisfaction, and retention to the profession. WPV may be triggered by a complex interplay of factors and insufficient structures and policies exist to prevent violence and support those affected. R.S. had the idea for the study and led study procedures. R.S. designed the survey that was revised by M.E. and A.R. and approved by the members of the Committee on Women Facing Crises. M.E. analyzed the data and wrote the draft for the manuscript. All authors have approved the final version of the manuscript. The authors have no conflicts of interest. Atziri Ramirez (Chair), Rubina Sohail, Lubna Hassan, Margit Endler (Vice‐chair), Denis Mukwege, Miguel Gutierrez, Anwar Marjan, Kristina Jariene, Priyankur Roy, Ana Patricia Gomez, Sarah Baffoe, Diana Galimberti, Shantha Kumari. Data S1. |
From virtual to reality: innovative practices of digital twins in tumor therapy | e549be2d-ef93-4a10-94f3-83332df6b556 | 11921680 | Medicine[mh] | Cancer is the second leading cause of death worldwide, accounting for approximately one-sixth of all deaths . This statistic is primarily due to the complexity of cancer and its high heterogeneity as a multi-cellular ecosystem . Such heterogeneity is evident not only between different cancer types but also in how a single cancer type affects different patients . Understanding the interactions between various cell types within tumors and their roles in tumor evolution is key to effective treatment . Despite recent advancements in cancer treatment, existing therapies still fail to deliver satisfactory outcomes for different patients, particularly within the context of precision oncology and molecular phenotyping (such as genomics and proteomics), underscoring the need for continued research and development . Additionally, current treatment strategies often follow a ‘‘one-size-fits-all’’ approach, lacking truly personalized therapeutic options . Against this backdrop, the emerging concept of digital twin (DT) technology has garnered widespread attention in oncology . Initially applied in aerospace and manufacturing, a digital twin refers to a mathematical model constructed using real-time data that provides a virtual representation of a specific physical object, enabling the prediction of its behavior under various external conditions and optimizing future decision-making [ – ]. When applied to healthcare, the comprehensive report Foundational Research Gaps and Future Directions for Digital Twins by the National Academies defines digital twins as 'computational models created for the human body or its parts, which establish a bidirectional connection with the patient's system and are calibrated through periodic data collection to dynamically predict the patient's health status' . The application of this technology in cancer treatment is particularly notable . Its advantages lie not only in integrating genetic information, clinical history, and imaging data to create highly personalized cancer digital twin models but also in its ability to precisely simulate tumor molecular dynamics and treatment responses through multiscale models and advanced machine learning algorithms . Based on this platform, medical experts can rehearse and optimize treatment plans in a virtual environment, track patient progress in real time, and significantly enhance treatment outcomes and quality of life . Although digital twin applications in oncology are still in their infancy, the integration of digital twin technology into tumor dynamics and personalized care is gaining attention as experimental technologies and advancements in mathematics and computer science continue to evolve . For example, the Gilbertson team is developing the world’s first computer model capable of spatially and temporally sensing the development of both normal and malignant tumors, aiming to identify efficient and low-toxicity treatment options for pediatric cancers. Wickramasinghe et al. , based on systems and mathematical modeling theories, proposed three digital twin models—gray-box, agent-based, and black-box—and explored the potential application of the black-box model in personalized care for uterine cancer. Additionally, a collaborative project initiated by the National Cancer Institute and the U.S. Department of Energy is focusing on the construction of patient-specific cancer digital twins, which provides a robust foundation for personalized cancer treatment . Meanwhile, an increasing number of empirical studies have demonstrated the potential application value of digital twin technology in cancer treatment. For instance, the model developed by Wu et al. integrates MRI data with biologically-based mathematical models, not only achieving precise prediction of neoadjuvant chemotherapy response in triple-negative breast cancer (TNBC) patients but also significantly outperforming traditional tumor volume measurement methods in predicting pathological complete response (PCR). Similarly, Kim et al. developed a two-stage model that combines digital twins with machine learning, successfully capturing patient-specific, spatiotemporal dynamics of TNBC response to neoadjuvant systemic therapy (NAST), providing highly accurate predictions of NAST response. These successful cases not only highlight the immense potential of digital twins in precision oncology and clinical decision support systems but also lay a solid practical foundation for the future development of personalized medicine. Given the immense potential of digital twins in cancer research, it is particularly important to comprehensively explore their scope of application. Therefore, this study aims to synthesize existing knowledge and analyze the extent of research on digital twins in the field of oncology. Through bibliometric analysis, we will examine trends in the total volume of publications, the distribution of research funding, active research countries, and collaborative networks to provide an overview of the current state and future directions of digital twin research in oncology. Furthermore, within the framework of a scoping review, this paper will evaluate the specific applications of digital twin technology in cancer treatment and explore the challenges and future research opportunities by analyzing emerging real-world cases. As an emerging personalized medical technology, digital twin technology shows great promise in oncology. To provide a thorough analysis of its application status, technical challenges, and future directions, this paper focuses on the following key questions: What are the research trends, active countries, collaborative networks, and funding distributions for digital twins in the oncology field? What are the characteristics of sample size distribution in current studies, and how does it relate to data sources and clinical applications? Is there a significant difference in sample sizes across different cancer types? How does digital twin technology utilize multi-source data (such as imaging data and omics data) in tumor therapy? What is the role of AI algorithms in this process? What are the main application scenarios and effectiveness of digital twin technology in oncology? What are the key challenges faced by digital twin technology in oncology, and what are the future research directions? Furthermore, to visually present the core logic and working mechanism of digital twins in oncology, a conceptual framework diagram of tumor digital twins (see Fig. ) has been designed. This diagram systematically illustrates the process from multi-modal data collection to personalized treatment feedback optimization.
This study employs a combination of bibliometric analysis and a scoping review to comprehensively assess the research status of digital twin technology in tumor therapy. In the bibliometric section, the framework proposed by Cobo et al. was followed. The scoping review was designed and conducted according to the PRISMA-ScR guidelines . Specific research methods include literature retrieval, screening, and analysis, as well as data processing and visualization. Data sources and search strategy Inclusion and exclusion criteria Data processing and analytical framework Analysis tools To ensure comprehensive retrieval and account for the interdisciplinary nature of tumor digital twins, we conducted a literature search across five major databases: PubMed, Web of Science, Scopus, Embase, and IEEE Xplore. Prior to the official search, all research team members participated in professional training based on the "Medical Literature Information Retrieval ’’ textbook. In collaboration with librarians, oncologists, and medical informaticians, we developed the search strategy. The search strategy was based on the definitions provided in the BEST vocabulary, with core search terms including "digital twin" and "tumor," combined using Boolean operators (such as AND, OR). To ensure comprehensiveness, we also reviewed the reference lists and cited articles of relevant literature. Furthermore, to avoid bias due to data updates, the cutoff date for all search results was set to October 13, 2024. Literature management and duplicate removal were performed using EndNote software. For the detailed search strategy, please refer to Supplementary Material 1.
Inclusion criteria Exclusion criteria Screening strategy Prior to the formal inclusion or exclusion of studies, three reviewers (SS, WQ, and XL) randomly selected 30 studies for preliminary screening to assess the reliability of the screening process. The final Cohen's kappa value was 0.89, indicating high consistency, and no adjustments were made to the inclusion/exclusion criteria or the reviewers. During the formal independent screening process, any discrepancies were ultimately resolved by SL, who intervened in the decision-making. The screening and verification process was completed on November 2, 2024, with 68 studies ultimately included. The detailed screening process is shown in Fig. , and the detailed information of the included literature can be found in Supplementary Material 2.
The literature must clearly research the application of digital twin technology in the tumor field (e.g., diagnosis, treatment, prognosis prediction), covering technical development or real-life cases. The included literature must focus on the technical framework, data integration methods, or algorithm optimization processes of digital twins, with at least one tumor-related experimental or clinical application. The literature must be peer-reviewed articles published in English.
Studies that cannot be accessed in full. Duplicate publications. Studies unrelated to tumors, such as those limited to engineering or industrial applications of digital twin technology. Literature that does not clearly provide research methods or data sources, particularly models or algorithms that cannot be reproduced.
o systematically analyze the research status and future prospects of digital twin technology in oncology, this study established the following analytical framework to ensure comprehensiveness and systematic coverage: Bibliometric analysis Scoping review This section employs bibliometric methods, involving data cleaning and analysis, to examine the research trends and development of digital twin technology in the oncology field. As digital twin technology is still in the early stages of exploration in oncology, the number of relevant publications is limited. Therefore, the analysis focuses on the following aspects: Research Initiation and Trends The number of publications from 2020 to 2024 was analyzed, exploring the characteristics and early development trajectory of digital twin technology as it extends from general fields to oncology. Geographical Distribution By analyzing the geographical information of authors and institutions, the primary research centers and active countries in the early stages of digital twin technology in oncology were identified. Funding distribution The funding sources reported in the literature were analyzed, particularly the funding support from governments and multinational research organizations during the early stages of the technology.
This section synthesizes existing literature to discuss the application and development trends of digital twin technology in tumor therapy. Unlike the bibliometric analysis, this section does not involve complex data analysis or statistical methods but focuses on the following aspects: Sample Size Analysis Based on existing literature and the specific needs of oncology , the studies were classified into small sample size (< 50 cases), medium sample size (50–500 cases), and large sample size (> 500 cases). Sample size directly affects the reliability and generalizability of digital twin research in oncology . Data Processing and AI Application Analysis Digital twin applications rely heavily on multi-source, multi-modal data integration and AI algorithms .This section systematically analyzes the use of medical imaging, omics data, and AI algorithms in digital twin technology. Theme Classification Analysis Based on the specific application scenarios of digital twins, the research was categorized into seven major areas: diagnosis, treatment decision-making, prognosis prediction, surgical planning, drug development and virtual trials, tumor microenvironment analysis, and personalized management.
Given the large volume of data, human error in statistical calculations may occur, and manual analysis often faces challenges. Furthermore, traditional single-tool analysis often suffers from low granularity . To ensure completeness and high granularity, a multi-tool bibliometric analysis strategy was employed. Specifically, the analysis utilized tools such as VOSviewer (Version 1.6.19), Gephi (Version 0.10.1), and Cortext (Gustave Eiffel University). In terms of layout and enhanced visualization, ScimagoGraphica (Version 1.0.16) was used. Data processing, analysis, and visualization were performed using Origin2024, networkD3, and ggplot2 R software packages. Detailed analysis strategies and data cleaning methods are provided in Supplementary Material 1.
Annual publication trends Country analysis Funding analysis Sample size analysis Sample size distribution and classification Data sources and application scenarios Sample size distribution by cancer type Data processing and integration in tumor treatment Role of artificial intelligence in digital twin technology Digital twin applications in oncology The temporal distribution of the literature reflects the research trend of digital twin technology in the oncology field. The number of publications increased annually from one in 2020 to 27 in 2024, with a significant rise after 2022. This trend indicates that with the advancement of technology and policy support, digital twin technology in oncology is receiving increasing attention and investment. In terms of research topics, early studies were more focused on concepts and theoretical exploration. For example, Zhang et al. (2020) proposed a framework for applying digital twins in lung cancer diagnosis, combining physical rendering technology and deep learning models to provide dynamic simulation and prediction tools for precision medicine. Béthencourt et al. designed a digital twin-based connected device to monitor the occurrence and development of post-operative lymphedema in breast cancer patients, providing a technical reference for early detection and dynamic monitoring in oncology. Later studies, however, have been more focused on practical applications and clinical validation. For example, Wang et al. integrated patient data and interview information to develop a digital twin for ovarian cancer patients and caregivers, optimizing the health information recommendation system and addressing the issue of insufficient clinical data in the early stages. Obergfell et al. used a target trial simulation framework to compare planned and unplanned resections and their effects on recurrence and survival in sarcoma patients, providing a real-life example of digital twin application in surgical decision-making.
Figure A shows the geographical distribution of digital twin technology publications in oncology, with 27 countries publishing related research. The size of the circles in the figure represents the publication volume of each country, while the colors differentiate the publication quantities. The map illustrates that, despite being in its early stages, the field exhibits a certain degree of global distribution. The United States (n = 28) leads the field, indicating its dominant role in combining digital twin technology with oncology research. Germany (n = 14), Switzerland (n = 10), and China (n = 10) have also contributed significantly, reflecting the research strength and interest in this emerging field within these countries. From a regional perspective, North America, Europe, and Asia are the primary research hubs. While the United States remains dominant in international research, European countries such as the United Kingdom and Germany have formed a robust research cluster that supports the widespread development of this technology. Figure B shows the network diagram of international cooperation, further revealing the distribution of collaborative relationships. The thickness of the lines represents the strength of collaboration. The diagram indicates that the U.S. is not only the leading country in publication volume but also a central hub in the global research cooperation network, with strong ties to Italy, the United Kingdom, Germany, China, and several other countries. European nations (such as the UK, Germany, and Italy) also exhibit close collaboration, forming stable research networks. China's collaborations with major Western countries (such as the U.S. and Germany) are prominent, showing China's increasing integration into the global research landscape.
A total of 49 studies (49/69, 71.01%) received funding, with a total of 104 instances of funding. On average, each funded project received 2.12 funding sources. These funds came from 66 different sources. As shown in Table , the major funding source was the U.S. National Institutes of Health (NIH), which provided 10 funding instances (10.4%), followed by the OPO Foundation, Margrit Weisheit Foundation, and Parrotia Foundation from Switzerland, each contributing four funding instances. Among the top eight funding agencies, four are U.S. government departments, such as the NIH and National Science Foundation, indicating the prominent role of U.S. government funding in this field. Additionally, we categorize all funding sources into government grants, funding from non-profit organizations and foundations, corporate funding, support from international organizations, funding from universities and research institutions, and individual contributions. As shown in Fig. A and 4B, government funding is the primary source of financial support, accounting for 52.88% of the total funding instances. University and research institution funding accounted for 18.27%, while corporate and private funding represented a smaller portion of the total funding.
To comprehensively evaluate the scope, representativeness, and application trends of digital twin technology in oncology, we conducted a sample size analysis of the included studies and visualized the correlation between sample sizes, data sources, and clinical application scenarios using a Sankey bubble chart (Fig. ). Detailed sample size information for each study can be found in Supplementary Material 2.
The included studies showed significant variation in sample sizes, ranging from individual cases to tens of thousands of patients. We classified studies into small sample sizes (< 50 cases, 20.59%), medium sample size (50–500 cases, 23.53%), and large sample sizes (> 500 cases, 27.94%). Additionally, about 27.94% of the literature did not clearly report sample sizes, often relying on non-traditional data sources such as virtual patient data or experimental data, or due to data privacy restrictions. This phenomenon reflects the need for improved reporting standards and transparency in current research. Small Sample Size Studies (< 50 cases) Small sample size studies are primarily used for technology development and preliminary validation, relying mainly on clinical trial data [ , , ], medical imaging data [ – ], and animal model data . These studies typically focus on personalized diagnosis and treatment [ , , , ] and technical precision testing [ , , , ]. For example, a study involving 20 breast cancer patients developed a digital twin-assisted health device measurement method for lymphedema prevention . Medium Sample Size Studies (50–500 cases ) Medium sample size studies often integrate multi-dimensional data, such as genomic, metabolomic, and medical imaging data [ – ], to support model validation and early clinical testing. As shown in Fig. , these studies are primarily focused on diagnosis (25%), prognosis prediction (25%), and treatment decision-making (approximately 25%), reflecting the wide applicability of medium sample sizes in relevant clinical fields. For instance, a study involving 116 breast cancer patients integrated multi-omics data to construct a digital twin model, which was used to optimize personalized treatment strategies . Large Sample Size Studies (> 500 cases) Large sample size studies often rely on public databases or comprehensive tumor datasets, covering broad application scenarios, especially in diagnosis [ – ] and personalized management [ , , ]. The primary data sources are medical imaging data (50%) and clinical data (30%).For example, a study involving 887 lung cancer patients utilized generative adversarial networks (GANs) to generate synthetic data, enabling clinical applications and data sharing while preserving patient privacy .
The Sankey bubble chart (Fig. ) illustrates the flow relationship between data sources and clinical application scenarios, revealing the patterns of data usage in tumor research. Among the data types, medical imaging and clinical data are the most common sources, primarily directed towards diagnosis and treatment decision-making. Virtual patient data and laboratory data are more commonly used for technology validation and personalized management, reflecting the significant role of non-traditional data sources in early-stage technology development. The major application scenarios of digital twin technology include diagnosis, treatment decision-making, and prognosis prediction, where diagnosis heavily relies on large sample sizes, while personalized management and surgical planning tend to utilize small and medium sample size studies. Notably, only eight studies utilized public datasets, suggesting that despite the increasing application of digital twin technology in oncology, the use of publicly available datasets remains limited, likely due to concerns over data privacy and quality.
The studies included in this research cover common cancer types such as breast cancer [ , , , – ], head and neck cancer [ – ], brain tumors [ , – ], lung cancer [ , , , ], as well as specific subtypes and experimental cell-level data, such as high-grade gliomas and triple-negative breast cancer . Among these, breast cancer research represents the largest proportion, with sample size distributions ranging from small case numbers (e.g., 20 cases ) to large-scale cohorts (e.g., 80,000 cases ), indicating a strong clinical demand for digital twin applications in this cancer type. In contrast, brain tumors and other specific cancer subtypes are predominantly studied with smaller sample sizes, focusing on precision medicine or exploring underlying mechanisms, such as the study of high-grade gliomas with about 100 cases .
Digital twin technology in tumor treatment relies on the integration and processing of multi-source, multi-modal data . These data provide essential support for constructing precise patient models. Medical imaging data Multi-omics data integration Additional data sources In addition to imaging data, various other data types have been progressively integrated into digital twin models. Specifically, physiological data were widely applied in 16 studies, primarily used to dynamically adjust personalized treatment strategies [ , , ]. Pathological slide data were mentioned in eight studies, mainly for enhancing the precision of diagnosis and treatment decisions by combining genomic information . Three studies involved animal model data, providing experimental support for tumor surgical planning, optimizing surgical approaches, and predicting treatment outcomes [ , , ]. Additionally, unstructured data were mentioned in two studies, further enriching the input for digital twin models .
Among the literature included, imaging data accounted for 36.76% (25 studies). MRI and CT imaging were the most commonly used, with 17 studies applying them for tumor segmentation, 3D modeling, and treatment optimization. For instance, Tai et al. used radiological images for tumor segmentation and treatment optimization, while Sainz-DeMena et al. reconstructed a 3D mesh model of neuroblastoma based on CT images, aiding personalized treatment and surgical planning. Additionally, 15 studies involved dynamic imaging data, covering dynamic contrast-enhanced MRI (DCE-MRI), time-series CT, PET, and SPECT imaging. DCE-MRI was the most frequently used dynamic data source (n = 4, 26.67%), employed to monitor tumor size changes and therapeutic response modeling [ , , , ].
The integration of multi-omics data (including genomics, proteomics, metabolomics, etc.) has provided strong data support for digital twin models, enhancing their ability to adapt to individual differences . Among the included studies, over 15% focused on the application of multi-omics data, aiming to enhance the model's ability to analyze the tumor microenvironment, predict treatment responses, and optimize decision-making. Specifically, the applications are as follows: Genomic Data Genomic data accounted for more than 14.29% of all relevant studies, primarily used to identify tumor-driving gene mutations, support personalized treatment decisions, and precision-targeted strategies, as well as provide a basis for personalized vaccine design . By identifying cancer-driving genes, digital twin models can predict the response of different genotypes to specific treatments. Proteomics and Metabolomics Data Seven studies explicitly mentioned the application of proteomics and metabolomics data. Proteomics data are mainly used to analyze intracellular signaling pathways and protein interactions in tumor cells, while metabolomics data reveal the phenomenon of metabolic reprogramming in tumor cells. Both play key roles in tumor microenvironment analysis and treatment response evaluation . Single-Cell Omics Data Single-cell omics data were applied in approximately 5.71% of studies, primarily to uncover tumor cellular heterogeneity and the dynamic changes in the immune microenvironment. Single-cell analysis can capture cellular differences within the tumor and their response to treatment, particularly with regard to temporal changes in immune therapy. Through single-cell-level data, researchers can obtain more precise tumor dynamic information, which aids in advancing personalized and refined immune therapy . Although the integration of multi-omics data and dynamic modeling is on the rise, only six studies have achieved comprehensive integration of multiple omics data types [ , , , , , ], with a primary focus on the combination of genomics and proteomics data. The integration of metabolomics and single-cell omics data remains relatively low, indicating that the integration of these data faces certain technical challenges.
rtificial intelligence (AI) plays a central role in digital twin technology, enhancing data processing, model construction, and predictive analysis through advanced algorithms and models . Widespread use of core algorithms Multimodal data fusion application In terms of multimodal data integration for digital twin tumor treatment, 22 studies explored the fusion of multimodal data. For example, Chaudhuri et al. and Gilbertson et al. successfully enhanced tumor treatment model accuracy by combining imaging and physiological data. Wang et al. further combined imaging, omics, and patient characteristic data for virtual patient modeling in immunotherapy. Furthermore, six studies [ , , , , , ]discussed the comprehensive integration of multimodal data, covering a wide range of data fusion methods, though overall research in this area remains limited, indicating that the field is still in the exploratory stage.
Among the 68 studies included, 64 involved the use of algorithms. Deep learning was mentioned in seven studies, mainly for image segmentation and feature extraction. For instance, Islam et al. applied several deep learning algorithms to classify kidney CT images, while Batchden et al. used convolutional neural networks (CNNs), attention-enhanced CNNs, and recurrent neural networks (RNNs) to detect metastatic diseases from continuous radiological reports and generate metastasis maps. Generative adversarial networks (GANs) were applied in six studies to generate high-resolution imaging data , addressing the issue of insufficient rare cancer data and supporting dynamic image generation and surgical navigation . Additionally, four studies used support vector machines (SVMs) for disease classification tasks [ , , , ]. Hybrid models integrating cross-domain algorithms provided key support for tumor dynamic simulation and personalized treatment optimization [ , , ].
Digital twin technology in tumor therapy exhibits multi-layered and synergistic characteristics, covering key application scenarios such as diagnosis, treatment decision-making, prognosis prediction, surgical planning, drug development, virtual trials, tumor microenvironment analysis, and personalized management. Each application scenario incorporates advanced modeling methods and techniques to optimize precise and personalized medical plans. The following sections analyze the functionality, technical features, and specific examples of each application scenario. To clearly demonstrate the hierarchical structure and functions of these scenarios, Fig. uses a hybrid pyramid diagram and a central radiating mind map to intuitively present the seven application scenarios and their respective functions. The pyramid is divided into three layers: the top layer includes prognosis prediction and treatment decision-making, which directly serve to optimize patient treatment plans through multimodal data integration and dynamic modeling; the middle layer includes diagnosis, surgical planning, and personalized management, which provide support for tumor detection, treatment planning, and patient care across the entire treatment cycle; the bottom layer includes drug development, virtual trials, and tumor microenvironment analysis, reinforcing the overall system with technical support and data foundations. Diagnosis Personalized management Surgical planning and training Tumor microenvironment analysis Drug development and virtual trials Prognosis prediction Treatment decision-making Digital twin (DT) technology in tumor treatment decision-making focuses on dynamic modeling and personalized optimization. A key approach is the Deep Q-Learning (DQL) model, which uses a reinforcement learning framework to simulate multi-step interactions between physicians and patients, predicting optimal treatment pathways. By inputting extensive historical treatment data, the DQL model creates a learning system that dynamically optimizes chemo-radiotherapy regimens to meet the specific treatment needs of individual patients . Notably, Bayesian optimization frameworks excel in handling uncertainties, particularly in designing personalized radiotherapy plans for high-grade gliomas. By probabilistically modeling longitudinal MRI data, Bayesian optimization generates tailored treatment plans while balancing efficacy and toxicity. This approach not only enhances treatment outcomes but also reduces damage to normal tissues . Mathematical models have also played a critical role in optimizing chemotherapy regimens for acute myeloid leukemia (AML). By analyzing white blood cell counts, drug metabolism parameters, and patient toxicity thresholds, these models simulate the physiological impact of different treatment strategies. Through systems of equations describing pharmacokinetics (PK) and pharmacodynamics (PD), mathematical models provide scientific evidence for optimizing chemotherapy doses and timing . Furthermore, multi-modal data integration is indispensable in treatment decision-making with DT technology. For instance, DT-enabled visualization platforms such as the myCMIE system integrate patients’ molecular profiles, genomic data, and clinical records to generate personalized treatment recommendations in real time. Compared to traditional methods, the myCMIE system offers a comprehensive perspective by deeply integrating various data types, enabling treatment plans that better align with individual patient needs . Table the primary models, data sources, and research outcomes of DT technology in tumor treatment decision-making, showcasing recent advances.
In the field of tumor diagnosis, digital twin technology significantly enhances diagnostic accuracy through the integration of multimodal data and deep learning methods, particularly in imaging analysis and early screening. Generative adversarial networks (GANs), as a powerful tool, have shown great potential in medical image processing. Through its generative and discriminative mechanisms, GANs can generate high-quality synthetic medical images, effectively denoising images and reconstructing low-resolution images, especially in tumor early detection. In cervical cancer screening, for instance, GANs achieved an accuracy of 98.91% in classification , demonstrating their potential for early tumor detection. In terms of improving diagnostic accuracy, three-dimensional acoustic modeling technology has also demonstrated unique advantages. Based on the Navier–Stokes equation, this modeling method precisely simulates the propagation behavior of sound waves in soft tissues, providing important evidence for accurate diagnosis and surgical planning of liver diseases . This technology not only enhances diagnostic accuracy but also offers new pathways for early liver disease detection. However, due to its high computational demand, further optimization is required for its clinical application. The Vision Transformer (ViT) model, a recent advancement, has significantly improved the efficiency of CT image feature extraction due to its attention-based mechanism. In automated detection of kidney tumors and stones, ViT demonstrated superior performance compared to traditional convolutional neural networks, especially in optimizing global information capture and data dependence . However, the complexity of training the ViT model and its high computational resource requirements may pose challenges in real-time applications. Table summarizes the main models, data sources, and research findings in digital twin-based tumor diagnosis, highlighting key advancements.
In tumor personalized management, digital twin technology drives the development of virtual patient models through multimodal data integration and advanced modeling methods. These models precisely simulate patients' physiological characteristics, genomic data, and treatment responses, enabling dynamic adjustments to personalized treatment plans. For instance, a physics-based digital twin model, integrating pharmacokinetics (PK) and pharmacodynamics (PD) models, successfully predicted patients' drug responses under different physiological conditions [ , , ]. This model provided scientific support for optimizing drug treatments, significantly improving individualized treatment outcomes. Additionally, digital twin technology has shown potential in managing cancer-related chronic pain. By combining Markov Chain Monte Carlo (MCMC) methods, researchers generated virtual patient populations and simulated the impact of variables like age and gender on fentanyl absorption and efficacy. This approach significantly enhanced the precision of drug dose regulation, avoiding the shortcomings of traditional trial-and-error dosing methods . Furthermore, a physics-driven thermal control model was used to optimize the transdermal fentanyl delivery process, reducing the standard deviation of blood drug concentration by 37.5% . Real-time data analysis platforms, such as Sarconnector®, seamlessly integrate multi-modal data to support continuous optimization of personalized treatment paths, ensuring flexibility in treatment plans . However, one of the main challenges in personalized management is the high complexity of patient physiological and genetic differences, which leads to varying treatment effects among different patients . Furthermore, ensuring the computational efficiency of real-time drug dose adjustments remains a challenge in the practical implementation of these technologies [ , , ]. Table summarizes the main models, data sources, and research findings on digital twin technology in tumor personalized management, showcasing key progress. Figure illustrates the application framework of digital twin technology in tumor personalized management.
In tumor surgical planning and training, digital twin technology leverages various modeling methods and techniques to achieve high-precision surgical simulation and dynamic support. For example, deep convolutional generative adversarial networks (DCGANs) are applied to generate 3D anatomical structures. By combining high-resolution image reconstruction with finite element analysis (FEA), DCGANs simulate the physical responses of bones and soft tissues, especially useful in surgeries like vertebroplasty that require high precision for bone tissue manipulation . This technology not only improves pre-surgical preparation but also offers strong support for intraoperative dynamic adjustments. Augmented reality (AR) technology combined with dynamic navigation models has further enhanced surgical planning accuracy. By overlaying virtual images onto surgical fields in real time, AR supports dynamic calibration and path planning during surgery, reducing risks and increasing precision . Additionally, advancements in 5G and Internet of Things (IoT) technology have enabled breakthroughs in remote surgical simulation. High-bandwidth, low-latency connections allow real-time remote guidance and training . Virtual reality (VR) combined with 3D modeling has provided innovative tools for surgical training. This approach builds virtual anatomical and pathological models, offering medical students and junior doctors a risk-free, repeatable environment to practice operations, enhancing their skills and ability to handle emergencies . However, the realism and interactivity of VR still require further research and optimization for different types of surgeries . Table summarizes the detailed model types, data sources, and related research metrics of digital twins in the field of tumor surgical planning and training, highlighting key achievements in current studies. Figure illustrates the application framework of digital twin technology in tumor surgical planning and training.
Digital twin technology in tumor microenvironment (TME) analysis employs advanced modeling methods to simulate complex intercellular interactions and dynamic characteristics, providing deeper insights into tumor development and treatment. Multiphase reactive poromechanics models, for instance, describe the interactions among tumor cells, interstitial fluid, and the extracellular matrix. Using such models, Urcun et al. successfully replicated the mechanical environment inside tumors, revealing the relationship between stress distribution and drug diffusion, offering theoretical support for anti-tumor drug development. Hybrid models combining partial differential equations (PDEs) and ordinary differential equations (ODEs) focus on simulating chemical concentration gradients in the TME, particularly interactions between immune and tumor cells. These models dynamically simulate immune cell migration behaviors, providing critical insights into tumor immunology and optimizing immunotherapy strategies . Cellular automata (CA) models, known for their ability to simulate cell behaviors, are widely used in studying tumor cell migration and metastasis pathways. These models efficiently simulate the diffusion characteristics of tumor cells in the microenvironment and have significant applications in understanding metastasis mechanisms . However, their capacity to capture complex intercellular interactions and heterogeneity needs improvement . Integrating genomic, proteomic, and metabolomic data into hypermodels further extends the application of digital twin technology in TME analysis, providing more accurate support for developing personalized treatment strategies . Table provides a summary of the primary models, data sources, and research outcomes of digital twin applications in tumor microenvironment analysis, highlighting key advancements in the field. Figure illustrates the multi-level application framework of digital twin technology in tumor microenvironment analysis. Relevant references are indicated in the figure legend.
In tumor drug development and clinical trials, digital twin (DT) technology enhances personalized treatment by precisely simulating individual biological processes and therapeutic responses. This optimization of drug screening, dose adjustment, and efficacy evaluation accelerates the advancement of tailored therapies. For instance, quantitative systems pharmacology (QSP) models are renowned for their precise prediction of dose–response relationships. By integrating pharmacokinetics with tumor biology, QSP provides a mechanism-driven tool for dose optimization and biomarker identification . Unlike the mechanism-driven nature of QSP models, multi-omics integration models expand the capabilities of tumor microenvironment (TME) modeling by incorporating genomic, proteomic, and metabolomic data. These models are particularly suited for assessing the complex efficacy of immunotherapies . Meanwhile, cellular dynamics simulations explore the behavior of immune cells (e.g., T cells) within the TME, dynamically describing processes such as cell proliferation, differentiation, and apoptosis. This provides valuable data and mechanistic support for optimizing cell therapies . However, individual variability among patients presents challenges to model generalization and personalized dose adjustments . Furthermore, constructing virtual cohorts and ensuring data consistency pose obstacles to the implementation of virtual trials. Variations in efficacy metrics and observation periods across studies can render some data incompatible with virtual trial frameworks, hindering drug development progress . Table summarizes the main models, data sources, and research outcomes of DT technology in tumor drug development and virtual trials, highlighting recent key advancements.
In prognosis prediction, digital twin (DT) technology models individual patient characteristics, dynamically simulating tumor growth and treatment response to forecast therapeutic outcomes and long-term survival rates. Biomathematical models, such as reaction–diffusion equations or partial differential equations (PDEs), are widely used to simulate tumor spread, proliferation, and treatment response . Given the high computational demands of such models, reduced-order modeling (ROM) has been introduced to create representative subsets in low-dimensional spaces, significantly reducing computational requirements and making these models more feasible in resource-limited environments . Simultaneously, machine learning methods have enhanced the accuracy of survival and disease progression predictions by integrating imaging, biomarkers, and clinical data . For example, Salimi et al. introduced the concept of "Organomics," which incorporates radiomic features of healthy organs into prognosis models for non-small cell lung cancer. The study found that integrating healthy organ data significantly improved the predictive accuracy of machine learning models compared to using tumor data alone . This approach provides a more holistic perspective on tumor prognosis prediction and highlights the advantages of DT technology in multi-modal data integration. Additionally, network science models, such as SynTwin, leverage graph structures to connect data from similar patients, generating virtual patient profiles. This enhances the applicability and generalizability of models across diverse clinical scenarioprognosis prediction, showcasing recent key advancements.
is study systematically explores the innovative applications of digital twin (DT) technology in tumor therapy, analyzing its current state, research trends, and key challenges while proposing future directions. Using bibliometric analysis and a scoping review, the rapid growth of DT applications in oncology since 2020 was revealed, particularly in leading countries such as the United States, Germany, Switzerland, and China. The United States' prominent publication output underscores its pivotal role in advancing the integration of digital twin technology with cancer treatment. Germany and Switzerland follow closely, reflecting Europe's robust growth in this area. Notably, large-scale initiatives such as the European Union–funded Ecosystem Digital Twins in Health (EDITH) project and the Swedish Digital Twin Consortium are driving significant progress in the application and innovation of digital twin technology across Europe. China has made substantial research contributions, highlighting its sustained focus and active participation in this field. Moreover, international collaboration has become increasingly close, particularly between the United States, European countries, and China. This cross-border cooperation has facilitated the rapid development of the technology, especially through synergies in data sharing, standardization of technologies, and global clinical trials, which will accelerate the application of digital twin technology in cancer treatment. As international cooperation deepens, the global prospects for the application of digital twin technology in oncology continue to expand. The analysis of funding sources reveals that government agencies, particularly the U.S. National Institutes of Health (NIH), play a leading role in advancing research on digital twin technology in the oncology field. Among the top eight funding institutions, apart from those in the United States and Switzerland, only China’s National Natural Science Foundation provided three instances of funding. This highlights a certain degree of imbalance in the global funding landscape for digital twin research in oncology. Additionally, in October 2024, the National Science Foundation (NSF), the National Institutes of Health (NIH), and the U.S. Food and Drug Administration (FDA) jointly launched the Biomedical Digital Twin Technology Innovation Program (FDT-BioTech), providing over $6 million in funding for 7 projects, further demonstrating the U.S. government's leading role in the field of digital twin technology and its continued support in advancing technological development . In terms of funding structure, government support accounts for 52.88% of the total funding instances, followed by universities and research institutions at 18.27%, while corporate and private funding represent a relatively small share. This indicates that current research heavily relies on public funding, with government support ensuring a degree of independence and the sustained development of fundamental science. However, the limited contributions from corporations and private sponsors may hinder the commercialization and clinical translation of research outcomes . Future efforts should focus on strengthening public–private partnerships, attracting more corporate investment, and fostering deeper integration between research and application. Additionally, the average number of funding sources per study is 2.12, but government-funded projects average only 1.53 sources, slightly below the overall mean. This suggests that current research funding is relatively dispersed, potentially requiring higher levels of coordination and focused investment to address key challenges in digital twin technology for oncology, such as data standardization and interdisciplinary collaboration . Swiss foundations (e.g., OPO Foundation, Margrit Weisheit Foundation) have shown consistent support in this field, providing critical backing for long-term research and innovative exploration. This model offers a valuable reference for other countries and regions: establishing dedicated funds to encourage free exploration and high-risk, high-reward research may be a viable strategy. Overall, although the funding landscape is diverse, it remains predominantly driven by government support. Issues of regional disparity and insufficient investment in applied research remain significant. Future progress may depend on fostering international collaboration, integrating academia and industry, and establishing dedicated funds to accelerate the global development and clinical translation of digital twin technology. Key findings and significance Comparison with existing studies Challenges Limitations of the study Future research directions This study reveals the diversity and application trends of digital twin (DT) technology in the oncology field through an analysis of sample size distribution. The results demonstrate significant variation in sample sizes across studies, ranging from a few cases to tens of thousands of patients. This phenomenon reflects the dual development trajectory of DT technology in oncology: While emphasizing robustness and universality in large-scale clinical applications, equal importance is placed on achieving technological innovation and breakthroughs in specialized fields. Firstly, studies with small sample sizes (< 50 cases) primarily focus on technology validation and early development stages, relying on clinical trial data, medical imaging data, and animal model data. These studies lay the foundation for personalized treatment and precision testing of DT technology. However, the limited sample size may constrain the generalizability of these findings, failing to fully address the clinical needs of broader populations. Future research should aim to expand sample sizes and conduct multi-center, long-term clinical trials to validate the widespread applicability of DT technology. Secondly, studies with large sample sizes (> 500 cases) showcase the broad potential of DT technology in areas such as tumor diagnosis and personalized management. These studies leverage multi-center databases and large-scale datasets, ensuring the representativeness of results and the generalizability of clinical applications. The high clinical translation potential of large-sample studies makes them pivotal in DT research, particularly in widely studied tumor types like lung and breast cancer. From the perspective of data sources and application scenarios, medical imaging and clinical data dominate in diagnostic and treatment decision-making contexts, while virtual patient data and laboratory data are predominantly used for technology validation and personalized management. The Sankey bubble chart illustrates the flow and role of these data types in tumor research, providing a clear direction for future studies. Additionally, the relationship between sample size and tumor type is noteworthy. Breast and lung cancers feature prominently in DT research with large sample sizes, reflecting the broad clinical demand and research focus on these tumors. Conversely, brain tumors and certain specific subtypes are more often studied with small sample sizes, focusing on precision medicine and micro-mechanism exploration. This differentiation highlights the tailored application of DT technology across various tumor types. In summary, the diversity in sample size distribution and data sources underscores the broad application potential and ongoing challenges of DT technology in oncology. Future research should prioritize the integration of large-sample, multi-center data while improving reporting standards and transparency to advance the clinical application and widespread adoption of DT technology. Furthermore, this study systematically analyzes the data processing and artificial intelligence (AI) applications in tumor treatment using DT technology. Clinical and genomic multi-source data provide a realistic foundation for model construction, while AI algorithms play a core role in data processing, dynamic modeling, and multimodal integration. Multi-omics analysis enhances the models' adaptability to individual differences. This synergy between technologies not only facilitates innovative applications but also establishes a solid foundation for achieving precision oncology . For example, a DT-based classification system grounded in systems and mathematical modeling theories and a DT framework for predicting cancer progression in prostate cancer patients both demonstrate the critical significance of data-driven approaches and algorithmic integration in improving model performance and clinical applications. Our study demonstrates that multi‐omics data play an indispensable role in constructing digital twin tumor models while simultaneously exposing several pressing challenges. Specifically, genomic data play a pivotal role in relevant applications by capturing driver gene mutations, which provide a robust foundation for personalized treatment and targeted therapies . Meanwhile, although proteomic and metabolomic data have been explored in seven studies to elucidate signaling pathways and metabolic reprogramming within tumor cells, their integration remains insufficient. The heterogeneity of the data and the lack of standardized protocols limit their potential in the detailed analysis of the tumor microenvironment and treatment response predictions . Therefore, improving data preprocessing and integration methods will be a key focus for future work. Additionally, although single-cell omics currently represent only about 5.71% of the field, they offer unique advantages in revealing tumor cell heterogeneity and dynamic changes in the immune microenvironment . For example, the single-cell electrophysiological model developed by Baumgartner et al. provides a novel perspective for capturing micro immune responses, suggesting that this data type could be further expanded for clinical evaluation in the future. Overall, multi-omics data offer multidimensional support for digital twin models, enhancing their ability to simulate tumor biological characteristics and guide personalized treatment. However, challenges such as data standardization, heterogeneity handling, and integration algorithms remain urgent issues that need to be addressed.
As a scoping review, this study systematically examines the current applications, research trends, and major challenges of digital twin (DT) technology in tumor therapy. Compared with existing review articles, this study presents several notable innovations and advantages: Firstly, Most existing reviews focus on the broad application of DT technology in healthcare or specific tumor types. For instance, Katsoulakis et al. reviewed the potential and challenges of DT technology in healthcare , while Fuchs et al. discussed how digital health technologies and AI promote value-based precision sarcoma care . However, reviews specifically addressing the overall application of DT technology in oncology, encompassing multiple tumor types and application scenarios, remain limited. This study fills that gap by providing a comprehensive overview of DT applications across a wide range of tumor types and scenarios. Secondly, This study offers an in-depth analysis of sample sizes and data sources, revealing application trends, research disparities, and future development directions for DT technology in oncology. Existing reviews often lack such detailed examinations, particularly concerning the relationship between sample size distribution, data sources, and application scenarios. Additionally, Through a systematic review of medical imaging data, multi-omics data integration, and advanced AI algorithms, this study highlights the precise applications of DT technology in tumor therapy. We summarizes the models, technical challenges, and advancements across various application scenarios, includingThis multi-layered and multidimensional analytical perspective provides a richer understanding of DT applications in oncology compared to existing reviews. Finally, by incorporating bibliometric analysis, this study uncovers the global research distribution and funding models of DT technology in oncology. It identifies the rapid growth of DT research and highlights differences in funding support and international collaboration among countries and regions. This global perspective offers new insights into the dynamics of DT technology development. In contrast, existing reviews often lack systematic analyses of global research distribution and funding models, limiting their ability to reflect the role of international collaboration and funding in driving technological advancements. In conclusion, Through the exploration of sample size distribution, data processing and integration, and global research dynamics, this study addresses the limitations in comprehensiveness and detail found in existing reviews. It provides a more systematic and complete picture of the applications of DT technology in tumor therapy. This contribution not only helps the academic community better understand the current state and future trends of DT technology but also offers scientific evidence and guidance for implementing precision oncology in clinical practice.
Global challenges of data diversity and sample size insufficiency Barriers to technical integration Privacy and ethical challenges Challenges in clinical translation and applicability Current status and issues Analysis and recommendations research indicates that the development of digital twin (DT) technology heavily depends on the diversity and adequacy of data samples . However, sample size analysis in this study reveals significant imbalances in tumor-related datasets. Large sample data are primarily concentrated in common cancers such as lung and breast cancer, while rare tumors (e.g., high-grade gliomas) suffer from severe data scarcity. Furthermore, discrepancies in distribution and quality among different data sources (e.g., imaging, omics, and clinical records) limit the generalizability and reliability of DT models .
To address the limitations in model generalization caused by insufficient sample sizes, previous studies have proposed expanding data representativeness through multi-center collaborations. For example, some studies integrated data from multi-center clinical trials and real-world datasets to construct virtual patient cohorts, successfully simulating disease diversity and the effects of various treatments . Other research has utilized generative adversarial networks (GANs) and DT models to merge heterogeneous data from multiple medical centers, thereby enhancing model robustness and predictive capability . Notably, Virtual patient technology has also emerged as a promising approach to extend real patient datasets. This method evaluates the effectiveness of complex treatment strategies by simulating diverse clinical scenarios, significantly improving model validation efficiency. However, uncertainties in input parameters and potential differences between simulated physiological responses and real patients may affect result reliability . To address this, an increasing number of studies have combined real patient data with multi-omics information (e.g., genomic and imaging data) to calibrate virtual patient models, improving their credibility and clinical relevance . Additionally, multi-modal data integration based on DT technology has proven effective in overcoming challenges associated with data heterogeneity and diversity. For instance, Moztarzadeh et al. proposed a DT framework that integrates machine learning and metaverse technologies to construct real-time digital replicas of patients, improving diagnostic and treatment precision and providing new avenues for rare disease research. Similarly, Keller et al. developed a Digital Medical Twin (DMT) platform that supports personalized treatment for complex diseases by integrating and dynamically invoking multi-modal data, offering a feasible framework for multi-center collaboration in rare disease research. At a higher level, the American Society of Clinical Oncology’s subsidiary, CancerLinQ, developed a global large-scale data integration platform that aggregates extensive cancer patient data to guide optimal treatment and improve clinical outcomes . CancerLinQ's success highlights the critical role of large-scale data integration in enhancing DT model performance and the profound impact of institution-driven data utilization models on the industry.
Current Status and Issues Analysis and recommendations While DT technology demonstrates significant potential in data processing and AI applications for tumor therapy, the comprehensive integration of multi-modal data remains a major challenge [ , , ]. Only a limited number of studies [ , , , , , ] have achieved full integration of multi-modal data, primarily due to two major challenges in interdisciplinary collaboration: Insufficient standardization of different data types (e.g., imaging, genomics, and biomechanical information), which severely restricts cross-platform collaboration . The scarcity and incompleteness of available multi-modal data, further hindering the efficient integration of patient-specific information . Moreover, current research tends to focus on genomic and proteomic analyses, while metabolomics and single-cell omics integration remain underutilized. This not only limits the biological depth of modeling but also restricts the full potential of multi-modal integration. Additionally, most studies prioritize optimization within single domains, lacking a comprehensive cross-domain integration framework.
To overcome these barriers, future research should prioritize the integration of metabolomics and single-cell omics data to enhance the biological depth of models, providing multi-dimensional support for precise modeling in tumor therapy. Several innovative tools and frameworks have already demonstrated the potential for technical integration. For example, Xu and Kowalski developed an interactive platform that integrates known and unknown genetic variations to create a comprehensive ‘‘total molecular profile,’’ significantly improving data-sharing efficiency and optimizing multi-modal data integration processes. Similarly, Sainz-DeMena et al. developed the im2mesh tool, which combines medical imaging, AI, and engineering modeling to automatically generate personalized 3D network meshes. This tool not only enhances the efficiency of transitioning from imaging to DT model construction but also offers a modular design with high interoperability, providing a concrete solution for complex diseases such as tumor microenvironment modeling. Merchant et al. proposed a framework to address delays and synchronization issues in large-scale parallel simulations, enabling dynamic behavior modeling and real-time interactive exploration of complex systems. Beyond these technical advances, addressing integration challenges requires robust public–private partnerships and international research alliances to promote standardization. In the near future, global digital twin alliances—bringing together industry, government, academia, and practitioners—will be essential for standardizing digital twin methodologies and establishing interoperability protocols. Initiatives such as the Swedish Digital Twin Alliance , the DigitWins Alliance , and the broader Digital Twin Alliance ® exemplify how collaborative networks can foster shared standards and accelerate cross-domain integration. Moreover, developing a universal digital twin design and development platform is crucial. Early steps in this direction are evidenced by initiatives like Boeing’s virtual shared workspace, which creates a global collaborative environment to harmonize practices among industry partners .
Current status and issues Analysis and recommendations The application of DT technology in oncology involves highly sensitive patient data, such as genomic and imaging data , making privacy protection and the lawful use of data critical ethical challenges . Furthermore, the fairness and transparency of existing algorithms across different populations remain unresolved . Hernandez-Boussard et al. noted that Cancer Patient Digital Twins (CPDT) are susceptible to biases when learning from skewed datasets, reflecting systemic inequities in existing healthcare systems.
Existing studies indicate that GANs can effectively anonymize sensitive patient data by generating synthetic datasets, which not only protect privacy but also expand data availability in data-scarce areas [ , , ]. Similarly, federated learning-based models improve algorithm performance by mitigating risks associated with centralized data sharing . In addition to these techniques, adopting robust privacy-by-design practices—such as strong encryption, secure transmission protocols (e.g., HTTPS or VPN), and regular anomaly detection—can further safeguard sensitive information . Integrating blockchain technology into DT systems also offers a promising means of enhancing data integrity and transparency by enabling decentralized consent management and efficient auditing. Moreover, addressing system-level vulnerabilities, as demonstrated by Zhang et al. with models that capture bidirectional contextual relationships in IoT environments, can mitigate risks like model manipulation. On the ethical front, frameworks such as that proposed by Moztarzadeh et al. , which build real-time, reliable cancer DT models, not only optimize diagnostic and treatment decisions but also reduce inherent biases in clinical applications. For a broader regulatory context, readers may also consult initiatives like the EDITH project’s Strategic Plan, which outlines comprehensive guidelines on data collection, privacy, and ethical standards for deploying DT technology in healthcare .
Current status and issues Analysis and recommendations To address resource constraints in clinical settings, Merchant et al. proposed the use of high-performance computing (HPC) for large-scale agent-based simulations, optimizing distributed computation for real-time interaction. Christenson et al. demonstrated that simplified modeling techniques, such as proper orthogonal decomposition (POD), reduce computational demands, offering solutions for low-resource environments. Lorenzo et al. emphasized the need to quantify numerical approximations and uncertainties in tumor growth modeling to enhance model robustness and generalizability. Wentzel et al. developed the DITTO platform to improve model interpretability. By incorporating visual tools such as time-risk curves, feature contribution analysis, and patient comparisons, DITTO significantly enhances the transparency of model predictions and clinical trust. This approach provides valuable references for promoting DT technology in clinical practice, particularly in building trust and improving model applicability.
Although DT technology shows remarkable potential in experimental studies, its translation from lab to clinic faces numerous challenges. High computational complexity and hardware requirements hinder its adoption in small and medium-sized healthcare institutions [ – ]. Additionally, the transition of complex models to clinical applications demands extremely high reliability and robustness . The lack of interpretability in current models undermines trust from clinicians and patients, impacting their practical application in clinical decision-making . Most research remains in experimental stages, with insufficient external validation and cross-institutional application, raising concerns about the reliability and generalizability of models in real-world clinical settings .
Despite providing a comprehensive analysis, this study has several limitations. First, the exclusive use of English-language sources may have underestimated the breadth of relevant literature in this field. Second, the inclusion of studies relied on subjective judgment by the researchers, which could introduce selection bias. Additionally, as the application of digital twin (DT) technology in tumor therapy remains in its early stages, the limited number of related studies may affect the comprehensiveness and representativeness of the analysis. Lastly, the pragmatic approach to identifying studies explicitly labeled as “digital twin” might have excluded relevant papers describing predictive models without explicitly using this term, potentially underestimating the scope of published literature on DT applications in oncology.
Based on the findings of this study, future research should prioritize the following areas: Data Diversity and Shared Platforms Through international multi-center collaboration, a diverse and high-quality tumor data-sharing platform can be established to ensure the representativeness and adaptability of sample distributions. This not only enhances the generalization capability of digital twin models but also fosters technological exchange and collaboration between different countries and regions . For example, a global tumor data repository could be developed to integrate clinical trial data and real-world data from various countries, supporting broader model training and validation. 2. Standardization of Tools and Frameworks To address the challenges of technical integration, future research should focus on driving the standardization of technical tools and data frameworks, particularly in unifying data formats, interface standards, and validation methods. This will improve the efficiency of multi-modal data integration, provide more accurate support for model validation, and resolve the fragmentation of imaging, omics, and dynamic physiological data, thereby enhancing the applicability and comparability of digital twin technology. Furthermore, developing highly interoperable platforms is critical for promoting interdisciplinary and cross-institutional collaboration, as it will help accelerate the development of standardized technical methods and interoperability protocols, especially for the global implementation of digital twin technology. Strengthening public–private partnerships will not only drive the implementation of these standards but also foster cross-domain collaboration, ensuring the effective integration of technical tools in complex issues like tumor treatment. 3. Data Privacy and Ethical Standards Strengthen research on data privacy protection and ethical standards to ensure the lawful use and privacy protection of patient data during the application of digital twin technology. Building on current practices, future efforts should focus on refining privacy-by-design strategies, including robust encryption, secure transmission, and blockchain-enabled consent management. Additionally, developing mechanisms to address data biases, enhance algorithmic transparency, and ensure equitable representation across diverse patient populations will be essential. Research should also explore ways to integrate real-time ethical decision-making frameworks into DT systems to further align with evolving healthcare regulations and maintain patient trust. 4. Clinical translation and validation Accelerating the clinical translation of DT technology requires multi-center clinical trials to validate the applicability and reliability of models. Large-scale trials should test the efficacy and utility of DT models across different tumor types and treatment stages. Enhancing the training of clinicians and technical staff will improve their ability to integrate DT technology into clinical practice . Meanwhile, developing user-friendly tools with intuitive interfaces will enable non-technical users (e.g., clinicians) to adopt these technologies more easily. Additionally, Regulatory bodies such as the FDA (U.S.) and EMA (Europe) must also establish clear guidelines for using DT technology in clinical trials . 5. Optimization of virtual patient modeling Future advancements should focus on developing virtual patient modeling technologies with higher biological fidelity to better reflect the complex physiological states of patients. Meanwhile, optimize multimodal data integration methods to enhance the accuracy and real-time performance of the model.
Digital twin technology demonstrates immense potential in tumor therapy by optimizing personalized treatment plans through multi-modal data integration and dynamic modeling. However, challenges such as insufficient data diversity, imbalanced sample sizes, limited interoperability of technical frameworks, and issues related to data privacy and ethics remain unresolved. Future efforts should focus on fostering international collaboration, building data-sharing platforms, standardizing cross-disciplinary frameworks, and strengthening ethical guidelines to promote the widespread application of DT technology in precision oncology. These measures will improve treatment outcomes and the quality of life for patients. As technology advances and research deepens, DT technology has the potential to transition from the ‘‘virtual’’ to the ‘‘real,’’ becoming a critical tool in precision medicine and contributing to the global fight against cancer. This study provides a systematic analysis of DT applications in tumor therapy, offering a comprehensive perspective and outlining directions for future research. It is hoped that this study will attract academic attention and facilitate the further development and application of DT technology in precision oncology.
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Effectiveness of mDiabetes intervention in enhancing diabetes awareness and promoting healthy lifestyle changes among the general population in rural India | 2cc9285a-6efe-41bf-b48f-ea49542c2794 | 11818752 | Health Promotion[mh] | India has emerged as a major hub of diabetes cases, affecting both urban and rural regions. The study conducted by the ICMR-INDIAB group has revealed that in India, more than 101 million individuals aged 18 and above are living with diabetes. In addition, there are about 135 million adults who are considered to be in a pre-diabetic state ( ). A recent pooled systematic review and meta-analysis of 1.7 million adults showed prevalence of diabetes increased in both rural and urban India from 2.4% and 3.3% in 1972 to 15.0% and 19.0%, respectively, in the year 2015–2019 ( ). These increasing trends of narrow urban and rural divide and equal gender affliction are alarming and can be attributed to changes in dietary and physical activity habits, along with economic growth patterns ( ). The rising diabetes burden has led to significant morbidity, mortality, and economic implications due to associated micro and macrovascular complications ( , ). Moreover, the indirect cost from premature death due to diabetes was over 64% in Low middle Income countries (LMICs) and 60% in high Income Countries [HICs; ( )]. Individuals with diabetes in LMICs tend to die at a younger and more productive age than do people with diabetes in HICs ( ). Urgent measures are required to address this growing health crisis and its socio-economic consequences. Community-based programs using technology have proven to be highly effective in reaching a diverse audience, especially in areas with limited healthcare resources and restricted access to medical services ( ). Global experts in diabetes prevention recommend the establishment of diabetes prevention programs led by non-physician personnel, utilizing technology, and community education ( ). Mobile health (mHealth) technology, particularly in the form of text messaging, has shown great promise in preventing diabetes and its complications, facilitating behavior change in low/middle income countries ( , ). With a significant number of mobile users in India, mHealth technology (mobile interventions) for the prevention of diabetes and diabetes-related complications is a promising option. Diabetes prevention messages delivered via SMS, personalized text messages, mobile apps, television-based lifestyle interventions, and weekly coaching calls have demonstrated a significant positive impact on the primary prevention of diabetes and the monitoring of diabetes control ( – ). However, these studies predominantly focused on users from urban locations. There is a need to study the effectiveness of such mHealth interventions in rural areas. Additionally, community health education, implemented through community health workers in LMICs, has demonstrated positive impacts on diabetes prevention and control ( ). Combining these approaches, the present study aims to assess the effectiveness of a combined mHealth (diabetes prevention messages over voice calls) and community health education intervention, delivered by community health workers, in raising awareness about diabetes, promoting dietary and lifestyle changes among a rural population cohort in the southern state of Andhra Pradesh, India.
Study design Study population Intervention design and implementation Data collection: KAP assessment Statistical analysis mDiabetes program is a 1 year, community-based intervention with a pre-test and post-test, quasi-experimental design. The study was approved by the institutional review board (LEC-BHR-P-06-21-665). Each participant provided verbal informed consent, and the study followed the Declaration of Helsinki.
The baseline assessment, conducted in October and November 2021, involved 1,019 subjects (The study recruitment period was between October 1st 2021–November 30th 2021). Among these participants, participants who expressed willingness to continue were included in the endline assessment and follow-up survey, conducted in November 2022 and February 2023, respectively. The inclusion criteria included participants who were 18 years or older, mobile users from the LV Prasad Eye Institute (LVPEI) patient database within the project area, and who were willing to participate by providing informed verbal consent. We initially approached 1,019 subjects from a larger database of 103,538 individuals, anticipating a minimum of 500 participants to complete all three surveys (baseline, endline, and follow-up). The endline survey ( n = 710) was conducted immediately after the intervention at 6 months, while the follow-up survey ( n = 625) was conducted 3 months after endline survey. Analysis compared data from 545 subjects who participated in all three surveys. Comparative analysis of baseline responses between participants ( n = 545) and non-participants ( n = 474) showed similarity, with no significant differences observed ( ).
Arogya World's mDiabetes program is an mHealth solution designed to provide diabetes prevention and control information through messages sent to the mobile phones of people-regardless of their risk status. In collaboration with the Rollins School of Public Health at Emory University, 56 mDiabetes messages were developed in 2011 based on the transtheoretical model of behavior change ( ). The content was made available in 12 languages and was reviewed by Arogya World's Behavior Change Task Force, which consisted of national and international experts in diabetes research, public health and behavior change. These 56 messages are delivered as automated voice calls (twice a week) for 6 months in the current study. These messages, categorized into five key themes, focused on medical information (20 messages), lifestyle changes (8 messages), nutrition (13 messages), fitness and physical activity (9 messages), and motivational content (6 messages). The messages provided practical advice on managing diabetes complications, adopting healthy habits such as balanced nutrition, quitting smoking, and engaging in daily physical activity like walking and yoga. Each message was concise, with a character range of 84–160 characters, ensuring clarity and accessibility. The content was designed in simple, actionable language to encourage participants to make sustainable lifestyle changes and improve diabetes awareness (examples of message content— ). During the registration process, participants were instructed to save the mobile number from which they would receive voice calls under the name “Arogya mDiabetes” on their phones to ensure they recognized the calls. If a mobile phone was unreachable or a participant did not respond, the scheduled voice call was reattempted at two different subsequent times.IMI Mobile was the technology partner for delivering automated mDiabetes voice calls. Pre-recorded content, tested for accuracy and reliability, was sent in Telugu between 4 and 7 p.m. over 6 months. If a call was missed, two retries were made at 5 min intervals. The decision to send messages twice a week was based on balancing effective intervention delivery with participant engagement and adherence, particularly in rural settings. Research indicates that overly frequent messaging can cause fatigue, reducing intervention effectiveness while infrequent messaging may fail to sustain behavioral changes ( ). This frequency also considers participant time constraints, mobile data usage, and communication preferences, making it an optimal choice for maintaining engagement without overwhelming recipients. The mDiabetes project employed mHealth technology to disseminate 56 diabetes prevention messages in the regional language (Telugu) via voice calls to over 1,03,538 individuals over a 6 month period. Study participants were recruited from the pool of mDiabetes project subjects in Andhra Pradesh's districts of Krishna, Nellore, East Godavari, and Krishna ( shows the project area map). The study methodology is comprehensively illustrated in : Study Flow Chart. To ensure effective dissemination, around 400 frontline workers (ASHA (Accredited Social Health Activists) were trained in delivering diabetes prevention messages. The training of frontline workers (ASHA) was conducted systematically on online platforms due to the COVID-19 pandemic, equipping them with the knowledge and skills to deliver diabetes prevention messages effectively. The content was structured into four modules: Module I focused on understanding diabetes, its types, risk factors, and symptoms; Module II addressed diabetes-related complications (retinopathy, neuropathy, nephropathy, and cardiovascular diseases) and their management; Module III emphasized healthy eating, promoting a balanced diet with whole grains, proteins, fruits, and vegetables while limiting sugar, salt, and fat; and Module IV highlighted the importance of physical activity, recommending 150 min of weekly exercise and strategies to overcome barriers. Training materials, including flip charts and educational leaflets, were translated into regional languages and validated for accuracy to ensure effective communication. A total of 136 community health education meetings were conducted by trained frontline workers to reinforce key diabetes prevention messages, reaching 5,681 participants (2,393 males and 3,288 females). The sessions focused on practical strategies, including understanding diabetes, its risk factors and symptoms, preventing complications, adopting management strategies, promoting healthy eating habits, and encouraging regular physical activity while addressing barriers. Flip charts and translated educational materials were used to enhance engagement and understanding, and 10,000 educational leaflets on diabetes prevention (sample leaflets enclosed— ) were distributed across the project area to further reinforce the messages. Events were held to celebrate World Diabetes Day in the project area, further promoting awareness about diabetes and its potential complications. Diabetes prevention videos were thoughtfully played in LV Prasad Eye Institute centers targeting patients and their attendants as a part of the education campaign. The field investigators collected periodic feedback from the residents in the field area, ensuring valuable insights for continuous improvement.
Field investigators have been provided with phone numbers and addresses of participants from the LVPEI patient database (General population) who received M DIABETES m-health intervention in the form of audio messages. Investigators employed a convenience sampling strategy, reaching out to participants at their study locations and recruiting those who expressed willingness to participate in the study. A convenience sampling strategy was chosen for logistic reasons and to accommodate the constraints of public health settings. The questionnaires administered at three time points: baseline (before the intervention), endline (immediately after the intervention at 6 months)aft and follow up (3 months after endline survey) collected demographic information, knowledge, attitudes, and practices related to diabetes, physical activity, and dietary habits. The endline questionnaire included additional questions about the impact of diabetes prevention awareness messages on lifestyle changes. The follow-up questionnaire aimed to assess the effect of community educational interventions. Field investigators received comprehensive training on the proper administration of questionnaires to ensure consistency and reliability in data collection, which involved the use of Google Forms with timestamps. Furthermore, to verify the accuracy of the responses, a validation process was implemented, wherein entries from each investigator were systematically audited for correctness and completeness. The collected data provided insights into the effectiveness of diabetes prevention awareness messages and their impact on lifestyle modifications among the study participants.
Statistical data analysis was conducted in 2023 through rigorous coding and cleaning of the collected datasets. One thousand and nineteen participants responded to the baseline survey, and 545 of them consistently responded in the two subsequent post-intervention surveys. Participant responses were analyzed using descriptive statistics (frequency and proportion) and inferential statistical methods in IBM SPSS Version 21 software. We applied a paired t -test to assess the statistical differences between the pre- and post-intervention endline and follow-up survey responses. We used independent-test for the age and χ 2 test for dichotomous variables to observe the baseline characteristics of follow-up visit participants and non-responders group ( ). Additionally, we ran ANOVA test to observe the influence of demographic and socio-economic factors (i.e., gender, age, education, and occupation) on intervention outcomes. A significance level of p -value set at 0.05 to interpret the intervention outcomes.
Study population characteristics Awareness—DM Impact of intervention (diabetes prevention messages) Practice Influence of socio-demographic factors on mDiabetes intervention outcomes Pre- and post-intervention responses did not show any significant gender and age-specific differences, However, education level and occupation influenced some survey outcomes. Participants with education levels of class 10 and above demonstrated greater knowledge of diabetes risk factors (e.g., hypertension and family history) and secondary complications (e.g., foot disease, nerve damage). They also reported notable improvements in healthy behaviors s such as increased consumption of fruits and vegetables, reduced intake of high-fat foods, and greater use of stairs ( P < 0.05) between baseline and post-intervention surveys. In terms of occupation, business owners, skilled laborers, and employed participants exhibited higher awareness of secondary complications (e.g., foot disease, heart failure, nerve damage), and more frequent engagement in healthy practices, including increased fruit consumption, avoidance of high-fat foods, and engagement in physical activities like taking short walking breaks and assisting with household tasks compared to unskilled workers and unemployed participants ( P < 0.05). These trends were consistent across baseline, endline, and follow-up surveys.
The final analysis involves a comprehensive comparison of data from the same 545 subjects who actively participated in the baseline, endline, and follow-up surveys. The study cohort consisted of 45.5% males and 54.5% females, with an age range spanning from 19 to 85 years, and a mean age of 55.42; SD 10.3 years. Regarding education, 7.9% ( n = 43) completed higher education, 47.7% ( n = 260) completed 10 years of schooling, and 44.4% ( n = 242) either had no formal education or it was not applicable to them. Occupationally, 52.2% ( n = 286) were employed in office jobs, 18.3% ( n = 100) were skilled laborers, 22.2% ( n = 121) were unskilled laborers, 4.6% ( n = 25) were business owners, and 2.7% ( n = 13) were unemployed.
During the baseline assessment, a notable 82.75% of the participants demonstrated awareness of diabetes. However, following the intervention, there was a significant increase in awareness levels. In the endline assessment, 97.43% reported being aware of information related to diabetes prevention and this awareness further improved to 99.63% in the follow-up survey. illustrates the results of the analysis, focusing on respondents who indicated a positive response (i.e., “Yes”) for each question. The intervention had a significant impact on enhancing awareness about diabetes causes, particularly in relation to overweight/obesity, hypertension, poor eating habits, inactive lifestyle, and family history. However, it is noteworthy that awareness of the role of lack of regular exercise remained relatively unchanged despite the intervention. illustrates the level of awareness among study subjects regarding diabetes complications. The intervention resulted in a substantial increase in awareness of diabetes-related complications, particularly evident in endline and follow-up surveys compared to baseline. Notably, there were significant improvements in awareness regarding complication such as loss of vision (90% to 98%), heart stroke (45% to 78%), and kidney disease (60% to 83%) p < 0.001. Additionally, awareness levels of other complications also increased. presents the attitudes and perceptions of study participants related to diabetes. The comparative analysis shows significant increases in agreement on key statements across different time points. The belief that diabetes can be prevented saw a remarkable rise from 25.5% (baseline) to 61.4% (endline) and further to 69.5% (follow up). Understanding lifestyle changes as the best way to control diabetes also witnessed a notable increase from from 72.6% (baseline) to 90.3% (endline) and 80.9% (follow up). Moreover, the recognition of regular blood glucose monitoring as essential for better diabetes management showed substantial growth, increasing from 79% (baseline) to 95.2% (endline) and 85.1% (follow up).
Over 90% of the participants recalled receiving messages about diabetes prevention in both the endline and follow-up groups ( ). Among them, 83% in the endline and 73% in the follow-up group reported making necessary lifestyle changes. The messages had a particularly strong impact on promoting healthy eating habits, encouraging lifestyle changes, and fostering the adoption of medical information adoption, resulting in certain beneficial modifications in the participants' behaviors in both endline and follow-up assessment. In the follow-up group, it was observed that 21.5% of the study participants expressed limitation in their ability to engage in regular walking.
The practice section involved assessing the dietary habits and physical activity of the study participants. Practice (Dietary habits) Practice (Physical activity) shows change in dietary habits of the study participants. The endline and follow-up groups showed improvements in healthy eating habits compared to the baseline group. In the endline group, the percentage of participants who reported daily fruit consumption increased from 28.4% at baseline to 51.4%. Additionally, the proportion of participants avoiding high-fat foods rose from 36.8% to 69.9%. In the follow-up group, daily fruit consumption increased from 28.4% at baseline to 78.5%, and the avoidance of high-fat foods increased from 36.8% to 67.7%.
shows comparison of physical activity patterns of study participants among baseline, endline and follow-up groups. The endline and follow-up groups demonstrated significant improvements in physical activity compared to the baseline group. The data highlights significant changes in behavior across the baseline, endline, and follow-up groups. Daily participation in yoga, running, gym, and aerobics increased notably in the endline (38.7%) and follow-up (38.7%) groups compared to the baseline (7.3%) group ( p < 0.001). Similarly, there was a significant increase in outdoor sports engagement in the endline (15%) and follow-up (15%) groups compared to the baseline (0.5%) group ( p < 0.001). Notably, lifestyle behaviors like regular stair usage (59.8%), walking for daily chores (84.7%), and taking short walking breaks (93%) all demonstrated significantly higher percentages in the follow-up group compared to the baseline and endline groups ( p < 0.001). These findings indicate a clear positive trend in physical activity and lifestyle choices for the Follow-up group, with particular emphasis on their preference for stairs, walking, and short breaks during work activities. Majority 482/545(88%)/subjects in follow-up group attended community awareness meetings conducted by community health workers in study locations.
study findings showcased a significant and positive transformation in awareness, attitude, perceptions, and practices related to diabetes prevention among endline and follow-up groups, surpassing the baseline. The combination of mHealth and community health education interventions had a significant impact in increasing awareness about diabetes among the rural population in India. The intervention's success is evident from a marked improvement in diabetes awareness. In the endline assessment, 97.43% of study participants reported awareness, and in the follow-up survey, the figure increased to 99.63%, compared to the baseline rate of 82.75%. In a prior survey in South India, only 47% of the rural population was found to be aware of diabetes ( ). The current study's higher awareness levels at baseline, endline, and follow-up indicate a substantial improvement in study participants awareness. The intervention had a notable impact on how participants in different groups understood the causes of diabetes. At the baseline (before the intervention), only 51.7% of participants were aware of the link between being overweight or obese and diabetes. After the intervention, this awareness significantly increased to 95% in the endline group and 95.2% in the follow-up group. Similarly, recognition of hypertension as a cause of diabetes rose significantly from 64.9% at baseline to an impressive 88.6% in the endline group, followed by 83.8% in the follow-up group. Awareness of other factors like poor eating habits, inactive lifestyle, and family history also showed considerable increases in all three groups, highlighting the intervention's effectiveness in enhancing diabetes-related knowledge among participants. However, it's worth noting that awareness of the importance of regular exercise in preventing diabetes saw minimal change across all three groups despite the intervention efforts. In the ICMR-INDIAB study, knowledge of diabetes risk factors indicated that 59.8% recognized consuming more sweets (eating habits), while overweight or obesity was identified by only 35.5%, family history of diabetes by 17.7%, high blood pressure by 23.2%, lack of physical activity by 16.5%, and mental stress by 12.2% of the general population. Importantly, awareness about risk factors for diabetes was higher in people with known diabetes compared to the general population. In contrast, our study's findings demonstrate a more substantial improvement in diabetes-related knowledge compared to the INDIAB study ( ). The intervention led tos such as loss of vision (90% to 98%), heart stroke (45% to 78%), and kidney disease (60% to 83%). Additionally, awareness levels increased for other complications as well. In the INDIAB study, the most commonly reported affected organs were the feet (54.0%), eyes (52.3%), kidneys (36.3%), heart (33.6%), and nerves (18.7%). Other reported complications included lung problems (19.6%), brain diseases (26.6%), and stomach disorders [16.9%; ( )]. The intervention's positive impact extended to participants' attitudes and perceptions associated with diabetes, with substantial increases in agreement on key statements. Participants' belief in diabetes preventability rose from 25.5% to an encouraging 69.5% emphasizing a hopeful mindset. Additionally, understanding the significance of lifestyle changes for diabetes control increased from 72.6% to 80.9%, while the importance of regular blood glucose monitoring saw a notable increase from 79% to 85.1%. In the INDIAB study, it was observed that 56.3% of participants exhibited an awareness of diabetes. Among these participants, 36.8% ( n = 1,185) indicated “diet” as a preventive measure, while 45.8% ( n = 1,474) acknowledged that both “diet and exercise” play a role in diabetes prevention ( ). The enhanced levels of awareness regarding diabetes control measures highlighted in our study could potentially be attributed to the implementation of mhealth interventions, as well as community education initiatives. These endeavors took the form of regular awareness meetings and the distribution of informative materials within the project area. These findings highlight the behavior change intervention's effectiveness of the in promoting positive attitudes toward diabetes management and improving knowledge among the study participants, which is in cognizance with several national and international studies ( , – ). For instance, a study in rural Bangladesh demonstrated that mHealth messaging improved T2DM knowledge and awareness, with messages being actively discussed and disseminated. However, sustained behavior change was challenged by social norms and habits, with participants expressing a preference for group discussions over messaging alone. This highlights that while mHealth is valuable as part of multi-component strategies for diabetes prevention, it may be less effective as a stand-alone intervention for addressing complex, socially influenced behaviors ( , ). A study in South East India found mobile phone messaging to be effective in reducing the incidence of type 2 diabetes among men with impaired glucose tolerance. Diabetes developed in 18% of the intervention group compared to 27% in controls (HR 0.64, 95% CI 0.45–0.92; p = 0.015), demonstrating that messaging can be a practical and acceptable method for supporting lifestyle modifications in high-risk individuals ( ). These examples further validate the positive impact of mHealth interventions in enhancing diabetes prevention and management efforts. The study's results unveiled the significant influence of diabetes awareness messages on behavioral changes.participants' healthy eating habits, lifestyle choices, and medical information adoption were positively influenced, leading to beneficial behavioral modifications. While the majority showed impressive progress, it is important to acknowledge that the follow-up group encountered certain challenges, with 21.5% of participants reporting limitations in their ability to go for a walk, suggesting potential obstacles in implementing certain behavioral changes in this subgroup. This finding underscores the importance of addressing individual barriers and customizing interventions to accommodate diverse needs ( – ). The study participants in both the endline and follow-up groups demonstrated remarkable improvements in healthy eating habits compared to the baseline group. Daily fruit consumption increased significantly, with the endline group showing an increase from 28.4% to 51.4% and the follow-up group reaching an inspiring 78.5%. Additionally, consistent avoidance of high-fat foods also saw improvements, with the endline group rising from 36.8% to 69.9% in the follow-up group reaching an 67.7%. These findings reveal the intervention's positive impact of the intervention in fostering healthier dietary choices among the study participants. Lin et al. demonstrated a notable decrease in HbA1c levels and an improvement in healthy diet after the 6 month health coaching. Patients in the intervention group reduced their daily intake of whole grains, fruits, meats, and proteins, as well as fats and oils, while increasing their intake of vegetables ( ). Similar health coaching sessions involving goal setting, community education, and telephone and mobile app interventions have demonstrated a positive impact in various studies ( – ). Lifestyle medicine is emerging as a pivotal discipline in the management of chronic diseases like diabetes. The practice of lifestyle medicine necessitates proficiency in addressing multiple health risk behaviors and enhancing self-management. This entails targeting areas such as diet, physical activity, behaviors change, body weight control, adherence to treatment plans, stress and coping, spirituality, mind-body techniques, and tobacco and substance abuse ( ). Our study findings shed light on some of these key areas. Physical activity levels showed a notable increase in both the endline and follow-up groups when compared to the baseline group. Participation in a variety of physical activities, including exercise, yoga, running, gym, aerobics, and outdoor sports, increased significantly, with p -values indicating highly significant changes ( p < 0.0001). The follow-up group demonstrated additional positive behaviors, such as choosing stairs over elevators, walking for daily tasks, and incorporating walking breaks during work, indicating sustained and improved adoption of physical activity. These findings underscore the effectiveness of the intervention in promoting an active and healthier lifestyle among the study participants. The positive impact of physical activity interventions on diabetes prevention and management has been highlighted in diverse studies ( – ). Our results indicated that age and gender did not significantly impact the effectiveness of the mDiabetes intervention. However, intervention was more impactful among participants with higher levels of education and employment status, particularly in improving knowledge of diabetes-related complications, and lifestyle determinants such as dietary habits and physical activities as observed in both baseline and post-intervention surveys. Supporting evidence from a cross-sectional analysis of 44 LMICs underscores the critical role of education in diabetes prevention. Individuals with secondary schooling were 6.5% points more likely to receive dietary counseling and 21.3% points more likely to undergo blood glucose screening compared to those with no formal education ( ). Similarly, data from India's National Family Health Survey (NFHS-5, 2019–2021) reveal stark socioeconomic disparities, with individuals in the highest wealth quintile being significantly more likely to be aware of, treated for, and have their diabetes under control compared to those in the lowest quintile ( p < 0.001). Diabetes awareness, treatment, and control (ATC) remain notably low among poorer and less educated groups ( ). The mDiabetes program by Arogya World demonstrated a 15% cumulative improvement in diabetes risk behaviors among 1 million participants over 6 months through 56 text messages, promoting increased exercise, and fruit, and vegetable intake ( ). Similarly, the myArogya app implemented at Arogya World Healthy Workplaces in Bengaluru, led to significant reductions in HbA1c and blood pressure levels and increased physical activity among prediabetic individuals ( ). A television-based lifestyle intervention for high-risk T2D patients across three cities improved cardiometabolic risk factors, physical activity, dietary habits, and weight loss, with higher engagement in video content, yielding better outcomes ( ). Furthermore, a randomized controlled trial on personalized text messaging for newly diagnosed T2D patients demonstrated reductions in HbA1c and LDL-c, highlighting the importance of sustained behavioral change ( ). These findings align with our study's results, demonstrating the effectiveness of mHealth interventions in improving diabetes awareness, addressing risk factors, and promoting behavioral changes, underscoring mHealth as a scalable tool for diabetes prevention and management.
The strengths of the present study encompass a comprehensive approach to creating awareness about diabetes prevention and its complications through a multipronged strategy. This encompassed utilizing m-health interventions, community education in the form of awareness meetings, and distribution of informational materials, effectively reaching a population of over 100,000 in the project area. By conducting interviews with the same participants at baseline, endline, and follow-up, the longitudinal data provided valuable insights into knowledge, attitudes, and practices related to diabetes prevention. However, the study also has its limitations. Generalizing the findings to regions with diverse socio demographics, such as urban areas or populations with varied cultural and ethnic backgrounds, may be challenging. Additionally, the absence of objective measurements like blood sugar levels, body weight, and body mass index limits the ability to fully assess the impact of the interventions. Digital literacy of participants was not assessed, which could have influenced the effectiveness of the mHealth interventions. Furthermore, the data collected is based on self-assessments and participant evaluations, lacking validation from health institutions or clinical records to confirm actual reductions in health issues. Finally, while “community awareness” is inferred from aggregated individual responses, direct measures of community-level awareness, such as qualitative assessments or public institution interviews, were not conducted.
The integrated mHealth and community health education intervention proved to be a game changer in diabetes prevention among the rural population in India. The significant improvements in awareness, dietary habits, and physical activity among the study participants showcase the intervention's effectiveness in fostering preventive behaviors and encouraging healthy lifestyle changes. The sustained and enhanced physical activity adoption observed in the follow-up group further highlights the potential long-term impact of the intervention. These study serves as a valuable blueprint for future public health initiatives aimed at tackling diabetes prevention resource-limited settings, offering hope and better health prospects for the communities involved.
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The opinions of GDPs on the challenges of managing tooth wear in primary dental care | f36a6ce7-e081-4361-b599-994f30ea2bb8 | 10037362 | Dental[mh] | Three general dental practitioners (GDPs), each with over 20 years' experience in a variety of healthcare settings, but predominantly in general dental practice, were interviewed about their opinions of how patients with tooth wear are managed in primary care. The interviewees were included on the basis that they worked in differing primary dental care settings in England, had significant professional experience themselves and worked with other colleagues of varying ages and experiences. Their practices models were: 1) majority NHS-funded; 2) majority private, fee-per-item; and 3) majority private, capitation and insurance scheme ( ). The interviewees had mostly worked in either the North or the South of England and their practice colleagues had experience in several other areas of the country. None had pursued post-graduate studies related to the management of tooth wear, which was important to attempt to avoid opinions of those which are likely to differ from most GDPs. A series of ten questions to be discussed was developed in advance, with contribution from and agreement with the interviewees. They were encouraged to discuss the questions and likely responses with other colleagues working at their practices before the interviews. Two weeks later, an initial semi-structured group discussion with the three interviewees was held via a virtual meeting, with the questions presented and discussions moderated by the authors and the dialogue recorded for later transcription. The transcription was shared with the interviewees. Two weeks later, a second group discussion was held via a virtual meeting, with each question and previous response discussed again. The transcription was again shared within the group, to ensure the responses correctly represented their views.
Are you seeing younger people with a lot more tooth wear presenting or is it older people who are just keeping their teeth for longer? What would prompt you to start discussing your patients' tooth wear with them? How do you and your colleagues monitor a patient's tooth wear? If you thought the patient may have an undiagnosed gastroesophageal problem, would you contact their general medical practitioner? What would prompt you to start offering treatment? Is tooth wear something your practice team has a focus on? It is important that we as professionals still focus on an issue such as tooth wear, even if it is probably irrelevant for many patients. Our experience normally tells us who are the 10% of patients we need to ask more questions to. What is an acceptable standard of dental care for tooth wear? What restrictions are there for managing tooth wear in your practices? What have been your experiences of referring tooth wear patients into a dental hospital for treatment? What is your confidence with maintaining these patients? Can tooth wear be effectively managed in primary dental care? 'There are two aspects to this: managing prevention of tooth wear and managing treatment of tooth wear. At present, neither of these are resourced well in this country and the biggest NHS focus is improving access for any dental care' 'I don't know if there are any healthcare systems in the world that have worked out how to fund or incentivise prevention. So, we either work out a way to improve the prevention side or we must accept we can only treat some patients who have an advanced condition. These patients must be motivated through either symptoms or appearance or have enough understanding of why intervening earlier is worthwhile. That makes it such a challenging area. The public has a better understanding of tooth decay and to some extent, periodontal disease. But not tooth wear' 'There isn't enough funding for Tier 2 services and Managed Clinical Networks for tooth wear. Under the UDA system, there would need to be a separate band for complex treatments, such as this. It needs something like the old "prior approval" system, where we do an additional, more detailed assessment, demonstrating why this is an advanced case, requiring extra UDAs and get that approved before starting' 'At a public health level, we need to consider what the public money is spent on. We need to ask: "should NHS dentistry only be for the essentials, such as tooth decay, gum disease, simple tooth replacement, mouth cancer screening etc?" If you happen to come across some tooth wear when examining a patient, is the dentist's only role to explain that it is happening and make the patient aware of the problem but not actually to try to fix it? Perhaps the NHS has got more important things to spend the public's money on than tooth wear' 'Let us be pragmatic about the budget required to manage tooth wear, the complexity of the treatments, the longevity of restorations and the burden of maintenance. Is it "only tooth wear" for most people? Most people don't complain about their ability to eat. Very few are really affected by sensitivity. It really is just a cosmetic problem for most of these patients'.
'People are certainly keeping their teeth longer, so they are wearing out more and we see some more tooth wear, sensitivity and facial pain' 'Over my 20-plus years in practice, there is a pattern of increasing numbers of younger patients and some worrying features, such as combined erosion and attrition with really damaged occlusal surfaces of their posterior teeth. The older patients tend to either have a mouth full of restorations or a lifetime of tooth wear. Tooth wear is a long-term condition for our older patients, with an occasional acute problem' 'In our practices, despite the majority of patients having at least some tooth wear, most just don't see it as a problem. If they have a cavity and toothache, they know they have something wrong. But the tooth wear is rarely an issue for them, until they have symptoms or an aesthetic concern'.
'I rarely consider low-level wear to be an issue and don't often mention it - it is usually just physiological tooth wear. For moderate and severe tooth wear, whether the patient has noticed a problem or not, it is important to mention this with them. I offer the opportunity to have further discussions about treatment, when the patient is ready to consider it' 'NHS practices aren't funded for working like this. We think about whether to get into a conversation about tooth wear, including explaining the diagnosis and how to prevent and treat it. Considering how quickly we all have to work in general practice, I have to just ask myself "am I worried by what I'm seeing?" due the extent of the damage or because of their symptoms: "is this active tooth wear or are we seeing the long-term gradual changes in a stable mouth?" Most patients are usually just not that bothered by tooth wear. The challenge is to explain the condition and to go through the prevention and consequences of treating or not treating it, with the minority who are concerned' 'I ask myself that if I don't talk about the tooth wear and it deteriorates, will I be in trouble? Secondly, is there going to be a reasonably easy way to treat this, or will I wish I'd never mentioned it?'
'I don't have a structured, "scientific" way of monitoring it. For example, I don't take serial study models. If someone has significant attrition, then the only treatment I would feel capable of giving is a soft splint. I don't think I'm qualified to do anything more than that. The only time I intervene is when people have an aesthetic issue with their teeth' 'We don't repeatedly monitor tooth wear at all. We are a mainly UDA-based [Units of Dental Activity] practice. We struggle to do everything we need to do in a check-up under one UDA, never mind monitoring tooth wear. It is mostly based around highlighting and informing the patient about the condition. Rarely, if everything was done and we had a very interested patient, you might offer to do some study models and photographs for their own awareness and to be kept for monitoring' 'We don't often use the Smith and Knight index, BEWE [Basic Erosive Wear Examination] or anything similar. I just describe it as early, moderate or severe and comment whether it is into dentine, pulp seen or at gingival level. There is just no funding or resource for it under UDAs, unfortunately'.
'Perhaps, especially if the teeth are showing signs of erosion but the diet isn't particularly acidic' 'Only if I am really concerned and just advise them to discuss it next time they attend with their GP. I wouldn't refer them myself unless I was concerned that they weren't capable of doing so themselves' 'I wouldn't write to the GP myself. Whether it is reflux problems for tooth wear or assessing diabetes in a patient with periodontal disease, I would just advise the patient to speak to their GP'.
'The likelihood to intervene is much higher in a younger person. I'd be more worried about a 17-year-old with erosion and attrition that a 70-year-old with buccal abrasion cavities. We are less inclined to intervene with the older patient for two reasons: they don't see it as an issue and they question whether it is going to make a significant difference to them keeping their teeth' 'If a patient isn't interested, motivated or even aware when we mention it, they are not normally going to want to do anything about it. I feel uneasy talking to them about treatments that they have said they feel are unnecessary or unimportant for them. However, when they are aware of an aesthetic issue, it is much easier to discuss treatment' 'My initial treatment step would be preventive, to stop the tooth wear getting worse. I would ask about diet if I was worried about erosion. The only other intervention I might do for a bruxist is to give a splint. If the patient is not motivated, if they don't see the benefit of the splint, they are never going to wear it. The only tooth wear patients who seem to wear a splint really well, are either those with facial pain or those who chose cosmetic treatment and want to protect it' 'If I am considering offering treatment to a patient, I am also thinking about what I will do when this fails in the future. For a younger patient, this is the first step in their cycle of treatment through their life. It is a big issue when treating a chronic condition such as tooth wear. Who has responsibility to maintaining a case?'
'No, it is low on our list of priorities. There are no national policies to push practices to think about it. We do have assessment of tooth wear on our practice template. Our patients have other problems, such as caries and periodontal disease. I don't know of any NHS practices that focus on tooth wear, although some private practices take specialist referrals for patients with tooth wear and occlusion problems' 'I agree. Tooth wear is probably the last thing we would focus on, at the bottom of the list of other priorities' 'This week we had our first ever practice meeting in over 20 years related to tooth wear and that is because I was discussing this with you all. We have sometimes spoken about a case we are struggling with, but not about tooth wear as a subject' 'From my point of view, when I see tooth wear, apart from making a soft splint, there isn't really anything I can do. I will restore teeth for a patient who has an issue with aesthetics. I don't know anything about things such as deprogrammers etc. I wouldn't know where to start with complex occlusal problems and severe tooth wear'.
'This isn't spoken about enough. There needs to be some level of public health understanding. Not everyone is going to be perfectly healthy. People are going to have all sorts of chronic conditions and it is then just a case of making them aware. As long as the patients are aware and have some input into how to manage it, we shouldn't feel a pressure to fix everything. Some patients are always going to grind their teeth, they are not going to wear a splint and they're always going to break most of what we use to restore their teeth. As long as they know their options and agree with our approach, we are practising safely and professionally' 'Many dentists are trained with a big medico-legal worry that if we can't fix it, we are not treating the patient correctly. We have got to accept that there are a lot of people who will be affected by tooth wear, for the rest of their life' 'From a medico-legal perspective, we are much more focused on periodontal disease. Diagnosing it, making sure the patient is aware of it, treating it or referring the patient' 'Record keeping for tooth wear isn't as comprehensive as is it for periodontal disease, soft tissue assessment and medical history'.
'I am not sure that many GDPs have the experience and knowledge of what to do for their patients. The cost of treatment and the time required to complete the treatment are also problems. Our associates said that as they can't do any comprehensive treatment for tooth wear under UDAs, they feel less confident and are probably deskilled. They wouldn't want to try some treatment they're not going to do very often. If treating tooth wear cases with composite and using direct Dahl approaches was more commonly performed or was an allowable thing within our NHS contract, for a severe tooth wear case, they would be more confident to do it' 'I think we have the same concerns and I'm sure many of our younger dentists will also be worried: "once I start this, where am I going with it?" Managing tooth wear just doesn't fit with the NHS UDA system at all' 'I'm quite patient-led with tooth wear cases. Someone is only going to have that treatment if they feel their tooth wear is an issue for them, especially a cosmetic problem. If it is a complex case, even from the perspective of private practice, most aren't likely to be able to spend that sort of money on their treatment' 'My practice is mainly private, and we have more time with our patients, but I still don't treat tooth wear as you may think we would do, without the NHS UDA constraints. I would only treat them if they didn't like the appearance of their teeth. I wouldn't even talk about a splint unless they were a severe bruxist' 'We use a capitation system for our patients. This lends itself better to managing some issues such as tooth wear, as we have more time and fewer financial restrictions'.
'Over the years since I qualified, I have had some good experiences and usually just expect a treatment plan to be sent back. More recently, most referrals are now returned, and the patients are rarely, if ever, taken on for treatment. We've not been given a lot of help' 'For a patient treated elsewhere, I would usually offer minor repairs myself. Anything more than that and I have to consider the consequences of what will I become responsible for, if I do any more. I usually advise the patient that they need to go back to whoever provided their treatment, as that colleague will know what was planned, in case the first treatment approach failed' 'We don't have a dental hospital nearby for that sort of referral, so I can't really comment. Our nearest hospital would be at least an hour away. We only refer privately to a local specialist prosthodontist' 'My practice colleagues said they have almost given up referring. Since COVID-19, just about everything gets rejected. Even before COVID, the patient would be returned with a treatment plan that was beyond what they are capable of doing or that was impossible to do in primary care, within the UDA system. Primary dental care is meant to be for prevention and for more simple and achievable treatments'.
Even by involving experienced colleagues, it is not possible for these discussions to accurately represent the views of all GDPs, especially those much more recently qualified. However, each of those that contributed, work alongside several other colleagues and have awareness of a wider view within the profession. The participants all confirmed that their patients are affected by tooth wear, but in general, justify their decisions in how they manage tooth wear by their belief that: The large majority of their patients have no concerns and express no symptoms related to tooth wear A smaller proportion of patients present with tooth wear that requires intervention, due to their age, the extent of tooth wear, or their concern about their symptoms and appearance Dentists have other, more important priorities to focus on, such as patient access, caries and periodontal disease Other public health issues may be of a greater priority for public funding Current practice business models and remuneration systems limit the clinical management of tooth wear Dentists may lack clinical knowledge and confidence in treatment methods Inadequate capacity for support from secondary care services. Other authors have identified similar themes related to management of tooth wear in primary care. O'Hara and Millar in 2020 evaluated currently available methods for assessing and monitoring tooth wear in a general dental practice environment. They concluded 'dentists do not seem to be aware of the current guidelines but do make reasonable attempts to monitor tooth wear'. Condon and Eaton in 2020, recognised that 'restoring complex tooth wear cases is technically challenging and not well-remunerated under the NHS general dental service contract. Therefore, numbers of referrals to secondary care are increasing, but these are often rejected as dental hospitals have a high workload. This may make it difficult for patients with tooth wear to access appropriate care unless paying privately, which may be costly for them. Their study found low confidence in restoring complex tooth wear cases: only 21% of practitioners stated they would treat complex cases under the current NHS contract and 62% reported that they had experienced difficulty referring these cases to hospital. In 2018, O'Toole and co-workers assessed charting, risk assessment and treatment-planning of tooth wear between four recently qualified and seven experienced dentists in general dental practice. Their findings identified that: there are significant differences between patient management between recently qualified and experienced dentists; improvements are required in recording (48% versus 5%), risk assessing (51% versus 1%) and preventive treatment planning (62% versus 1%) of erosive tooth wear; and experienced GDPs may benefit from re-training in this area. In 2020, Mehta and co-workers assessed the habits of tooth wear risk assessment and charting using a tooth wear index, by UK and non-UK dental practitioners. Based on a sample of 297 responses, 81% agreed to the need to undertake risk assessment for all dental patients attending for a first-time consultation. In total, 59% undertook risk assessments for patients previously identified with signs of severe tooth wear. The routine use of a clinical index to perform tooth wear charting was described by 13.5%, with 5% documenting the frequent use of the BEWE tool. The paper found that specialist dental practitioners or those with further post-graduate training were more likely to use a tooth wear index.
Understanding the opinions and perceptions of experienced GDPs is important when the management of patients presenting with tooth wear is considered, as almost all patients will be either initially or only ever seen in the primary care environment. A number of challenges to delivery of treatment are identified and discussed. There is a large body of academic work related to tooth wear, including text books and guidelines from the Royal College of Surgeons of England, much of which explains and recommends what would be considered 'best practice'. The opinions expressed by the interviewees in this study and other recent studies suggest that a notable and perhaps alarming gap exists between the management of tooth wear in general dental practice in England compared to the published guidelines. The opinions of the interviewees in this study can also be interpreted as suggesting, controversially, that, as the management of most patients with tooth wear is not prioritised within commissioned NHS funding models, by GDPs, by dental hospitals receiving referrals or by most patients, perhaps it is not currently of importance outside academic environments. Further, cross-profession discussions are required, related to both commissioning NHS primary and secondary care services and to addressing the lack of dentists' confidence to manage patients affected by tooth wear.
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Study of Sexual Dimorphism in Metatarsal Bones: Geometric and Inertial Analysis of the Three-Dimensional Reconstructed Models | dc9c44f8-c0fd-4a82-bb75-b9686c280151 | 8551807 | Anatomy[mh] | “Virtopsy” is a term introduced in forensics and bio-archeology to describe the application of the three-dimensional (3D) cross-section imaging (CSI) analysis of human remains. These techniques include organs’ 3D reconstruction and precise quantitative measurements based on multi-slice computed tomography or magnetic resonance imaging data ( ). Indeed, the replacement of traditional postmortem techniques with CSI examination has been recommended by scientific, cultural, and humanitarian groups due to its non-invasiveness, digital nature, and 3D reconstruction opportunities ( ). Literature data provide numerous evidences that CSI reconstruction is useful for the analysis of the neuronal morphology, bones, and teeth ( – ). For instance, the possibility to estimate a person’s age by assessment of dental pulp volume or sex by calculating long bone’s metrical quantities has been documented ( – ). Sex estimation of skeletal remains is a basic element in creating a biological profile in archeology and forensics ( , ). The estimation relies heavily on the analysis of the pelvic and cranial features ( , ). However, in reality, it is not rare that the discovered skeletal remains are incomplete and consequently investigators have to focus on the remaining bones ( ). In this context, metatarsal bones with small quantity and surface area are more likely to be preserved intact and in some cases they present the only option for sex estimation ( ). The prevalence of metatarsal bones at archeological sites ranges between 43% and 89%, considerably more than that of other bones. For example, in seven forensics cases in Northern Italy 97.1% metatarsal bones were present, including 100% first metatarsal bones ( ). Research has already proven the accuracy of virtual sex assessment using CSI of metatarsal bones. For instance, metatarsals’ linear measurements such as maximum length, width of head, and width of base are shown to provide accurate sex estimation ( ). In addition, the volume of the first metatarsal bone and the torsion of the second metatarsal bone were employed to establish successful sex and aging protocols ( , ). The accuracy of currently available geometric protocols is approximately 80%, which gives room for further improvements ( ). Sex estimation can be accurately performed using the DNA analysis, which highly depends on the quantity and quality of DNA samples; however, it is not an applicable method to accurately identify sex of human skeletal remains, since the DNA begins to degrade immediately after the cells die ( ). Although bone and teeth can provide some protection against DNA degradation, the environment for preservation is highly demanding, such as temperature, moisture levels, oxygen levels, soil composition, and pH value ( ). Y chromosome deletions or mutations in the priming or binding sites can lead to incorrect estimation of sex and reduce the accuracy of DNA analysis ( – ). Studies show that the accuracy of DNA analysis in determining the sex of ancient human remains ranges from 52% to 95% ( – ). For bone fossils, the older the fossil, the lower the amount of extractable DNA. No extractable DNA was left in bone fossil between 200,000 and 500,000 years ago ( ). Furthermore, the process of DNA extraction is destructive ( , ), which is not feasible when preservation of ancient skeletal remains is required ( ). Therefore, when we attempt to determine the sex of bone fossils, the advantages of morphology and inertia variables will be more obvious. The current investigation seeks to test the hypothesis that 3D reconstruction of metatarsal bones might present a promising alternative to traditionally employed methods for forensics and archeological sex estimation. More precisely, this study aims to define physical metatarsal bone quantities of interest for the discrimination between male and female subjects. To this purpose, 60 subjects’ metatarsals (n = 600) were scanned using computed tomography and virtually analyzed with an intention to identify quantitative measurements referring to the sex of the individual. In addition to normalizing bone’s sexual identifying geometric variables (bone length, width, height, surface area, and volume), this study was specifically designed to increase the sex determination accuracy by including the inertial variables—three principal moments of inertia (PMI) relative to their principal axes of inertia (PAI) ( x , y , and z ).
2.1 Subjects 2.2 Scanning Procedure 2.3 Definition of Coordinate System of Metatarsal Bones 2.4 Extracting Biometric Sex Estimation Identifiers 2.5 Parameter Setting of the Reconstruction 2.6 Statistical Analysis We recruited subjects from our university who volunteered to participate in this program. Sixty healthy adults (30 males and 30 females) from Fujian Normal University were selected. Their mean age was 20.9 ± 3.0 years, mean height 170.9 ± 9.9 cm, and mean weight 62.5 ± 10.6 kg. The detailed characteristics of male and female subjects are shown in ( ). The study received approval from the Ethical Committee of Fujian Normal University. The subjects provided fully informed consent to participate in the study by signing a written consent form. Then, a questionnaire was distributed to volunteer students to exclude those with lower limb injury history. Each potential subject’s annual medical report was checked to exclude those with disease or trauma in their nervous and/or musculoskeletal system.
Subjects were scanned using Aquilion One 320 Slice CT Scanner (Toshiba, Japan). The scan settings were as follows: tube voltage of 120 kV, tube current exposure time of 50 mAs, layer distance of 0.45–50 mm, pixel size of 0.46 ± 0.02 mm, and automatic threshold between -1024 and 4145 Hounsfield units (HU). The scanning was conducted along the transect of both feet, from top to bottom. The scanning posture of 60 subjects is shown in ; .
The 3D models were constructed using Mimics software system (Mimics Research 17.0 for X64; Materialise, Leuven, Belgium). The reconstructed metatarsal bones are shown in ; . Software solution was employed to position the 3D models of 600 metatarsals ( ). In brief, software includes setting the direction and order of three coordinate axes of the metatarsal. Specifically, by going through the center of mass (COM) of the metatarsal, the PAI set to go from metatarsal head to base was the z axis, with the direction from the head to the base as the positive direction; the PAI set to go from plantar to dorsal was the x axis; and the PAI set to go from the medial metatarsal body to lateral was the y axis. The bone length, width, height, bone density, surface area, volume, and three PMIs around the x , y , and z axes were obtained from the positioned metatarsal bones ( ; ).
The bone length, width, height, surface area, and volume were extracted automatically from Mimics software ( ; ) and from the 3D models of metatarsal bones described in Section 2.3. 2.4.1 Normalization of Linear Variables 2.4.2 Normalization of Inertial Variables 2.4.3 Calculation of Surface-Area-to-Volume Ratio 2.4.4 Calculation of Bone Density The bone density of the 3D reconstructed metatarsal bone was calculated, shown in Equation (4): (4) d = ∫ 1 N d i N where d i = g i g w , g i stands for the gray value of the volume element, g w that of water. The equipment has been calibrated; the gray value of the air is set to 0, and that of the water is 1024. N refers to the number of bone’s volume elements.
Equation (1) was used to normalize the linear measurements and to eliminate the effect from subjects’ body height difference, possible sub-voxel scanning accuracy ( , ), and possible voxel order of magnitude errors from segmentation accuracy derived from both segmentations alone or non-detected subject micro-movement during the scanning procedure ( ): (1) { L n = L p L p + W p + H p × 100 W n = W p L p + W p + H p × 100 H n = H p L p + W p + H p × 100 where Lp , Wp , and Hp refer to the length, width, and height of the positioned bone, and Ln , Wn , and Hn those of the normalized, respectively.
The Hounsfield number of a CT scan is a product of radiation dose and attenuation coefficient (derived from density and atomic number) of the scanned material ( ). In our case, it is influenced by bone density, body mass and size, and again possible micro-movement of the subjects during scanning procedure, which can create voxel order of magnitude geometric dimension errors on the 3D model derived from the scan by altering the valve of HU and thus threshold and segmentation procedures. As we derive the mass of 3D models of metatarsal bones from the HU used for the segmentation procedure to calculate the PAI, Equation (2) was employed to eliminate those effects ( ) and to normalize inertial variables: (2) { P M I x = P M I x P M I x + P M I y + P M I z × 100 P M I y = P M I y P M I x + P M I y + P M I z × 100 P M I z = P M I z P M I x + P M I y + P M I z × 100
The surface-area-to-volume ratio (SA: V) of 3D reconstruction of the metatarsal bone was calculated, as shown in Equation (3): (3) S A : V = S V where S refers to the surface area of metatarsal bone, and V to the volume of metatarsal bone.
The same parameter settings were used to reconstruct all metatarsal bones. Specifically, in the Mimics software, the “Predefined Thresholds Sets: Bone (CT),” “Fill holes,” and “Keep largest” options of “Thresholding” were not selected. The operations of “Local Thresholding,” “Region Growing,” and “Dynamic Region Growing” were not performed. In “Morphology Operations,” the operation was set to “Close” to operate the selected metatarsal.
To test the influence of the reconstruction parameter setting on the consistency of reconstructed metatarsal geometric measurements, intraclass correlation coefficient (ICC) analysis was performed on the length, width, height, surface area, volume, and SA: V of 60 metatarsal bones from previous research ( ), where the reconstruction parameter settings were the same as this study, and 60 metatarsal bones were scanned and reconstructed twice. The assumption of normality and homogeneity of variances were tested by the Shapiro–Wilk test and Levene’s test, respectively ( ; – ). The comparisons of measurement values between sexes were evaluated with the independent sample t -test analysis when data were normal distribution and homoscedasticity. The Mann–Whitney U test was performed when data were non-normal distribution, and the results of Welch’s test would be accepted when data were heteroscedasticity. The statistical level was determined as p < 0.05. The sexual dimorphism index (SDI) was determined as (X m - X f/ X m + X f ) × 100, where X m and X f are the mean values of the male and female groups, respectively ( ). SDI represents the degree of variation between sexes. When males’ variables were larger than those of the females’, the SDI value was positive; otherwise, it was negative. The closer it got to zero, the less significant the difference between the male and the female was. The correlation between subjects’ characteristics and bone variables was evaluated by Spearman’s correlation coefficient ( ). To determine the best sex-discriminatory variables, the stepwise discriminant function analysis (SDFA) (Wilk’s lambda) was performed for each left and right metatarsal bone. The assumption tests including multivariate normality, multicollinearity, multivariate outliers within groups, homogeneity of variances/covariances, and linearity were conducted prior to the performance of the SDFA. Multivariate normality was assessed by Mardia’s skewness and kurtosis ( ). A Mahalanobis distance test was used to detect multivariate outliers ( ). The Pearson correlation test was performed to test multicollinearity among variables ( ). Homogeneity of variance–covariance matrices and linearity were evaluated by Box’s M test and matrix scatter plots, respectively. Data analyses were processed with SPSS 23.0 (IBM Corp.). Prior probability was set as “all group equal” for all analyses. The smaller value of the function’s Wilks’ lambda indicates greater discriminatory ability of the function. The standardized canonical discriminant coefficients imply contribution of each variable. The higher the value, the greater the contribution of the variable. The structure matrix demonstrates the correlation between each variable and the discriminant function. The closer the value of the variable to 1, the stronger the correlation. Unstandardized coefficients are utilized to form the discriminant function and calculate the discriminant function score (Y). The form of function is Y = a 1 x 1 + a 2 x 2 +···+ a n x n + C (a 1 - a n = unstandardized coefficients, x 1 -x n = variables, n = the number of variables, and C = the constant value). A “leave one out classification” procedure is performed in order to estimate the accuracy rate of the original sample and the sample created by cross-validation.
The original and positioned scanning postures of the investigated metatarsal bones are shown in . shows the process of bones’ alignment in the x , y , and z planes. Shown in are the ICC analysis results of the length, width, height, surface area, volume, and SA: V of the reconstructed metatarsal bones from two scans. The best consistency is shown in bone length, up to 1.00. The volume of the third metatarsal presents the lowest ICC, i.e., 0.81. The ICC values of the remaining measurements range from 0.91 to 0.99. It is worth noting that the ICC values of SA: V are between 0.93 and 0.98, higher than those of surface area and volume. – reveal the descriptive results and SDI values of eight variables of both sides of the investigated metatarsal bones between sexes. The highest SDIs were found in SA: V (-6.055% – -7.656% and -6.227 – - 7.949% from the left and right sides, respectively), PMI z (3.797%–5.455% and 2.564%–7.692% from the left and right sides, respectively), and height (1.292%–2.389% and 1.026%–2.235% from the left and right sides, respectively). SA: V and PMI z show greater sexual dimorphism than linear variables. Of note is that the SDI values of bone length, SA: V, PMI x , and PMI y were negative, indicating a larger value of females than that of males. – present independent sample t -test and Mann–Whitney U test results of eight variables of both sides of the investigated metatarsal bones between sexes. Highly significant sexual differences were found in SA: V of all metatarsals and in PMIs of the first to fourth metatarsals; in length of the first, third, and fourth metatarsals; in height of the first, fourth, and fifth metatarsals of both sides; and in width of the first metatarsal of the right side (p < 0.01). Sexual differences were found in length of the second and fifth metatarsals, in height of the third metatarsal, and in PMI z of the fifth metatarsal of both sides (p < 0.05). Sexual differences from the left side were found in width of the first metatarsal, in height of the second metatarsal, and in PMI y of the fifth metatarsal (p < 0.05). Significant differences from the right side were found in width of the fourth metatarsal and in PMI x and in bone density of the fifth metatarsal (p < 0.05). The first metatarsal was the most sexually dimorphic of five metatarsals, showing significant sexual differences of all variables except bone density, followed by the third metatarsal with statistical difference in bone height, length, SA: V, and PMIs. The subjects’ body height and weight showed low correlations (| r s | < 0.40) with variables except SA: V (0.60 <| r s | < 0.81). Length presented multicollinearity with some variables (| r p | > 0.80), and PMI x , PMI y showed a high correlation with PMI z (| r p | > 0.80) ( ; – ). Considering the high SDI and significant sexual difference by the independent t -test, the width, height, PMI z , bone density, and SA: V were selected as independent variables for the SDFA. The probability of variables was more than 0.001, indicating the absence of outlier in the samples. Two multivariate outliers were identified and removed in our study, which were found in the first metatarsal bone of the left and right sides, respectively. The homogeneity of variance matrices was evaluated by Box’s M with p > 0.001 for both sides in our analysis ( ; ). The multivariate normality of variables was found in the first metatarsal of the left side and in the second to fourth metatarsals of the right side ( ; ). Linearity among five variables was presented in matrix scatter plots ( ; ). , describe the SDFA results for sex determination. The results of standardized canonical discriminant coefficients and structure matrix show that SA: V has the highest correlation with discriminant functions and thus contributes most to sex estimation. The accuracies of discriminant functions based on original samples and cross-validated samples are reported in , . The sex determination accuracies of the original samples were between 88.3% and 98.3% of both sides. Moreover, the percentage of correct classification of cross-validated samples was also between 88.3% and 98.3% of both sides. No significant variance was observed in accuracies between the original samples and the cross-validated samples, revealing the steady predication ability of the discriminant functions. The highest accuracy was found in the third and fourth metatarsal bone of the right side—98.3% in our cases. The accuracy of the right side was slightly higher than that of the left side.
Parameters such as the voltage, parameter of field of view, and reconstruction settings influence the accuracy of measurements (length, width, height, surface area, and volume) during the 3D reconstruction of bone. The ICC values of bone length, width, and height presented high consistency of metatarsal measurements between two reconstructions while the ICC values of surface area and volume were lower than those of linear measurements. Volume is a higher-order quantity compared to length, width, and height. For example, the ICC value of the side length of a square is 0.91, while that of its volume is 0.75. The ICC value of SA: V was also calculated, and the lowest ICC values rose to 0.93, indicating that SA: V has higher consistency than surface area and volume. Therefore, SA: V instead of surface area and volume was used in this study. Parameters such as field of view and voltage were not set the same in the two scans, which also affected the ICC values of the reconstructed metatarsal measurements. From this view, the ICC values of metatarsals’ measurements between two reconstructions were high, indicating that the reconstructed 3D bone model was precise under these reconstruction parameter settings. It is reasonable to assume that the parameter setting mentioned in Methods generated an accurate reconstruction model. The sexual dimorphism in the human skeleton system is well studied ( , – ). Researchers keep on exploring the potential of bones in sex estimation, for instance, mandible ( ), long bones of the upper limb ( , , ), metacarpals and phalanges ( ), pelvis ( , ), tibia ( ), metatarsal ( , , ), and proximal foot phalanges ( ). The accuracy of sex estimation provided by different parts of the bone varied ranged from 66% to 99%. For metatarsal bone, this study showed for the first time that the SA: V and PMIz of metatarsal bone with significant sexual dimorphism may be successfully implemented for sex estimation. The discriminant accuracy of metatarsal’s geometric and inertial variables of Chinese samples in this study were between 88.3% and 98.3%, which were comparable to the accuracy reported in the Portuguese Caucasian population (83.0%–100.0%) ( ), the Greek samples (80.7%–90.1%) ( ), the Iranian population (82.6%–86.9%) ( ), and the Egyptian population (81.3%–97.5%) ( ). The accuracy variation can be found in different populations, indicating that the classification accuracy of metatarsal bone was population-specific. Gibelli et al. reported the superiority of linear measurements over volumetric measurements in sex estimation ( ). In our study, the SA: V and PMIz of five metatarsal bones showed greater sexual dimorphism than linear variables and SA: V contributes most to sex estimation. The discriminant function based on SA: V provided 91.7% accuracy (91.7% cross-validated). Studies found that SA: V would decrease with increasing body size as trabeculae became thicker ( – ). In our cases, high and negative correlations (0.60 <| r s | < 0.81) were found between SA: V and body weight as well as between SA: V and body height, indicating that high classification accuracy and significant sexual dimorphism of the SA: V value may arise from the differences in body size between males and females. On the other hand, normalized linear and inertial variables presented low correlations (| r s | < 0.4) with body height and weight, suggesting that normalized bone height and PMI z value were less likely affected by the differences in individuals’ height and weight in sex estimation. It is known from the natural principles that form follows the function. Loading can significantly modify bone shape and mass, and this influence is long-lasting ( ). Bone robusticity is generally considered as an important indicator of the magnitude and nature of the force that acts on the bone, providing information about the habitual behavior of organism ( – ). Some studies reported the sexual differences in robusticity of hand and foot bones ( , , ). In our study, high SDIs of bone height and PMI z may reflect differences in genetics and physical activity level between sexes. In linear measurements, higher SDI values were found in bone height other than in bone width and length, which was consistent with findings of the literature ( , ). Ruff et al. found that the diaphyseal cross-sectional size changed significantly with the increase of mechanical load (body weight increase) ( ). Similar results were discovered by Lieberman et al., reporting a significant change in the diaphyseal cross-sectional geometry of limb bone of sheep after 3 months’ moderate exercise ( ). PMI is associated with bone morphology and mass distribution. Significantly higher PMI z values in the male than in female population presumably come as a consequence of the increased physical activities of the male subjects. Namely, tarsals and metatarsals, with ligaments and tendons from the foot arch, could resist impact and maintain stability during walking, running, or jumping. Metatarsals mainly bear the longitudinal pressure from its base to head, which explains the greatest variation in ratio of PMI z values—the load bearing along the z axis has presumably undergone adaptability changes ( ). The effect of age on bone dimension and robusticity should be noted. No consensus has been reached. It was found that the subperiosteal diameter increased with age ( – ). However, some studies found that mechanical loads during adulthood had little effect on the external dimensions of long bone diaphysis and the age-related changes in diaphyseal cross-sectional size of bone were not evident ( – ). It may indicate that the diaphyseal cross-sectional properties of bone were mainly affected by physical activity before skeletal maturity ( ). The effect of age and mechanical loads on the geometric and inertial characteristics of metatarsal bone needs further in-depth studies of larger sample size of different ethnical/racial groups. How to best implement the CSI analysis for sex estimation in forensics and archeology can be discussed, but some methodological issues have to be resolved. Firstly, the measurements that differ in the male and female populations should be identified. Those variables may be geometric (length, width, height, SA: V) and inertial (PMIs). The present paper paves the way for sex estimation by introducing the concept of PMI-oriented bone coordinate system normalization. PMI z is the attribute of the rotational movements; it is an analog of the mass of the translational movements. The advantage of the inertial analysis is the evidence that such analysis does not depend on factors such as nutrition and genetics, as is the case for linear variables. It quantitatively assesses the foot bone physical properties, providing more accurate data than qualitatively measured pelvis and cranium or metrical approaches focusing on a single bone element ( , ). Secondly, the method that yields precise measurements based on 3D models should be determined. The present paper introduces a bone positioning method. The body coordinate system sets COM as the origin, achieving bone location, and sets three PAIs of bone as the body coordinate axes, positioning bone posture to avoid measurement error caused by different scanning positions, which ensures the high accuracy of dimensions along the axis (bone length, width, and height). One additional advantage of this method is that the dimension of bone along the axis can be obtained automatically, reducing the possible error caused by manual measurement. This study is the continuation of the ongoing scientific efforts to employ virtual 3D reconstruction in determination of individuals’ sex and age ( , ). Foot bones were chosen purposely due to their wide availability in both archeological and forensic context owing to more resistance to the rigors of time than long bones ( ). The accuracy of virtual analysis of the metatarsal bone is proven in a previous investigation that evaluated the efficacy of a radiological method to estimate the individuals’ sex using measurements of the first and second metatarsals of a Portuguese Caucasian population ( ). The high prevalence of metatarsal bones at archeological and forensic sites justifies that the proposed method may be widely applied in archeology and forensics. The wide application of 3D CSI in forensics is constrained because of ethical issues, which also has limited the establishment of populations’ databases. However, the data obtained during routine medical examination may be stored and subsequently employed in forensic analysis. The analysis of metatarsal bones using radiography is rapid and noninvasive. The advantages of 3D CSI forensic analysis include, besides the potential for sex estimation, precise documentation and 3D demonstration of forensic findings for the court, reduction of trauma, and decreased risk of transmission of disease ( ). It is interesting to note that the CSI analysis of the fourth metatarsal even allowed the scientists to explain the ground-dwelling biped walking pattern of Australopithecus afarensis dating back to 3.2 million years ago ( ). Some weaknesses of virtopsy-oriented skeletal assessments should be noted though. The quality of the CSI can be affected by many factors, including the scanning posture, error aggregation, resolution, and dose, resulting in the inconsistency in the 3D reconstruction models ( – ). Studies have shown that 3D bone models can achieve high accuracy at the sub-millimeter scale, while increasing the voxel resolution (from 0.3 to 0.15 mm) does not improve the accuracy of the models ( ). Our previous study ( ) compared the accuracy of 3D bone models reconstructed with different anisotropic voxels (different pixel sizes) and found no significant differences in the linear, volume, and surface area measurements of the models. In particular, the linear measurement values remained highly consistent, indicating that pixel size had no significant influence on model accuracy at the submillimeter scale. Micro CT scanning, providing scans at the few-micron level for small size samples, is commonly used to evaluate the trabecular bone microstructure ( – ). However, the effects of multi-detector CT and micro CT with different resolutions on the accuracy of 3D bone models are still unclear and need to be further studied. The standardization of the body coordinate system of bone is able to avoid the adverse effects arising from different scanning postures, while rating those quantities by percentage could reduce the effect from resolution and dose. The same bone can be reconstructed by a different operator or it can be reconstructed by the same operator for many times, so the parameter setting of the reconstruction process should be taken into consideration when comparing the results of different studies on the CSI analysis of the skeletal tissue. For statistical results, some assumptions were not confirmed, such as multivariate normality of the second to fifth metatarsals of the left side and the first and fifth metatarsals of the right side. The accuracy of formulae based on these metatarsal variables should be treated with caution, although discriminant function analysis is relatively robust against deviations from multivariate normality ( ). It should be further highlighted that the accuracy of estimation may be influenced by the characteristics of the selected bone, population, sample size, and age. The promising results (cross-validated accuracy ranges) of this study may in part be driven by the small sample size as well as the same young age-group. The potential future application of this new proposed method for sex determination on unidentified individuals would not be as accurate as suggested in this study. Therefore, the methods should be tested (or independently developed) for distinct population groups, before being widely applied in individuals of unknown population origin (i.e., unidentified skeletons in forensic and bioarcheological contexts).
This study demonstrates that sexual dimorphism was found in both metatarsal bones’ geometric and inertial variables. A profound analysis of 60 subjects’ metatarsals revealed that discriminant functions based on geometric and inertial variables of metatarsal bones generated accuracies of 88.3%–98.3% in sex estimation. The ongoing studies are under way to test the potential of the proposed method on the sex determination of archaeological remains and of larger sample size with different population groupEthical Committee of Fujian NormFF and MD conceived the study. YL, DA, RL, YXF, KD, MM, and GY collected and analyzed the data. YFF, YL, DA, and ZL wrote the manuscript, and all authors revisgrant numbers 11672075, 11972119), Natural Science Foundation of Fujian Province (grant number 2019J01429), and Ministry of Education, Science and Technological Development of the Republic of Serbia (grant number III 4500 |
Bilateral ankle dorsiflexion force control impairments in older adults | 91ac4d34-2e45-42fa-9aad-346f4f43dc0f | 11925285 | Musculoskeletal System[mh] | Aging induces progressive impairments in the motor system, which increase the prevalence of functional disabilities in the lower extremities such as slower walking speed [ – ]. These motor deficits interfere with independent living in older people . Age-related ankle joint function impairments can induce a loss of static and dynamic balance control , further previous studies have suggested that strength and fine motor control during ankle dorsiflexion are crucial to minimize the risk of falls in the aging population [ – ]. Thus, estimating altered ankle dorsiflexion functions may effectively quantify the progression of neuromuscular deficits in the lower extremities of older adults. Previous studies have indicated that aging typically reduced flexibility (e.g., less range of ankle dorsiflexion motion), impaired mobility (e.g., delayed ankle dorsiflexion during the gait swing phase), and caused muscle weakness (e.g., lower strength in the tibialis anterior muscle) of the lower extremities [ , – ]. Moreover, many studies have used an isometric force control paradigm to assess age-related changes in fine motor control functions during ankle dorsiflexion while processing online visual information [ – ]. Given that age-related changes in the central and peripheral nervous systems may affect the ability to simultaneously correct and maintain isometric force outputs near a targeted level , isometric ankle force control capabilities were impaired with aging [ – ]. The older group showed greater force error and variability at lower targeted force levels (e.g., 5%–10% of maximal voluntary contraction [MVC]) because of altered visuomotor networks in the brain and activation of motor neuron pools . However, these ankle dorsiflexion force control impairments were mostly unilateral (e.g., dominant leg). In fact, age-induced movement control deficits were frequently observed in walking, postural control, and sit-to-stand that require bilateral actions in the lower extremities . Given that many daily activities for living independent life consist of fundamental motor skills related to interlimb coordination between feet , investigating bilateral force control and coordination in the lower extremities is necessary for understanding age-induced functional impairments. Studies on bilateral ankle dorsiflexion force control capabilities in older adults may provide additional information on how aging influences cooperative fine motor control between feet. Further, these findings can be used for developing new rehabilitation programs (e.g., robotic exoskeletons for improving lower limb coordination) targeting functional recovery of bilateral ankle movements in aging population . Bilateral force control capabilities in the lower extremities can be estimated by quantifying the accuracy, variability, and complexity (i.e., temporal structure of variability) of the total summed forces produced by the left and right sides of extremities [ – ]. Greater force accuracy, decreased force variability, and higher complexity normally indicated better bilateral force control capabilities affected by organism, environment, and task constraints (e.g., different populations, visual feedback, and targeted force level conditions) . Moreover, estimating interlimb force coordination patterns can reveal the performer’s motor control strategies that require more complex neural involvements to optimize bilateral force control performances [ – ]. Interlimb force coordination strategies have two aspects to consider: (1) coordination pattern within-trial analysis and (2) coordination pattern between-trial analysis. A former approach can estimate the strength of interlimb force coordination according to in-phase and anti-phase actions between feet so that more anti-phasic force coordination within a trial predominantly appeared with better bilateral force control performances . A latter approach can assess how a performer successfully coordinates interlimb motor actions synergistically across repetitive trials . The uncontrolled manifold (UCM) analysis is a representative between-trial approach, and an increased index of bilateral motor synergies (i.e., a ratio of good variability relative to bad variability) from the UCM approach denotes better interlimb force coordination across multiple trials, which contribute to overall task stabilization indicating improvements across multiple force control trials . This study aimed to examine bilateral ankle dorsiflexion force control and coordination patterns in older adults. To the best of our knowledge, this is the first study to investigate bilateral ankle dorsiflexion force control capabilities in aging population. Both older and younger participants executed isometric force control tasks across two experimental conditions: (1) visual feedback (vision vs. no-vision) and (2) targeted force level (10% and 40% of MVC). Force accuracy, variability, and complexity were measured, and interlimb force coordination patterns between feet were determined using within-trial and between-trial approaches. Considering the potential age-induced progressive degeneration in lower limb functions , we hypothesized that older adults would reveal a lower accurate, more variable, and less complex ankle dorsiflexion forces produced by feet while generating impaired interlimb force coordination patterns within a trial and across multiple trials than younger adults.
Participants Experimental procedures Data analysis Statistical analysis Participants were recruited between January 2, 2019 and February 15, 2024. The study enrolled 25 older adults (age: M ± SD = 64.9 ± 3.8 years; physical activity: M ± SD = 222.8 ± 105.2 minute/weak; 15 females) and 25 younger adults (age: M ± SD = 23.0 ± 2.4 years; physical activity: M ± SD = 283.6 ± 182.1 minute/weak; 15 females). All subject had no cognitive impairments, vision disorders, and musculoskeletal deficits in their lower extremities. The dominant leg of all participants was the right leg based on the ball-kicking test . shows specific demographic details. Before starting the experiment, all participants read the study protocols and signed informed consent approved by the University’s Institutional Review Board (No. 7007971-201810-002A).
A customized isometric foot-force measurement system (SEED TECH Co., Ttd., Bucheon, South Korea) was used for executing bilateral ankle dorsiflexion force control tasks. Participants sat 80 cm in front of a 54.6 cm LED screen and placed their feet on the customized platforms. Then, we directly adjusted the chair and instructed them to maintain proper joint positions following joint angle ranges: 90°–95° of hip flexion, 90°–100° of knee flexion, and approximately 90° of ankle dorsiflexion ( ). During tasks, we continuously monitored and ensured that participants maintain proper joint positions. Two force transducers were embedded to the left and right sides of the platforms (Micro Load Cell-CZL635-3135, range = 330 lbs, Phidgets Inc., Calgary, Canada). Force signals were collected at 100 Hz of sampling rate using a 16-bit analog-to-digital converter (A/D; ADS1148 16-Bit 2kSPS; minimum detectable force = 0.0192 N) and amplified with an excitation voltage of 5 V using an INA122 (Texas Instruments Inc., Dallas, TX, USA). All experimental procedures were administered using a customized Microsoft Visual C++ program (Microsoft Corp., Redmond, WA, USA). To minimize effects of different ankle dorsiflexion strength across participants on force control tasks, we normalized targeted force levels based on each participant’s MVC including 10% and 40% MVC . Initially, participants performed two MVC trials by producing isometric ankle dorsiflexion forces by feet (trial duration = 3 s and 60 s of rest between trials), and a peak force value (i.e., the maximal value of the sum of the left and right foot forces) was identified for each trial. Then, 10% and 40% of the mean of two peak force values were calculated to set two submaximal targeted force levels. During bilateral ankle dorsiflexion force control tasks, participants tried to generate and match their sum of the left and right foot forces around a targeted force level for 20 s. Four different experimental blocks were randomly administered including a combination of two targeted force levels (i.e., 10% and 40% of MVCs) and two visual information conditions (i.e., vision and no-vision; ). In the vision condition, participants received real-time visual information describing a targeted force level (i.e., white horizontal line) and the sum of bilateral forces (i.e., red trajectory line). In the no-vision condition, visual information displaying the sum of bilateral forces was unavailable after the initial 5 s. Before starting an experimental block, we provided one practice trial for familiarization. Participants completed ten consecutive trials for each experimental block. To minimize potential fatigue effects, a 30 s rest between trials and a 60 s rest between conditions were provided [ , , ].
Offline analyses were performed using the MatLab program (MathWorks TM Inc., Natick, USA). To minimize the initial adjustment and early termination effects, the initial 5 s and the final 1 s of force signals for each trial were eliminated. The remaining 14 s of force signals were also low-pass filtered with a 30-Hz cutoff frequency using a bidirectional fourth-order Butterworth filter. Bilateral force control performances were estimated by calculating force accuracy, variability, and complexity. We used relative variables, including relative root-mean-square error (rRMSE), relative bias error (rBE), and coefficient of variation (%CV), to minimize potential distortions by individual force levels . Force accuracy was quantified using rRMSE ( ) consistent with previous studies [ – ]. To specify the direction of force errors, rBE ( ) was also estimated . More positive values of rBE indicate overshooting errors, whereas more negative values mean undershooting errors relative to a targeted level. r R M S E = ∑ i = 1 N F i − T a r g e t e d f o r c e 2 N × T a r g e t e d f o r c e (1) r B E = ∑ i = 1 N ( F i − T a r g e t e d f o r c e ) N × T a r g e t e d f o r c e (2) F i = bilateral force at sample i , N = number of data samples Force variability was estimated by quantifying the %CV ( SD / mean force × 100 ) of the force outputs . r force complexity, we used the refined composite multiscale sample entropy (rcMSE) consistent with the following procedures : (1) create a coarse-grained time series using k refer to nonoverlapping windows that range from 1 to the scale factor τ , (2) use m (number of similar vector lengths) and r (tolerance range; usually 0.2 × SD ) parameters, and calculate the number of matched vector pairs n k , τ m + 1 and n k , τ m in each coarse-grained time series, (3) the rcMSE value at a scale factor of τ was defined as the logarithm of the ratio of n ¯ k , τ m + 1 , to n ¯ k , τ m ( ), and ( ) the index of force complexity was calculated as the sum of rcMSE values with scales from 1 to τ Max ( ). r c M S E t i m e s e r i e s , τ , m , r = − ln ∑ k = 1 τ n k , τ m + 1 ∑ k = 1 τ n k , τ m (3) C o m p l e x i t y i n d e x = ∑ τ = 1 τ M a x r c M S E t i m e s e r i e s , τ , m , r (4) In this study, m to 2, r to 0.2 × SD of force data, and τ Max to 10 were used. A higher complexity index means a more complex force control pattern. Within-trial bilateral force coordination patterns in the lower extremities were evaluated using the vector coding approach that can estimate the frequency of coordination patterns between two segments . In scatterplots that reflect the left and right foot force outputs, the direction of the trajectories over time is quantified by the coupling angle. Specifically, coupling angles are calculated using a vector for two successive data samples relative to the right transverse axis of the scatterplot ( ; ). C o u p l i n g a n g l e θ = tan − 1 R F i + 1 − R F i L F i + 1 − L F i (5) L F i = left foot force at sample i , R F i = right foot force at sample i The coupling angle ranges from 0° to 360°, and coordination patterns were categorized as follows: (1) in-phase (22.5° ≤ θ < 67.5°, 202.5° ≤ θ < 247.5°) and (2) anti-phase (112.5° ≤ θ < 157.5°, 292.5° ≤ θ < 337.5°). More frequency in-phase patterns denote impaired interlimb force coordination, whereas greater frequency in anti-phase patterns indicates complementary coordination between feet . Furthermore, the UCM analysis was conducted to estimate bilateral force coordination in the lower extremities across multiple trials . First, the mean of the left and right foot forces for each trial was calculated, and the mean force values relative to a targeted force level (i.e., 100 × left mean force / targeted force level and 100 × right mean force / targeted force level ) were normalized. For each trial, two normalized mean force values were considered as a pair of elemental variables, and the same calculations for ten trials were performed to generate ten pairs of elemental variables. Then, all pairs of elemental variables were projected to the UCM sub-space and orthogonal to the UCM sub-space (i.e., ORT sub-space). Good variability (V UCM ) is a variance of elemental variables projected to the UCM sub-space. Although altered levels of V UCM do not influence task performances, greater V UCM is considered a flexibility of interlimb coordination strategies (e.g., various motor solutions) across multiple trials. Bad variability (V ORT ) is the variance of elemental variables projected to the ORT sub-space, and increased levels of V ORT interfere with task stabilization leading to lower task performances ( ). Finally, bilateral motor synergies (V Index ), which is a ratio of V UCM to V ORT ( ) were calculated, and V Index values were Z-transformed for additional parametric statistical analyses ( ). Thus, greater values of V Index indicate improvements in bilateral force coordination patterns across multiple trials. V I n d e x = V U C M / d f U C M − V O R T / d f O R T V T O T / d f T O T (6) V T O T = V U C M + V O R T d f = d e g r e e s o f f r e e d o m d f U C M = 1 , d f O R T = 1 , a n d d f T O T = 2 V I n d e x Z − transformed = 0.5 × ln 2 + V I n d e x 2 − V I n d e x (7)
The normality of distribution for all dependent variables was confirmed by the Shapiro Wilk tests. All dependent variables were analyzed by the three-way mixed model (Group × Force Level × Vision Condition; 2 × 2 × 2) analysis of variance (ANOVA) with repeated measures on the last two factors. For the post hoc analysis, Bonferroni’s pairwise comparisons were used. For older adults, Pearson’s correlation analyses were conducted to investigate the potential relationships between age and bilateral force control outcome measures in the lower extremities. IBM SPSS Statistics version 28 (IBM Corp., Armonk, NY, USA) was used for all statistical analyses, and the alpha level was set at 0.05.
Bilateral force control performances Bilateral coordination: Vector coding and UCM analysis Correlation finding between force control variables and age in the older group For the older group, Pearson’s correlation analyses reported that significant correlation between force complexity and age ( and S3 Table): (1) increased age versus lower rcMSE at 10% of MVC in the vision condition ( r = − 0.432; P = 0.031; ) and (2) increased age versus lower rcMSE at 40% of MVC in the no-vision condition ( r = − 0.409; P = 0.043; ). These results indicated that decreased force complexity was related to increased age for the older group.
For force accuracy, three-way mixed ANOVA on the rRMSE revealed a significant Group × Force Level × Vision Condition interaction [ F (1, 48) = 9.013; P = 0.004; partial ƞ 2 = 0.158; ]. Bonferroni’s pairwise comparisons revealed that the older group produced higher rRMSE values than the younger group at 10% of MVC for vision ( P < 0.001) and no-vision conditions ( P < 0.001), respectively. The analysis of the rBE showed a significant Group × Force Level × Vision Condition interaction [ F (1, 48) = 5.755; P = 0.020; partial ƞ 2 = 0.107; ]. In the post hoc analyses, the older group showed greater overshooting than the younger group at 10% of MVC for vision ( P = 0.007) and no-vision conditions ( P < 0.001), respectively. These results indicate that older adults revealed higher overshot force errors in bilateral lower extremities, and these patterns increased when visual feedback was unavailable. The analysis of the %CV304; P = 0.026; partial ƞ 2 = 0.100; ]. In the post hoc analyses, the older group produced higher %CV values than the younger group at 10% of MVC in the vision ( P = 0.013) and no-vision conditions ( P = 0.012), respectively. At 40% of MVC, the older group showed higher %CV values than the younger group in the vision condition ( P = 0.045). Moreover, the analysis of rcMSE reported a significant Group × Force Level × Vision Condition interaction [ F (1, 48) = 5.106; P = 0.028; partial ƞ 2 = 0.096; ]. The older group showed lower rcMSE values than the younger group at 10% of MVC in the vision ( P = 0.036) and no-vision conditions ( P = 0.008), respectively. At 40% of MVC, the older group showed lower rcMSE values than the younger group in the vision condition ( P = 0.013). These results indicate that older adults produced greater force variability and lower force complexity than younger adults.
The three-way mixed ANOVA on the in-phase frequency indicated significant two interactions: (A) Group × Force Level interaction [ F (1, 48) = 4.783; P = 0.034; partial ƞ 2 = 0.091; ] and (B) Force Level × Vision Condition interaction [ F (1, 48) = 29.476; P < 0.001; partial ƞ 2 = 0.380]. However, no significant differences in in-phase frequency were found between younger and older groups ( ). The analysis of the anti-phase frequency showed significant two interactions: (A) Group × Force Level interaction [ F (1, 48) = 18.309; P < 0.001; partial ƞ 2 = 0.276; ] and (B) Force Level × Vision Condition interaction [ F (1, 48) = 6.614; P < 0.013; partial ƞ 2 = 0.121]. Bonferroni’s pairwise comparisons on Group × Force Level interaction findings reported that the older group revealed lower anti-phase frequency than the younger group at 40% of MVC collapsed across vision conditions ( P = 0.001). Taken together, these results indicate that older adults used compensatory anti-phase coordination less frequently than younger adults. The three-way mixed ANOVA on V Index revealed significant Group × Vision Condition interaction [ F (1, 48) = 4.863; P = 0.032; partial ƞ 2 = 0.092; ]. In the vision condition, the older group showed lower V Index values than the younger group collapsed across force levels ( P < 0.001). The analysis of good variability indicated a significant Force Level main effect [ F (1, 48) = 27.122; P < 0.001; partial ƞ 2 = 0.361], but there was no significant difference between the younger and older groups. Finally, the analysis of the bad variability showed Group main effect [ F (1, 48) = 4.117; P = 0.048; partial ƞ 2 = 0.079; ] and Force Level × Vision Condition interaction [ F (1, 48) = 26.330; P < 0.001; partial ƞ 2 = 0.354]. The older group produced greater bad variability than the younger group collapsed across force levels and vision conditions. These findings indicated that older adults showed lesser synergy with greater bad variability between feet than younger adults across multiple trials.
This study investigated bilateral fine motor control capabilities of lower extremities in older people using an isometric ankle dorsiflexion force control paradigm. In comparison with younger adults, older adults had increased force error with a tendency to overshoot at 10% of MVC across vision and no-vision conditions. Moreover, bilateral ankle dorsiflexion forces produced by older adults were more variable and less complex, as indicated by conventional and nonlinear approaches to quantifying motor variability. Regarding interlimb force coordination strategies, older adults showed less anti-phase coordination within a trial and a reduction of motor synergies across multiple trials. Finally, the correlation analyses found that increased age was related to lower force complexity in older adults. The lower accuracy of bilateral ankle dorsiflexion forces in older people expanded previous findings of increased bimanual hand-grip and wrist-extension force errors by demonstrating that aging may induce interlimb motor control deficits across the upper and lower extremities . Spedden and colleagues reported that tracing error at 10% of MVC increased for older adults during unilateral ankle dorsiflexion force control task . Further, the greater force tracing error was significantly associated with impaired bidirectional connectivity between the primary motor area (M1) and the premotor cortex (PMC) within the dominant hemisphere . Further, previous findings indicated altered interhemispheric connectivity patterns (e.g., bilateral sides of the M1 and PMC) in older adults because of potential structural changes in the corpus callosum . Specifically, higher neural connections in the bilateral motor network developed in older adults , and despite the unclear neural mechanisms underlying this relationship, certain patterns (e.g., resting state connectivity) were associated with poorer bimanual motor performances . Although cumulative findings were limited to the upper extremity motor control tasks, altered neural communication between hemispheres with aging appears to influence bilateral motor control capabilities in the lower extremities. Importantly, the current study did not directly measure brain activation patterns between older and younger adults during bilateral ankle dorsiflexion force control tasks. Thus, future studies should focus on brain activation changes to examine potential neurophysiological mechanisms underlying age-related changes in bilateral ankle force control. Moreover, the greater variability of bilateral ankle dorsiflexion forces at 10% and 40% of MVC support recent meta-analytic findings that age-induced loss of force steadiness was typically observed in smaller and distal muscle groups across various submaximal force levels . In particular, our results confirmed that the increased variability of ankle dorsiflexion forces in older adults emerge in bilateral conditions in addition to the unilateral condition [ , , ]. Prior studies reported that aging may induce greater temporal and spatial variability of locomotion [ , , ]. For example, older adults aged 60–86 years showed significant correlation between increased age and greater variability of gait variables (e.g., step length, width, and double support time) . Further, increased gait variability may be associated with higher risk of falls . Interestingly, significant correlation between gait variability and ankle dorsiflexion force variability at 10% of MVC was observed in the older group as well . These findings suggested that more variable gait patterns as well as higher fall risk in older adults may be related to increased variability of bilateral ankle dorsiflexion forces. Potential mechanisms underlying excessive force variability with aging involved the motor unit reorganization. Aging may cause a loss of motor units leading to the remaining alpha motor neurons reinnervated with some of the denervated muscle fibers. Because aging muscles may have fewer and larger motor units, these larger motor units are recruited early even at lower targeted force levels, leading to more variable muscle forces [ , , ]. This phenomenon may increase in distal muscle groups because of the greater effects of motor unit reorganization on smaller muscles [ , , ]. Older adults have a smaller number of motor units and reduced excitability in motor neuron pools in the tibialis anterior [ – ]. Potentially, age-related motor unit remodeling may increase motor variability, presumably resulting in higher task errors with more overshooting patterns at the lower targeted level in this study. The nonlinear method indicated bilateral motor control deficits in the lower extremities of older adults by identifying loss of complexity for bilateral ankle dorsiflexion forces across all vision conditions. In addition, a significant correlation was found between lower complexity of bilateral ankle dorsiflexion forces and increased age. According to the loss of complexity hypothesis , aging may interfere with the neuromuscular system, resulting in the progressive loss of complexity within the dynamics of physiological signals. Previous findings supported this assumption by demonstrating the lower complexity of isometric forces in small muscles of the upper extremities (e.g., index finger abduction force) at 5%–40% of MVC [ – ], and these patterns were also observed in the lower extremity muscles, such as the plantar flexors and knee extensors . These findings posited that a loss of complexity in the aging appeared in both small and large muscle groups because of the increased common synaptic input, leading to impairments in fine motor control and gait performances [ , , ]. To the best of our knowledge, this study is the first to demonstrate the loss of complexity in bilateral ankle dorsiflexion forces in older people and suggest that aging may progressively debilitate the ability of lower extremities to instantly adapt motor outputs in response to environmental changes. Decreased motor adaptability in bilateral ankle dorsiflexors may be responsible for loss of locomotion complexity in the aging population . During bilateral ankle dorsiflexion force control tasks, older adults revealed less anti-phasic ankle dorsiflexion coordination within a single trial, as indicated by the decreased frequency in anti-phase ratio. These findings are consistent with previous results that older people showed more in-phase coordination patterns while modulating force production between hands . These coordinative patterns in older adults typically represent less compensatory and adjustable motor actions between extremities . The present findings support that older adults may exhibit impairments in interlimb force coordination across the upper and lower extremities in comparison with younger adults. Previously, impaired movement coordination between legs during locomotion, as indicated by less anti-phase coordination (i.e., lower accuracy of relative step timing between legs), was observed in older adults . Moreover, a reduction of anti-phase coordination patterns may increase fall risk in older adults because these compensatory actions between legs are essential for maintaining balance and stability in response to altered environments . Despite the lack of kinematic data in this study, the potential relationship between bilateral kinetic and kinematic coordination patterns should be examined in the future studies because of the contribution of ankle joint force control capabilities to gross movement control, such as gait and postural control . In the vision condition, older adults produced lower bilateral motor synergies between feet with greater bad variability indicating poor interlimb coordination across multiple trials. According to the UCM theory, increased bilateral motor synergies indicate greater stabilization of task performances, which are highly affected by changes in bad variability (i.e., the variance of fundamental elements projected to the undesired space). Previous studies have revealed that bilateral motor synergies in healthy young participants increased when they performed bilateral knee and ankle force control tasks that required more challenging goals (i.e., intentionally asymmetrical force productions between legs) . These findings raised a possibility that people with more complex coordination such as anti-phasic interlimb force outputs within a trial would demonstrate greater synergetic interlimb motor actions across multiple trials. Like previous studies showing upper extremity deficits in age-related interlimb coordination (e.g., decreased anti-phase coordination and motor synergies during bimanual finger and wrist force control tasks) , our UCM findings also suggest that aging induces impaired lower limb coordination between trials as well as within a trial. The current findings suggest that older adults may experience deficits in bilateral ankle dorsiflexion force production potentially associated with impaired locomotion and fall risk. Potentially, exoskeleton robots may be a viable option for improving bilateral ankle motor control because these approaches can provide accurate support and resistance to strengthen targeted muscles contributing to functional recovery of ankles . For example, the application of ankle exoskeletons in older adults demonstrated positive effects as assistive devices for locomotion and effective rehabilitation tools for bilateral ankle functions . Specifically, exoskeleton assistance on bilateral ankles reduced metabolic cost and improved gait speed in older adults . A case study reported that four weeks of resistance training using bilateral ankle assistive robots increased left and right ankle muscle strength, gait speed, and soleus activations during gait for an older adult . Thus, future studies are necessary to determine whether robot-based ankle rehabilitation programs can improve bilateral ankle dorsiflexion force control and interlimb coordination in older adults. Importantly, the current findings have several potential limitations. First, a bilateral force control task of this study required participants to perform similar motor actions between feet (i.e., ankle dorsiflexion forces). Given that various activities of daily living often involve asymmetrical force production between feet, how older adults deal with these challenging force control tasks must be examined in future studies. Second, investigating the relationship between bilateral ankle dorsiflexion force control and movement control deficits in the lower extremities (e.g., a risk of fall) is necessary for the aging population. Third, impaired bilateral ankle dorsiflexion force control in the older group may be influenced by different weight or muscle mass that were not controlled for group comparisons. Finally, neuroimaging and motor unit analysis studies for motor control of the lower extremities are still insufficient. Thus, additional approaches that focus on bilateral lower limb force control paradigms while collecting brain and motor neuron activation data should be conducted for older adults.
In conclusion, we found that older people had deficits in bilateral ankle dorsiflexion force control performances, including a reduction of force accuracy, increased force variability, and loss of complexity. Moreover, interlimb coordination patterns between feet for older adults were altered with less anti-phase coordination within a trial and decreased bilateral motor synergies between trials. Given that bilateral ankle dorsiflexion force control may be crucial for postural control, walking, and climbing stairs [ – ], effective training programs are required to prevent fall risks and promote independent life in the aging population . Potentially, advancing the central and peripheral nervous system using neuromodulation techniques such as transcranial direct current stimulation (tDCS) and functional electrical stimulation combined with movement training may be viable options for older adults. For example, anodal tDCS on either M1 or cerebellar regions (i.e., key regions for fine motor control and coordination) showed large effects on improved balance control capabilities in older adults . Despite the limited number of studies that have examined the effects of neuromuscular electrical stimulation (NMES) on quadriceps muscles, the findings suggested potential improvements in muscle strength and mass in older people . Thus, future studies may investigate effective protocols of tDCS and NMES specific for older adults to optimize their fine motor control capabilities of lower extremities.
S1 Table Bilateral force control capabilities for the younger and older groupData are mean ± SD. Number sign (#) means a significant difference between force levels. Ampersand sign (&) denotes a significant difference between vision conditions. ( P < 0.05). (DOCX) S2 Table Bilateral force coordination for the younger and older groups3 Table Pearson’s correlation between age and bilateral force control capabilities for the older groupAsterisk (*) indicates P < 0.05. (DOCX) S1 Dataset Specific values of bilateral force control outcomes for each participant across force levels and vision conditions. MVC = maximum voluntary contraction, rRMSE = relative root mean square error, rBE- relative bias error, %CV = coefficient of variation, rcMSE = refined composite multiscale sample entropy. (PDF)
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A case report on the successful treatment of a specific type of open ankle fracture-dislocation called logsplitter injury | 8c440ac6-0ce4-494a-9234-572f5246acdd | 11877603 | Surgical Procedures, Operative[mh] | Logsplitter injury, a rare and challenging type of injury, is characterised by high-energy traumatic events and is associated with complex fracture lines and severe soft tissue damage. , This injury poses a significant challenge to clinical management. The existing literature on this subject is limited, with most reports consisting of case studies and small clinical studies. , These studies underscore the significance of multidisciplinary teamwork and the development of individualised treatment plans for the management of this injury. The objective of surgical intervention is to restore the anatomical structure and function of the ankle joint, while minimising the occurrence of complications. This case report delineates the intricate surgical management of an open ‘Logsplitter injury’ in a patient. The surgical intervention involved several critical steps, including debridement, internal fixation, bone block repositioning and lower tibiofibular union reconstruction, which was combined with the use of vancomycin-loaded polymethylmethacrylate bone cement and negative pressure wound therapy (NPWT).The patient's ankle function exhibited significant improvement as a result of the meticulous handling and comprehensive post-operative management by the surgical team. Notwithstanding the generally poor prognosis associated with such injuries, we present a favourable outcome following the provided treatment, which is substantiated by imaging. This case provides valuable clinical experience and reference for the management of this type of injury, and the reporting of this study conforms to CARE guidelines.
Patient information Clinical findings Timeline Diagnostic assessment Therapeutic intervention Follow-up and outcomes A discharge rehabilitation plan was formulated based on the specific needs and rehabilitation progress of the patient, and he was informed to return to the hospital on time for a follow-up examination to evaluate the healing of fractures. Three months after the operation, the Kirschner wire was removed, and the lower tibiofibular screw and antibiotic bone cement were removed. The X-ray of the left ankle joint was reviewed , and the patient was instructed to walk with weight gradually. One year after the operation, the quality of fracture reduction was evaluated in this patient based on the Burwell–Charnley fracture reduction evaluation criteria, which classify the quality of fracture reduction into three grades: anatomical reduction, acceptable reduction and poor reduction. An X-ray of the left ankle joint showed that the dislocation of the lower tibia was corrected, the anatomical reduction was performed, and the fracture line of the lower fibula was healed , so we completely removed the internal fixation. The range of motion of the ankle joint was as follows: dorsiflexion 15° and plantar flexion 30°. The AOFAS scoring system, developed by the American Orthopaedic Foot and Ankle Society, is one of the most widely used assessment tools in foot and ankle surgery. The patient's post-operative evaluation utilised a 100-point scale, with scores above 75 indicating favourable outcomes. The evaluation encompassed various parameters, including pain, ambulatory function, gait characteristics, joint mobility, ankle stability and joint alignment. The patient's AOFAS score was determined to be 85 points. In order to ascertain whether post-traumatic arthritis had occurred after surgery, we employed the Kellgren-Lawrence grading system, which assesses the patient's condition based on the radiographic findings. This system is graded from 0 to IV, with higher grades indicating more severe lesions. Ultimately, the patient's Kellgren–Lawrence grade was I, indicative of a suspected narrowing of the ankle joint space and the possible presence of osteophytes. It is evident that following the administration of emergency open reduction and internal fixation (ORIF) surgery, in conjunction with active infection prevention during the perioperative period and the implementation of appropriate rehabilitation exercises, the patient's ankle joint attained satisfactory anatomical alignment and functional recovery. This enabled the resumption of daily activities with minimal residual disability.
A Chinese-origin male in his early 20 s sustained a severe injury to his left lower limb in a traffic accident in December 2020. The patient exhibited significant swelling, pain, bleeding and limitation of movement. There was no other significant medical history and the patient denied any relevant family history or psychosocial influences. The emergency team arrived at the scene of the accident and used a small splint to temporarily immobilise the injured left calf. The patient was then transported to the emergency department of Shenzhen Pingle Orthopedic Hospital, which is located at No.15 Lanjinsi Road, Pingshan District, Shenzhen City, Guangdong Province, China.
The initial medical examination by the primary physician revealed marked swelling in the left leg and ankle, accompanied by an irregular transverse wound measuring approximately 6 cm in length on the inner ankle and exposure of the distal tibia. This was accompanied by significant bleeding . The patient was conscious and reported experiencing pain, but was unable to perform active or passive ankle movements. A decreased sensation was noted at the base of the left foot, normal pulsation of the dorsal pedis artery and normal peripheral blood flow. To further clarify the diagnosis and assess the extent of the fracture, the emergency physician performed a radiological examination of the patient's ankle.
presents a timeline that meticulously organises historical and current information relevant to the patient's care. This timeline encapsulates the key medical events, interventions and outcomes from the day of the accident to one-year post-operation, thereby highlighting the patient's recovery and rehabilitation process.
In addition to the physical examination of the patient, laboratory investigations were performed to assess the risk of infection and to determine the necessity for preoperative preparation. These investigations included blood counts and coagulation tests. In addition, a frontal and lateral X-ray of the ankle was performed to analyse the fracture and dislocation in detail. The results of these investigations revealed a significant fracture displacement of the distal fibula and medial ankle, with the talus embedded in the distal tibiofibular union . Fractures or dislocations not associated with open wounds and Pilon fractures were excluded and the final diagnosis was an open fracture of the left ankle in the ‘Logsplitter Injury’ category.
Pre-operative Intra-operative Post-operative Prior to undergoing surgery, interventions such as the administration of braking agents, reduction of swelling, analgesia, anti-tetanus immunoglobulin and empirical prophylaxis of infection were routinely administered. Following the completion of the preoperative examination and evaluation, the decision was taken to proceed with emergency surgical intervention. With the patient's informed consent, the surgical operation commenced at 20:30 h.
The patient was positioned supine on the operating table under spinal anaesthesia, with strict aseptic techniques employed for the preparation of the surgical area. The surgical procedure included the following steps: (1) Wound debridement and exploration: The wound at the left medial malleolus and the surrounding skin were thoroughly irrigated with saline to remove contaminants and clots. The surgical area was disinfected and draped with sterile towels. Exploration revealed an intact posterior tibial tendon, posterior tibial artery and tibial nerve, with a palpable pulse in the tibial artery. The anterior tibial artery and deep peroneal nerve were structurally intact, with a palpable pulse in the anterior tibial artery. Necrotic tissue was excised, and the wound was extensively irrigated with saline. (2) Reduction and fixation of medial malleolus fracture: The area was then subjected to a second cycle of disinfection, after which the use of sterile materials and instruments was replaced. The talocrural joint and medial malleolus were reduced and stabilised using cannulated screws, and the medial wound was temporarily closed. The area was then disinfected once more, and the use of sterile instruments, surgical gowns and gloves were replaced. (3) Reduction and fixation of lateral malleolus: A 15-cm incision was made between the peroneus longus and extensor digitorum longus tendons to expose the fibular fracture end and anterolateral tibia. A high transverse-oblique fracture of the lateral malleolus, syndesmotic separation and anterolateral tibial avulsion fragment were identified. The avulsion fragment was then reduced and fixed with the use of cortical screws, while the fibular fracture was aligned and fixed using a plate and screws. C-arm fluoroscopy was used to confirm satisfactory fracture reduction. (4) Syndesmotic reconstruction: A hole was drilled 2–4 cm above the syndesmosis at a 30° anterior angle, and the use of two 45 mm cortical screws was used for fixation. The talocrural joint was reduced and fixed with two 2.5 mm screws inserted from the plantar aspect. C-arm X-ray confirmed equal medial and lateral joint spaces and proper implant positioning . (5) Placement of antibiotic-loaded bone cement: Partial sutures of the medial wound were removed, and polymethyl methacrylate bone cement loaded with vancomycin at a 10:1 ratio was prepared and placed in the anterolateral ankle and medial tibial spaces. (6) NPWT: Vacuum Sealing Drainage sponges were placed in the anterolateral ankle and medial malleolus areas, secured with sutures and film and connected to a drainage tube for negative pressure suction. A sterile dressing was applied to the lateral malleolus. The operation was completed after 4 h, with an intraoperative blood loss of approximately 300 mL, and the patient was transferred to the recovery room in stable condition.
Following the procedure, the patient was administered routine post-operative analgesia and infection prevention measures. The affected limb was elevated to reduce swelling, and on the second day after the surgery, the morphine injection was discontinued, with the oral administration of diclofenac (75 mg once daily) being maintained. The patient was instructed to ambulate without weight-bearing. To gain a more comprehensive understanding of the position of internal fixators and the alignment of fracture, we conducted imaging examinations of the post-operative ankle joint, including X-ray and three-dimensional CT. The results showed that the fracture and dislocation of the left ankle joint returned to normal, the joint space of the lower tibiofibular joint did not widen significantly, the fracture block was in good alignment, the corresponding relationship of each joint was good, and the joint surface recovered smoothly . On the fifth day, following a stringent wound dressing change, the drainage tube was removed (with a lead flow of less than 10 ml), and the patient was instructed to commence passive ankle flexion and extension training.
The majority of ankle fractures and dislocations are caused by high-energy injuries and generally result in suboptimal functional outcomes. Logsplitter injury is one of the distinct types, which can be categorised into typical and atypical damage according to the injury mechanism. Research has identified that the majority of typical cases occur in the context of road traffic accidents or falls from height and that the fracture reduction rate and range of motion are poor, with a high incidence of post-traumatic ankle arthritis. Radiological Examination reveals that the medial malleolus fracture is located at the ankle joint level, the lower tibiofibular syndesmosis is separated, and the fibula fracture is located above the lower tibiofibular syndesmosis level. The presence of these signs indicates that the foot is in a pronation position when injured and is subjected to external rotation and axial force, thus indicating a typical injury. Currently, a clear consensus on the treatment of Logsplitter injury is lacking. However, the International Association for the Study of Internal Fixation asserts that the vast majority of displaced ankle injuries are unstable, necessitating reliable internal fixation through open reduction to ensure accurate anatomical reduction. It is therefore hypothesised that ORIF surgery is the optimal treatment for Logsplitter injury. The restoration of stability in the ankle joint can be achieved through anatomical reduction and fixation of the tibiofibular syndesmosis, in conjunction with the repair of the deltoid ligament. This approach has been shown to reduce the incidence of post-traumatic osteoarthritis to a certain extent and has been demonstrated to show significant advantages in the early stabilisation of the fracture, promotion of soft tissue healing and accelerated functional recovery. Post-operative results showed that the patient's AOFAS score was 85, indicating a favourable clinical outcome. Recent studies have focused on the management of open ankle injuries, with the traditional ‘Fix and Flap’ protocol emphasising delayed internal fixation after initial damage control. , However, studies have shown that under certain conditions, emergency definitive treatment can achieve similar results to delayed surgery, especially if the operator has a high level of skill and the patient's condition permits. The present case further validates this view, suggesting that emergency open reduction and internal fixation may serve as a viable alternative in young, uncomplicated and highly compliant patients. In the present case management, NPWT and antibiotic bone cement appeared to play a positive role in preventing infection. This assertion is further substantiated by extant studies that support their role in promoting wound healing and reducing the risk of infection. , However, it is also acknowledged that the positioning and technique of intraoperative internal fixation devices require further optimisation. For instance, it is noted that the elevated position of the transverse ligament screws may have an impact on post-operative healing outcomes, a matter that requires attention in future studies. With this case report, we recognise the importance of individualised treatment plan. This case report serves to underscore the significance of two factors in the management of complex open ankle injuries: individualised treatment plans and multidisciplinary teamwork. The findings of this study demonstrate that the precision of surgical technique and comprehensive post-operative management exerts a direct influence on the healing outcome of patients. Consequently, future studies should endeavour to optimise surgical techniques and enhance post-operative management, with a view to further improving treatment outcomes.
This study demonstrates the feasibility and efficacy of emergency definitive treatment in a specific clinical context and highlights the critical role of intraoperative technical details and post-operative management in achieving good functional recovery. The case provides a new perspective and valuable experience in the individualised treatment of open logsplitter injury.
The initial impact of the injury, as well as the severity of the accident, was significant. However, the emergency team's prompt response and professional demeanour provided immediate relief and reassurance, instilling a sense of confidence in the care I was receiving. During the surgical intervention, I was thoroughly informed about the procedures and the anticipated outcomes, which contributed to alleviating my anxiety. The surgical team's meticulous approach and the use of advanced techniques such as antibiotic bone cement and NPWT assured me that I was receiving cutting-edge care tailored to my specific needs. Post-operatively, the pain management plan was effective, allowing me to gradually regain mobility without undue discomfort. The rehabilitation process, although demanding, was crucial in aiding my recovery. The provision of comprehensive post-operative follow-up care and clear instructions regarding post-surgical exercises was also beneficial in restoring function to the patient's ankle. The multidisciplinary team's dedication and the personalised treatment plan were instrumental in the patient's recovery, and the support and expertise they provided enabled a return to daily activities with minimal limitations. This experience serves as a testament to the effectiveness of modern medical interventions and the importance of patient-centred care.
The patient has provided written informed consent for the treatment and procedures described in this case report, including surgical interventions, post-operative care and rehabilitation. The patient was thoroughly informed about the nature of the injury, the proposed treatment plan, potential risks and expected outcomes. Additionally, the patient consented to the use of their medical data and images for this publication, understanding that all identifying information will be kept confidential to maintain privacy. We have obtained the patient's written informed consent (Chinese version).
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A comprehensive study on MDP effects: Microshear bond strength and fatigue resistance in 4YSZ ceramics | a5d01855-65b7-49d4-90b8-d44dae897da1 | 11654019 | Dentistry[mh] | Over the years, zirconia ceramics have undergone modifications, evolving from being solely used as frameworks and copings to serving as monolithic restorations with enhanced aesthetics, particularly with 4 mol.% yttria-stabilized zirconia (4YSZ). However, due to the polycrystalline nature of zirconia, lacking a vitreous matrix and resistance to acid etching, establishing an effective adhesion protocol remains a challenge . The advantages of adhesive cementation, involving luting components with chemical adhesion to ceramic materials, are evident in improving the mechanical properties of glass ceramics, however, in a recent systematic review the same conclusion could not be drawn to zirconia ceramics . Besides the zirconia’s great mechanical resistance , its polycrystalline nature makes it unable to bond with a traditional silane agent without a proper air abrasion with silica coated aluminum oxide . One concern about this technique is that the restoration surface may not be uniformly air abraded, leading to low-adhesive zones that could compromise the overall bond strength . Yet, the application of adhesive cementation in polycrystalline zirconia-based ceramics presents a knowledge gap that requires investigation . Notably, the impact of luting systems containing 10-Methacryloyloxydecyl dihydrogen phosphate (MDP) or similar substances in their components have been considered, given MDP's ability to form strong chemical interactions with metallic oxides, such as zirconium oxide . This occurs by bonding P-OH groups, present in MDP, to zirconia crystals. Consequently, it has been speculated that components with high MDP concentrations would enhance effectiveness in bonding to zirconia . Recently, the market has seen the introduction of numerous luting systems incorporating MDP in various components. When opting for systems that feature this monomer, the clinicians have essentially three options, being one, systems with an MDP-containing primer or adhesive associated with an MDP-free cement. Another option is the use of an MDP-containing cement that does not require primers, and a third option with the use of an MDP-containing primer associated with an MDP-containing cement. Nevertheless, the optimal allocation of MDP within the luting components (primer, adhesive or cement) and whether the combination of different components containing it, yields additive effects when facing an environment of extensive challenges to the adhesive interface require further clarification . Moreover, when assessing the success of indirect restorations, crucial considerations include their adhesion and fracture resistance . In the context of adhesion studies, employing longer and more aggressive water aging protocols is believed to yield more pertinent data for investigating bond interfaces . Aging in water-based media, besides breaking chemical bonds, can induce changes in the mechanical properties of cements . This alteration in properties influences stress distribution, potentially resulting in stress accumulation in ceramic restorations and leading to premature failures . Additionally, stress accumulation occurs in fatigue regimes, where subcritical loads are applied over extended periods. In such scenarios, minor defects can grow and become critical, diminishing the material's resistance and contributing to restoration failures . Given the aforementioned, there is a lack of studies assessing luting strategies involving MDP at various steps with an intensive aging protocol concerning 4YSZ 's bond strength and mechanical fatigue behavior. Hence, this study investigated whether luting components featuring MDP at different stages of the cementation process and the combination of materials containing MDP could produce a favorable additive effect on microshear bond strength and fatigue behavior in simplified 4YSZ restorations bonded to an epoxy resin. The tested hypothesis were that luting systems containing MDP at the different components of the luting process, and the MDP additive effect on would improve microshear bond strength (first hypothesis) and fatigue behavior (second hypothesis) of simplified 4YSZ restorations bonded to an epoxy resin.
The sample size was determined using G*Power (Heinrich-Heine-Universität, Düsseldorf, Germany), with parameters set at a power of 80%, an alpha-type error of 0.05, and an effect size of 0.42. The considered sample units for the microshear bond strength tests were the cement cylinders, while for the fatigue resistance tests were the luted assemblies. Details on the materials utilized, including manufacturers and composition information, are provided in . The study experimental design is outlined in . Microshear bond stregth (µSBS) specimens Fatigue resistance specimens Cleaning and surface treatments Luting protocols and procedures Aging Microshear bond streagth (μSBS) Test Cyclic fatigue test Failure and fractographic analysis Statistical analysis The Kolmogorov-Smirnov test confirmed the normal distribution of the data. Bond strength data from adhesive failures underwent analysis through two-way ANOVA and Bonferroni post-hoc test (α= 0.05) using statistical software (IBM SPSS, Version 28.0, IBM Corp, Armonk, USA). Survival analysis, including Kaplan-Meier followed by Mantel-Cox post-hoc tests, was performed considering FFL and CFF (α= 0.05) using the same statistical software. Survival rates were calculated for all parameters at various testing steps. Qualitative analysis was employed for failure data.
Forty zirconia (4YSZ) square slices, measuring 19 mm by 19 mm by 2.5 mm (width × height × thickness), were cut from prefabricated blocks (IPS e.max ZirCAD MT A2, Ivoclar, Schaan, Liechtenstein) using a cutting machine (IsoMet 1000, Buehler, Lake Bluff, USA) with continuous water-cooling. Topographical standardization was achieved by polishing with a sequence of silicon carbide sandpapers (#400-, #600- and #1200-grit) using a polishing machine (Ecomet/Automet 250, Buehler) under refrigeration for 30 s each sandpaper. Subsequently, the slices were sintered (VITA Zyrcomat 6000 MS, Vita Zahnfabrik, Germany) at 1,500°C for 120 min, following the manufacturer's recommendations, resulting in final dimensions of 15 mm by 15 mm by 2 mm (width × height × thickness). Finally, these slices were embedded in polyvinyl chloride (PVC) cylinders using self-curing acrylic resin, leaving the surfaces to be bonded exposed (JET Clássico, Campo Lindo Paulista, Brazil).
Similar to the procedure for obtaining bond strength specimens, prefabricated 4YSZ blocks (IPS e.max ZirCAD MT A2, Ivoclar AG) were cut into squares measuring 13 mm by 13 mm using a cutting machine (IsoMet 1000, Buehler, Lake Bluff, USA) with water-cooling. These squares were then shaped into cylinders with a diameter of 12 mm using a polishing machine (Ecomet/Automet 250, Buehler). Subsequently, the cylinders were cut into 48 units of 1.2 mm thick discs using a cutting machine (IsoMet 1000, Buehler) under water-cooling, considering an estimated shrinkage rate of approximately 20% during the sintering process. Topographical standardization was achieved by polishing withwater-cooling for 30 s each sandpaper. The resulting discs were sintered following the same process as the slices, resulting in final dimensions of 10 mm in diameter and 1 mm thickness. Dentin analog discs (epoxy resin reinforced by glass fiber - Carbotec GmbH & Co. KG, Königs Wusterhausen, Germany), measuring 10 mm in diameter and 2.5 mm in thickness, were obtained through cutting large prefabricated cylinders through a similar process .
Following preparation, the specimens underwent cleaning in an ultrasonic bath (92% isopropyl alcohol - 5 min). The epoxy resin discs were etched with 10% hydrofluoric acid for 60 s, followed by thorough washing with water and drying using an oil-free air jet. All 4YSZ ceramic specimens underwent air abrasion with 45 μm Al 2 O 3 particles (Polidental, Cotia, São Paulo, Brazil) for 10 s under 2 bar pressure, employing oscillating movements at a distance of 1 cm between the surface and the blaster tip. Subsequently, the 4YSZ specimens were cleaned again in an ultrasonic bath and assigned to their respective experimental groups.
The protocols were implemented in accordance with the manufacturer's application recommendations and selected based on the experimental groups outlined in . The selection considered products available in the market, their compositions, and indications as follows: 1- Universal Primer: A universal primer containing MDP, disulfide, and silane (Monobond N, Ivoclar) was chosen. In this group, the primer was actively applied onto the 4YSZ surface for 15 s and volatilized for 45 s. Furthermore, only for the fatigue test, Primers A and B were mixed and actively applied to the etched dentin analogue for 30 s, followed by luting with Multilink N resin cement (Ivoclar AG). 2- Universal Adhesive: A universal adhesive containing MDP (Single Bond Universal adhesive; 3M ESPE Dental Products, St. Paul, MN, USA) was applied actively onto the 4YSZ surface for 20 s and volatilized for 5 s. Furthermore, only for the fatigue test, the adhesive was also applied on the etched dentin analogue. Luting was then carried out using RelyX Ultimate resin cement (3M ESPE). 3- Cement: A resin cement with MDP (Panavia F 2.0; Kuraray, Kurashiki, Okayama, Japan) was employed, by mixing equal amounts of pastes for 20 s before application onto the 4YSZ surface. Furthermore, only for the fatigue test, ED Primer II A and B were mixed and actively applied on the dentin analogue resin for 30 s, gently air-dried, and then luting was carried out with the resin cement. 4- Primer + Cement: This group utilized an VBATDT and MDP-containing primer (Alloy Primer; Kuraray) and resin cement (Panavia F 2.0, Kuraray). The primer was applied to the 4YSZ ceramics and left untouched for 45 s until the solvent was evaporated, followed by luting with the resin cement as described above. For μSBS specimens, following surface treatments and primer applications, ten starch tubes (height= 1 mm, internal diameter= 1.2 mm) (Isabela, M. Dias Branco S.A. Indústria e Comércio de Alimentos, São Caetano do Sul, Brazil) were affixed with wax 7 on the luting surface. The resin cement from each experimental group was manipulated, applied inside the matrices using a resin spatula and endodontic spacer (#25, Dentsply Maillefer, Switzerland), and light cured for 20 s at close range (1200 mW/cm², Radii-Cal, SDI, Bayswater, Australia) in accordance with the manufacturer’s recommendations. Resulting in ten resin cement cylinders per ceramic slice. For fatigue strength specimens, the resin cements were applied to the luting surface of the 4YSZ ceramic discs, which were then placed on the respective epoxy resin discs. Subsequently, a constant load of 2.5 N was applied, excess cement was removed, and the assembly was photoactivated for 20 s at close range (Radii-Cal, SDI) at each position (0°, 90°, 180°, 270°, and top).
After luting, the specimens were immersed in distilled water at 37 ºC for 24 h. Following this, the starch tubes were removed, and the resin cement cylinders were examined. Any defects or bubbles observed at the interface resulted in the discarding of the respective cement cylinder. Post-evaluation, five specimens from the same ceramic slice underwent the microshear bond strength test, while the other five were subjected to an aging condition. For the groups undergoing aging, storage occurred in distilled water at 37 ºC for 180 days, followed by exposure to 25,000 thermal cycles (5-55ºC, residence time 30 s, transfer time 5 s) using a thermal cycling machine (Nova Ética, Vargem Grande do Sul, SP, Brazil) . Regarding the fatigue test, half of the specimens were stored in distilled water at 37ºC for 24 h prior to testing, while the other half underwent the aging protocols described above.
The specimens were affixed to a universal testing machine (EMIC DL-2000, São José dos Pinhais, Brazil) and subjected to testing with a 100 N load cell at a speed of 1 mm/min using a stainless-steel wire (Ø= 0.2 mm) positioned on the adhesive interface. Bond strength (MPa) was calculated using the equation “S=L/A,” where “S” represents bond strength (MPa), “L” denotes the load at which the specimen failed (N), and “A” signifies the area of the adhesive interface (mm²).
The specimens underwent a cyclic fatigue test until failure using specialized equipment (Instron ElectroPuls E3000, Instron Corporation, Norwood, USA). In this test, cyclic loads were applied by a 40 mm diameter stainless steel hemispherical piston, with the sample immersed in water. To enhance contact and stress distribution, an adhesive tape (110 μm) was positioned between the piston and the luted assembly . A load frequency of 20 Hz was employed , with an initial 5,000 cycles at 200 N to adjust the piston/specimen contact. Subsequently, 15,000 cycles were performed at progressively increasing stress levels of 100 N, starting at 400 N, until crack detection by transillumination . The values for fatigue failure load (FFL) and the number of cycles to fatigue failure (CFF) were recorded for subsequent statistical analysis.
Following the bond strength test, all specimens underwent analysis using a stereomicroscope (Stereo Discovery V20, Carl Zeiss, Göttingen, Germany). Failures were categorized as adhesive (involving more than 50% of the adhesive area) or cohesive (involving more than 50% of one substrate). Only adhesive failures were considered for statistical analysis. Representative images of each failure type were captured using Scanning Electron Microscopy (SEM - Vega3, Tescan, Czech Republic) at 100× magnification. For the fatigue test, fragments from the failed specimens were highlighted to facilitate visualization of the failed region. Using a stereomicroscope (Discovery V20, Carl-Zeiss, Göttingen, Germany), the region of the crack and other fractographic characteristics were observed. Following this, a representative failed specimen was chosen for scanning electron microscopy analysis (Vega3, Tescan) at 250× and 1500× magnification to determine fracture origin, direction of crack propagation, and other pertinent fractographic features.
In the Microshear Bond Strength test, two-way ANOVA and Bonferroni post-hoc tests revealed a statistically significant decrease in bond strength values for the Primer + Cement group (p<0.001), particularly after aging. The Cement group appeared stable after aging (p=0.303), while the Universal Adhesive and Universal Primer groups showed an increase in bond strength values (p<0.05). After aging, the Universal Adhesive and Universal Primer groups demonstrated statistically higher bond strength values compared to other groups (p<0.05); and the Cement group exhibited superior results compared to the Primer + Cement group (p<0.001) but inferior to the other groups (p<0.05). Pre-test failures were observed only in the Primer + Cement Aged group, resulting in the loss of a whole ceramic slice. Failure analysis indicated a predominance of adhesive failures. The percentage for the occurrence of cement cohesive failures relative to adhesive failures in the groups was as follows: 0%/100% in Universal Primer Baseline, 2%/98% in Universal Adhesive Baseline, 4%/96% in Cement Baseline, 14%/86% in Primer + Cement Baseline, 0%/100% in Universal Primer Aged, 0%/100% in Universal Adhesive Aged, 6%/94% in Cement Aged, and 2.2%/97.8% in Primer + Cement Aged . In terms of load-bearing capacity under fatigue, Kaplan-Meier and Mantel-Cox tests showed no statistically significant differences among the tested groups for both fatigue failure load and cycles for failure (p>0.05). The aging process did not lead to a decrease in fatigue strength data (p>0.05). Regarding failure patterns, radial cracks were observed in all tested specimens .
This study investigated the adhesive and mechanical effects of using various luting systems containing MDP at the different components of the luting process, examining its additive impact on bonding to a 4YSZ ceramic. In the adhesive test, favorable behavior was observed in systems utilizing a universal primer or universal adhesive with MDP, combined with a non-MDP resin cement. However, the combination of different products with MDP did not show a favorable outcome. Thus, the first tested hypothesis was partially rejected. In contrast, the fatigue mechanical test revealed no differences among the used systems, indicating that the presence of MDP in a specific step or component, or its additive effect, is not a significant factor for this outcome. Consequently, the second tested hypothesis was rejected. The current study observed that the group using an MDP primer in conjunction with an MDP-containing resin cement exhibited the poorest bond strength results to a 4YSZ. Similar findings were obtained by Moura et al., (2018) for a different zirconia ceramic, where luting systems with an MDP-containing primer or adhesive and MDP-free cements yielded better results than a combination of components with MDP . Additionally, existing literature suggests a potential plateau in MDP concentration , challenging prior assumptions . Whether by successive MDP primer applications , or by the combination of a primer and cement containing MDP , it seems that the extra monomer quantity is, at least, ineffective. Perhaps in these scenarios, the excess of MDP components will not have available zirconia particles to bond, showing no additional or beneficial effect . The primer used in the Primer + Cement group also contains VBATDT, another chemical intended to bond to zirconia and appears to have a synergic effect with MDP when they are in specific concentrations . However, it is not clear by the manufacturer the specific concentration of each component in the used formulation. Furthermore, the presence of MDP in the Primer + Cement group in the present study was negatively affected by the aging protocols more than non-MDP resin cements with prior MDP-containing universal primer or universal adhesive application, which is also in accordance with the literature . When testing experimental MDP containing adhesives, no linear correlation was found between MDP concentration and bond strength . Furthermore, as the MDP concentration in each system varies, there is still no consensus on whether there is a limit for the MDP concentration over the zirconia surface. It is well established that the utilization of luting systems containing MDP is associated with improved adhesion to zirconia, as highlighted in the systematic review by Özcan and Bernasconi (2015) . Even though, it was previously demonstrated that the use of MDP-containing components still leads to a decrease in bond strength values over time when employing an experimental primer or luting components featuring this monomer . This demonstrates the possibility of degradation and a long-term reduction in performance. When considering the oral environment, humidity is an exposing factor that always must be considered. The constant saliva presence facilitates the adhesive layer degradation, compromising its chemical bonds and quality . This degradation probably occurred due to the breaking of the bonds via hydrolysis or due to an insufficient MDP concentration to sustain strong bonded chains , since the increase in the concentration of this monomer can lead to an improvement in the interactions between the resinous material and ceramics . In addition, when it comes to in vitro studies, it has been pointed out that long times and aggressive aging protocols lead to more relevant data considering adhesion outcomes , . One notable result from the present study is the increase in bond strength after aging in the Universal Primer and Universal Adhesive groups and possible reasons for this can be interpreted. The Universal Adhesive has in its composition a specific copolymer (Vitrebond copolymer) that is a methacrylate-modified polyalkenoic acid and is known to have a moisture-stabilizing effect. This was possible to provide enough time for the resin cement to continue its polymerization process and have a better interlocking effect in the ceramic topography, yielding better bond strength over time . In the Universal Primer group, a similar reason explains the bond strength improvement after aging. As the resin cement for this group has a greater degree of conversion than in the other groups , the provided micromechanical interlocking was improved over the interaction with high temperatures, providing additional polymerization and an increase in elastic modulus . Furthermore, both resin cements without MDP also have a lower Young’s Modulus (Multilink N: 6.3 GPa and RelyX Ultimate: 7.7 GPa) than the one containing MDP (Panavia F 2.0: 12 GPa) , , which make them easier to penetrate the ceramic’s irregularities and promote better micromechanical interlocking. Despite the aesthetic advancements in zirconia ceramics, their notable feature remains high mechanical strength. When examining the fatigue data, it becomes evident that the different luting systems, with variations in the MDP component, did not induce significant alterations. This can be attributed to the high elastic modulus of 4YSZ (210 GPa) . Consequently, there were no statistical differences among the tested groups, as the elastic modulus of the used resin cements (Multilink N: 6.3 GPa; RelyX Ultimate 7.7 GPa; Panavia F 2.0: 10.2 GPa) , (28,29)exhibited small variations when compared to the ceramic. Previous tests with 4YSZ discs in setups similar to the present study, with comparable thickness, have indicated that this ceramic is more influenced by the support material than by differences in resin cement properties . Additionally, while water aging can potentially alter the resin cement's elastic modulus by carrying away water-soluble monomers and affecting tension distribution over the restoration , in the current study, this was not sufficient to modify the fatigue behavior of the tested groups. Some limiting factors should be considered in the present study, such as the use of luting systems from different manufacturers, leading to variations in material composition and concentrations. Additionally, the Primer + Cement group employed components from the same manufacturer, although not indicated for use together. This was done to address the research question posed in the study. Furthermore, zirconia ceramics from different generations may exhibit varying responses to the proposed techniques and require further investigation. While the study employed water storage and thermocycling aging methods, the oral environment is dynamic and aggressive. Models incorporating pH variations could yield different results. Despite utilizing a simplified disc setup for the mechanical test, this research stands as the first to evaluate adhesive outcomes with a fatigue strength test, assessing different luting strategies containing MDP.
Luting systems with MDP-containing primers or adhesives paired with non-MDP resin cements demonstrated enhanced bond strength for 4YSZ in the long term. Despite that, all luting systems exhibited comparable fatigue behavior for 4YSZ bonded to an epoxy resin.
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Cushing’s Disease Management: Glimpse Into 2051 | 9f455a5d-516b-4339-81c0-8d71a011b26b | 9299426 | Physiology[mh] | 23 June 2051 00:00 : Sound exposure: limited to the breathing sounds & background noise Light exposure: no blue light exposure. Heart Rate: Normal Systolic Blood Pressure: Trending higher at night Diastolic Blood Pressure: Trending higher at night Glucose: Trending higher Hypnograph: Stage 3/Stage 4 sleep. Stress level: “High” Cortisol Level: “High” from 00:00 to 24:00 23 June 2051 06:00 : The alarm gently rings and plays “Good morning”. Sunlight creeps through the curtains and fills the room with warmth and light. Claire awakens, rubbing her eyes seeing the analytics of the previous day affirming good productivity and excellent sleep patterns. A red notification blares on the side indicating high stress levels throughout the night, tense muscles around the head and forecasting a feeling of headache that may prop up in the day, suggesting a dose of painkiller before work. The weather outside is reported as very good with no rains and work schedule is displayed after that. The living room and kitchen is spick and span; the bath water exactly 37 degrees Celsius, the day’s clothes laid out- creaseless and ironed. Breakfast table is laid with 2 eggs, sunny side up. The self-driving ride is on time with the first meeting on the way to work. The planned day runs smoothly. The bank account at 15:00 hours shows that the amount has increased as expected. The watch detects an exercise pattern in the evening, a slow walk for 60 minutes with increase in heart rate to warm up level. The 10,000 steps goal for the day was achieved. Bright red – the notification remains visible on the side and beams again indicating nocturnal trend of high stress in the evening. A reminder to watch the trend is added to her digital notes. 30 June 2051 06:00 Alarm beeps: Reminder: check the detailed analytics . Heart Rate: Trending higher Heart Rate Variability: Trending Lower More information: see detailed analytics Systolic Blood Pressure: Abnormal More information: see detailed analytics Diastolic Blood Pressure: Abnormal More information: see detailed analytics Glucose: Abnormal More information: Baseline trend higher; see detailed analytics Hypnograph: Awake. Stress level: “Very High” Cortisol Level: “High” from 00:00 to 24:00 More information: No Dip of cortisol levels at night; see detailed analytics 1 July 2051 06:00 Alarm beeps: The stress hormones are in dangerous high levels, please visit the nearest healthcare facility for deeper analysis . The morning ride slows at the entrance of a healthcare facility and signals -disembarkation. Claire alights and enters the reception grumbling about the ‘obvious glitch in the watch’. They take the wearable and download all the data to the nearest workstation. Indeed, very high cortisol levels with a loss of the usual circadian rhythm in all parameters is observed. A higher order specialist workstation (termed Endocrinology) is assigned to Claire. She waits for her turn; foot tapping impatiently and enters the room. An endocrinologist is seated at the computer table. She smiles serenely and asks about the day, general feelings, and emotions. The Heart rate monitor reflects an increase, as Claire feels visibly uncomfortable sharing deep thoughts with a stranger. A new watch is given to her with an additional chip which would measure the levels at increased intervals, more precisely, throughout the day for next week and upload results to her doctor’s database. The situation would be reviewed in one week to decide upon the next course of action. The week passes routinely. The only reminder is on the day of the appointment when the ride slows again in front of the healthcare facility. This time, the lady is whisked directly into her endocrinologist’s room. The data, uploaded live and pathways studied, shows high hormones -steroids with an active pituitary to adrenal pathway suggesting a pathology in the pituitary gland. The proposed diagnosis is explained, and a small chip is inserted underneath her collar bone. After the chip insertion, she is made to pass through a scanner and remain there for 5 minutes. The screen confirms the diagnosis: Cushing’s disease: microscopic hot spot in pituitary. The next screen recommends an available specific targeted treatment to target this region deliverable through a small nano-based therapeutic implant on the forearm. Claire is free to go after that. Daily circadian patterns, monitored through the watch, slowly returns to normal within a month. Every time a similar increase in cortisol levels over a threshold is seen, an additional dose is delivered remotely through the same implant. No further alarms are heard as life continues as usual.
In the 20th century, practice of medicine and healthcare benefited from significant scientific breakthroughs. We are at inflection point for another incredible breakthrough in healthcare – in the sense that digitization will enable the application of data technologies and artificial intelligence into healthcare. The term ‘digital biomarker’ has been introduced. FDA defines a digital biomarker as “characteristic or set of characteristics, collected from digital health technologies,� . This ability to derive biomarkers from daily patterns can potentially provide context to enrich normal values for the population, derive individual person-centric baseline values, and assess changes in health status over time to make clinical diagnosis. Modern-day wearables can be in the form of headbands, sociometric badges, camera clips, smart watches or sensors embedded in clothing and have the ability to monitor vital physiological measurements such as heart rate, electrocardiogram, heart rate variability, respiratory rate, oxygen saturation, temperature, pressure sensors, activity levels, sleep patterns, environmental sound and light exposure etc . The requirements for authorisation by the U.S. FDA or regulatory CE-marking, remuneration, and privacy/data security depend on the specificities of the product, its purpose, the technology, the risks and benefits, and the data it processes. Devices with capability to measure blood pressure, in the form of multi-parameter, miniaturized solutions for home environments are currently being pursued with great interest . Correlations of ambulatory blood pressure, especially high nocturnal blood pressure with cardiovascular risk has been observed and automated methods of blood pressure monitoring are being encouraged . Various techniques are being exploited for these measurements including miniaturization of cuff oscillometry, tonometry, pulse propagation techniques and pulse wave analysis . Pulse propagation techniques include using the PTT (pulse transit time) or the PAT (pulse arrival time) (time required for the pulse to travel between 2 arterial sites) is directly proportional to the blood pressure. Photoplethysmography (PPG) uses optical and inertial sensors to detect blood flow patterns. The technology indirectly measures the blood flow rate through the amount of light absorbed or reflected by blood vessels. Since the relationship between PPG and blood pressure is non-linear, a machine-learning algorithm is used to convert blood flow information to blood pressure measurement. As more data is collected, the algorithm will get more precise . Protocols for validation of these ambulatory blood pressure measurements are being developed and some of these devices will likely receive regulatory approvals in the near future. Please see for a summary of devices with FDA approval or CE mark. Currently, non-invasive methods for glucose measurements in a simple wearable like a watch, are under development. Methods using a subcutaneous wired enzyme glucose sensor inserted in the body which transmits data to a smart phone are available and approved by the FDA . These systems can be applied by self or need to be implanted by a healthcare professional. Cortisol rises early in morning and is highest before awakening, it falls naturally throughout the day and can spike in response to meals and to stress. Current methods for measuring cortisol concentrations is a laboratory-based blood test and is time consuming. Increasingly more rapid and direct plasma assays are being developed . Salivary and sweat cortisol concentrations reflect the systemic steroid concentrations . In terms of development, several independent researchers across the globe are working on systems which can be used to measure cortisol concentrations in body fluids and can thus be estimated on a superficial patch or wearable. A cortisol sensor has been formulated using extended gate-field-effect transistor . This has been developed as wearable contact lenses which can detect cortisol concentration in tears . This cortisol sensor is integrated with transparent antennas and wireless communication circuits to link with the smartphone . A similar sensing system applied on the wrist with capability to measure sweat cortisol levels has been developed and tested which shows promise . It is very likely that such a device will be developed and integrated into the traditional wearable watch as cortisol levels have applications in measurement of daily stress or allostatic load. With regards the percentage of population using a wearable, whether the utopian type of order written in this short story can be true, is also highly probable. Insurance companies or other healthcare payers are likely going to mandate wearing of a daily wearable, so to enable preventive care. It is likely that the premium rates may be higher in individuals refusing to comply in the beginning but in the long run when the population adopts this technology, it will become a mainstay. This lady above has ACTH-dependent Cushing’s syndrome secondary to a pituitary adenoma also called Cushing’s Disease (CD). Cushing’s disease was first described in a landmark monograph more than a century ago, in 1910 by Dr. Harvey Cushing. He described his first patient, Minnie G. to have “… syndrome of painful obesity, hypertrichosis, and amenorrhea, with overdevelopment of secondary sexual characteristics accompanying a low grade of hydrocephalus and increased cerebral tension. Pituitary, adrenal, pineal or ovary?” . Diagnosis and management of CD has evolved significantly in the last century. Despite the advances, significant pitfalls and challenges remain. The typical patient presents 5-10 years into the illness, when the high cortisol hormones lead to downstream multi-organ problems. They present to healthcare when frank symptoms and signs are visible which includes significant change in appearance (moon shaped facies, central obesity) and change in metabolic status (hypertension, diabetes mellitus) and body composition (central visceral obesity and osteoporosis). After clinical suspicion, multiple tests (1mg dexamethasone suppression test, 24 hrs urine free cortisol, midnight salivary cortisol, ACTH, cortisol assays) are required to confirm the diagnosis. Once diagnosis is confirmed, then localisation is extremely challenging and pituitary adenomas secondary to Cushings’ is detected on magnetic resonance imaging with sensitivity ranging from 42% to 85%. Early, small lesions <4 mm in size are even more difficult to localise. Functional imaging, in the form of 11c-methionine PET, is still under research development. The invasive inferior petrosal sinus sampling needs to be performed which can localise the lesion at best to the pituitary gland only . Many of the tumours are sent for surgery without localisation and are localised intraoperatively . Surgical treatment is the mainstay for pituitary adenomas but remains challenging and only a handful of patients go into remission (at best 60-70%) . Medical treatment has evolved with 2 FDA approved therapeutics (pasireotide and mifepristone). However, even these are not superior to curative excision . Early diagnosis in CD can be made through changes in heart rate and blood pressure dynamics . The hypothalamic-pituitary-adrenal axis (HPA), responsible for the circadian rhythm of endogenous cortisol secretion, contributes to the circadian rhythm of blood pressure . In CD, the typical dip in nocturnal blood pressure (lower by 10% from baseline) is absent and the daytime heart rate is higher . Heart rate variability shows a characteristic pattern in terms of circadian differences and the typical pattern of highest between 10-2 PM at night is attenuated in Cushings disease . Corticosteroids also affects insulin signalling pathways directly and through an increase in growth hormone and results in higher post prandial glucose and blunted circadian pattern . A characteristic pattern has also been reported in patients with acromegaly, a pituitary condition with high growth hormones even before it affects glucose tolerance . Differential diagnosis includes: Phaeochromocytoma, and primary aldosteronism. Periodic patterns would suggest phaeochromocytoma and similar pattern as CD with normal steroid concentrations suggest primary aldosteronism. Adrenal and pituitary incidentalomas are commonly detected during screening for non- related medical concerns. These may represent subclinical hypercortisolism in otherwise clinically asymptomatic patients. The wearables can potentially be used to ascertain subclinical disease and to differentiate from pseudo-Cushings’ syndrome (occurs in obesity, alcoholism etc). Conversely, with the advent of regular wearables incorporating cortisol, it is possible that such subclinical glucocorticoid excess will be detected more frequently and may even be the causative mechanism in some patients with metabolic abnormalities. We envision that initially, the wearables will be useful in patients with clinical suspicion like above. However, with time as more long term longitudinal data is collected in the population (over 10-20 years), big data analytics is set to uncover digital biomarkers (patterns) that can be used to make an early diagnosis before definite clinical signs appear. We envision that by 2051, preventive care with remote digital monitoring is highly probable at a population scale level. Next steps for localisation require the characterisation of CRH-ACTH-cortisol pathway . Cushings disease has a unique metabolomic signature and with advancement in omics platforms , and in artificial intelligence predictive analytics it is highly probable that the pathway can be used to identify the active areas. Pituitary lesions in Cushing’s syndrome are only detected by MRI in <60% of cases. Hybrid imaging combining PET and MRI such as 11C-methionine PET co-registered with volumetric MRI will likely improve the sensitivity and specificity in the near future . As novel data reveals more information on exact gene and protein expressions in these tumours, it will become possible to design advanced functional imaging methods which targets these areas to show “hotspots”. Molecular targeted therapies such as ACTH antagonists or melanocortin type 2 receptor (MC 2 R) , EGFR, retinoic acid receptors, CDK with specific inhibitors for CD, and cyclin E-Mediated Human Proopiomelanocortin pathway are being developed. Efficiency in targeted delivery can be achieved with the conjugation of drugs with target cell surface-targeting moieties and encapsulation of unique nanocarriers/nanoparticles . Studies evaluating the clinical efficacy of these therapeutics will bring some of these into clinical practice. While the above case vignette, appears to be a sci-fi fantasy and significant challenges in each area of diagnostics and therapeutics remain; the wearables and the massive data that will be accrued, will likely transform healthcare through predictive modelling and implementation of personalised care. One of the key factors for successful implementation is defining specific problems for targeted wearable solutions in specific disease states and establishing partnerships with clinician champions . We envision that these methods are set to bring about a major paradigm shift in the management of most endocrine related conditions. The practice of endocrinology is set to evolve significantly in the coming decades. At the turn of the 20th century, Dr. William Osler said: “Listen to your patient; he is telling you the diagnosis,” In the 21st century: “Look and analyse the digital physiological and behavioural trends; therein lies the diagnosis”RD and BB conceptualized the short story, performed literature review, critically reviewed and wrote the final draft. SB reviewed and critically evaluated the final draft. All authors reviewed the final |
Characterizing the variation in safflower seed viability under different storage conditions through lipidomic and proteomic analyses | 6340fb0d-5fe1-48ca-aa64-a6770c76970e | 11914450 | Biochemistry[mh] | Safflower ( Carthamus tinctorius L.), a member of the Asteraceae family, serves as a versatile cash crop. The dried tube flowers of safflower are utilized as Chinese medicinal materials and are known for their hot, bitter, and warm properties. In traditional Chinese medicine, they are commonly used to promote blood circulation and remove blood stasis. Historically, safflower was regarded as one of the raw materials for dye production before the advent of chemically synthesized dyes . Safflower seeds are of both medicinal and economic value due to their high content of unsaturated fatty acids. Specifically, their Linoleic acid (LA) content is approximately 74% . Safflower also has a rich history of use in Chinese medicine for treating cardiovascular diseases, showcasing notable anti-myocardial ischemia effects . Additionally, safflower exhibits various pharmacological properties such as anti-coagulant, antioxidant, and neuroprotective effects . Safflower was introduced to the mainland by Zhang Qian during the Western Han Dynasty, following his travels to the Western regions. It was further spread across the world during the Tang Dynasty, mainly through the efforts of Tang Dynasty ambassadors. Presently, safflower is extensively cultivated in China, with significant plantations in Xinjiang, Yunnan, Sichuan, and Henan provinces. Notably, Xinjiang boasts a large safflower planting area . Despite its global cultivation, safflower remains an underutilized cash crop in terms of its potential value, adaptability to high temperatures, drought, saline-alkali lands, and the changing environmental conditions observed in recent years. Seeds are the fundamental materials for agricultural planting and production, playing a crucial role in determining the level of agricultural output. However, seed aging presents a significant challenge to seed germination, production, and storage, resulting in considerable economic losses. It has emerged as one of the primary threats to seed vitality . Numerous factors contribute to seed aging, encompassing both genetic (internal) and environmental (external) factors. During the natural aging process, seeds generate reactive oxygen species (ROS), also known as free radicals. Excessive accumulation of ROS, primarily produced by mitochondria, induces abnormal cell metabolism and accelerates aging . Seed aging involves distinctive cellular, metabolic, and chemical changes, including chromosomal aberrations, DNA damage, reduced RNA and protein synthesis, alterations in enzyme and sugar reserves, and compromised membrane integrity ; . Our previous study demonstrated a significant decline in germination percentage of safflower seeds stored at room temperature for different durations (4 months, 16 months, and 28 months). Moreover, an increase in aging time was accompanied by a notable decrease in catalase (CAT) activity and a significant rise in malondialdehyde (MDA) content. Additionally, the aging process led to substantial modifications in both the surface and internal structure of the cotyledon. Therefore, investigating improved preservation methods for safflower seeds holds great significance. Low temperature and air isolation have been identified as effective methods to prolong the longevity of seeds. Research has demonstrated that Dendrobium officinale seeds, when dried and stored at different temperatures including room temperature, 4 °C, -20 °C, and in liquid nitrogen, exhibit varying germination percentages. Notably, when the seed water content reaches 20.63%, the germination percentage can achieve 94.15%. Additionally, a seed moisture content ranging from 10 to 20% can result in a seed germination percentage exceeding 90%. Optimal storage practice involves subjecting the seeds to 24 h of drying treatment followed by storage in liquid nitrogen . Furthermore, an investigation was conducted on the impact of different packaging methods on the storage stability of 89 wheat seeds in foil bags and non-vacuum aluminum boxes at -40℃ over an 11-year period in a low-temperature germplasm bank. This article examines storage effects under low-temperature conditions by analyzing three aspects: the difference in germination percentage between stored seeds and the initial germination percentage before storage, the difference in seed germination percentage between the two packaging methods across different years, and the difference in seed germination percentage between the two methods within the same year. The findings indicate that, after 11 years of low-temperature storage, 82% of the material exhibited an average germination percentage higher than the initial rate. However, a significant decrease in germination percentage was observed only after the 10th year of storage in comparison to the initial germination percentage. Additionally, the germination percentage decline in seeds stored in aluminum foil bag packaging was less pronounced than in those stored in aluminum boxes, with a statistically significant difference. Moreover, the storage effect of vacuum-sealed aluminum foil bag packaging was significantly superior to non-vacuum packaging . Nonetheless, further research is required to explore the specific mechanisms underlying the situation in safflower. To investigate the effects of temperature and oxygen levels on the membrane structure of safflower seeds, an array of storage conditions were employed, including: -18 °C, vacuum -18 °C, -4 °C, vacuum -4 °C, 4 °C, vacuum 4 °C, 15 °C, vacuum 15 °C, 25 °C, and vacuum 25 °C. The seeds were subjected to these conditions for a duration of one year. Initially, X-ray imaging technology was utilized to assess seed fullness and germination percentages. Subsequently, TTC staining and scanning electron microscopy (SEM) were employed to examine seed viability and cotyledon outer surface cell structures. Concurrently, catalase (CAT) activity and malondialdehyde (MDA) content analyses were conducted, accompanied by the determination of proteomic and lipidomic profiles of safflower seeds following one year of storage in varying environments. Additionally, the presence of differentially expressed proteins and lipid metabolite variations were scrutinized. Ultimately, the specific influence of temperature and vacuum on seed aging was analyzed. The objective of this study is to elucidate the molecular mechanisms underlying natural aging in safflower seeds under diverse environmental conditions and establish a scientific foundation for their preservation.
Experimental material X-ray imaging Determination of water content Germination percentage determination Staining of 2, 3, 5-triphenyltetrazole chloride (TTC) Scanning electron microscope (SEM) Activity of catalase (CAT) Content of malondialdehyde (MDA) Lipidomics data analysis Proteomics data analysis Safflower were planted in the Medicinal Plant Garden of Chengdu University of Traditional Chinese Medicine and all the safflower seeds used in the experiment were collected by ourselves. The seeds were kept in a clean kraft paper bag, then the part was removed and vacuumed, placed in sealed bags. The air was extracted with a vacuum machine (Starfish family, ZK-8815). Subsequently, they were stored together with untreated seeds at -18 °C, -4 °C, 4 °C, 15 °C, and 25 °C. After one year, 20 seeds were chosen from each seed bag kept under different conditions for X-ray examination and germination rate determination.
The experiment utilized the MX-20 Cabinet X-ray System (Faxitron, AZ, USA). X-ray technology is an efficient tool for examining the interior aspects of seed embryos, endosperms, and detecting mechanical damages, fractures, and presence of pests, while ensuring minimal seed damage , . Such analysis provides essential insights for determining seed viability, germination percentage, health status, and degree of fullness among others. Imaging was facilitated by a 12 cm x 12 cm DC-12 camera, which captured images of the seeds. The camera’s 20 μm nominal focal spot and up to 5 times geometric magnification yielded images with ultra-high contrast and high spatial resolution. Approximately twenty seeds were included in each image capture.
Seeds from an extensive variety of plant species conclude their developmental phase by shedding a significant portion of their cellular water content, achieving a state of anhydrobiosis. In this state, cellular functions and metabolic activities are halted, and the cellular contents are solidified. This desiccated state endows seeds with the remarkable ability to transcend time and space, a critical attribute for species dissemination and foundational for global agriculture and food security. It preserves the seeds’ inherent value, ensuring their viability for subsequent planting seasons . Put the clean aluminum tray into the oven for 2 h, weigh and record it as M0, then put 10 g seeds which were stored for 1 year under different conditions into a clean aluminum tray, weigh and record it as M1, put the aluminum disc and seeds into a constant temperature oven at 103 ± 2 °C for 8 h, then weigh and record it as M2. The experiment was repeated 5 times. The seed moisture content (%) = (M1-M2)/(M1-M0) × 100%.
Twenty seeds were randomly selected from ten sample sets that underwent natural aging for one year at different storage environments and temperatures. A sodium hypochlorite (NaClO 3 ) solution with a concentration of 1.66% was prepared, resulting in a total volume of 140 ml. Subsequently, the seeds are immersed in NaClO 3 solution, rinsed under running water for approximately three minutes, and then blotted with a paper towel. The seeds are then placed on Petri dishes with 1% agar. Observe and record germination percentages one day later, observation for 7 days. The experiment was repeated five times to ensure accuracy and reproducibility of the results.
Five safflower seed samples were isolated from each group asnd placed into cryogenic storage tubes, following which an adequate amount of distilled water was added. After a period of six hours, the samples were removed, de-shelled, and positioned on a Petri dish. Subsequently, a 1% 2,3,5-triphenyltetrazolium chloride (TTC) staining solution was prepared and introduced into the Petri dish, avoiding the seeds. The Petri dish was then sealed and subjected to a water bath regulated at 37 °C.
Following one year of preservation, the cellular structure of the outer surface of safflower seed cotyledons was examined utilizing scanning electron microscopy (SEM). Cotyledon outer surfaces were meticulously dissected using a scalpel and subsequently affixed onto an aluminum stub using double-sided adhesive tape. Following gold sputter coating, variations in cell structure were observed under an acceleration voltage of 6.00 kV.
The spectrophotometer was activated and preheated for a minimum of thirty minutes, its wavelength adjusted to 240 nm, and its baseline set using distilled water. Subsequently, 0.1 g of seed kernel was collected into an Eppendorf (EP) tube, to which 1 ml of catalase (CAT) extract and a steel bead were added. This mixture was homogenised then subjected to centrifugation (8000 g, 4 °C, 10 min) for 30 s. The reagent solution for CAT detection was immersed in a water bath regulated at 25 °C for ten minutes. A quantity of 10μL of sample supernatant and 190μL of reagent solution was introduced into the cuvette, promptly mixed and timed. The initial absorption value (A1) at 240 nm and the absorption value (A2) after one minute were recorded, with the difference (ΔA) calculated as A1—A2. The experiment was repeated five times.
The microplate reader was switched on and preheated for a duration of at least 30 min; the respective absorbance settings were established at 532 nm and 600 nm, with distilled water serving as the zero reference. Following this, 0.1 g of seed kernels were acquired and placed into Eppendorf (EP) tubes, to which 1 ml of malondialdehyde (MDA) extract and steel balls were included. The mixture was subjected to a homogenizer and then centrifuged (8000 g, 4 °C, 10 min) for 30 s. Assay tube reagents (consisting of 100μL supernatant, 300μL MDA working solution, 100μL Reagent III) and blank tube reagents (consisting of 100μL distilled water, 300μL MDA working solution, and 100μL Reagent III) were prepared accordingly. The assay tube was then positioned in a water bath held at 100 °C, later to be transferred to an ice bath for one hour for rapid cooling. Following centrifugation (10000 g, room temperature, 10 min), 200μL of each supernatant was transferred into a 96-well plate to determine the sample’s absorbance. From these readings, the respective Δ532 and Δ600 values were calculated. The experiment was repeated five times.
In this study, four experimental conditions were selected, namely -18 °C, 25 °C, vacuum -18 °C, and vacuum 25 °C, and were assigned the labels L1, L2, L3, and L4, respectively. Using the CD search library software for simple screening and peak alignment of raw data, peak extraction and quantification are performed based on parameters such as retention time deviation, mass deviation, signal intensity deviation, signal-to-noise ratio, and minimum signal intensity; predicting molecular formulas through molecular ion peaks and fragment ions, and comparing them with the Lipidmaps and Lipidblast databases; removing background ions with blank samples and normalizing the quantification results. Subsequently, multivariate statistical analysis is conducted using the metaX software, including Principal Component Analysis (PCA) and Partial Least Squares Discriminant Analysis (PLS-DA), to determine the VIP values of metabolites. Univariate analysis calculates the P-values and fold changes of metabolites based on t-tests, with the criteria for screening differential metabolites being VIP > 1, P-value < 0.05, and FC ≥ 2 or FC ≤ 0.5. Finally, the R package ggplot2 is used to draw volcano plots, Pheatmap for clustering heat maps, and the corrplot software package for drawing correlation graphs between differential metabolites.
Seeds stored at -18 °C, 25 °C, vacuum -18 °C, and vacuum 25 °C for one year were selected as lipid sequencing samples and labeled as P1, P2, P3, and P4 respectively. Proteomic sequencing was performed by the commissioned service of Beijing Nuohe Zhiyuan Technology Co., Ltd. The mass spectrometry data is analyzed using the Proteome Discoverer software for database searching, with a precursor ion mass tolerance set to 10 ppm and a fragment ion mass tolerance of 0.02 Da, considering both fixed and variable modifications, and allowing for up to 2 missed cleavage sites. The analysis results are filtered to retain only peptides with a confidence level above 99% and proteins containing at least one unique peptide, with a false discovery rate (FDR) validation to remove peptides and proteins with an FDR greater than 1%. Statistical analysis of the protein quantification results is performed using a T-test, defining proteins with p < 0.05 and |log 2 FC|> * as differentially expressed proteins (DEPs). Further functional annotation is conducted using the interproscan software for GO and IPR, as well as COG and KEGG pathway analysis ; . DEPs are subjected to volcano plot, cluster heat map analysis, and pathway enrichment analysis, with the STRING DB software used to predict protein–protein interaction networks.
Analysis of the activity and structure of safflower seeds after one year of storage under different storage conditions Physiological and biochemical changes of safflower seeds after one year of storage under different storage conditions Lipidomic analysis of safflower seeds after one year of storage under different storage conditions Proteomic analysis of safflower seeds after one year of storage under different storage conditions To investigate the relationship between the vitality of safflower seeds and different treatments, as well storage environments, we stored safflower seeds for one year under various conditionsWe measured their moisture content and germination percentage. To ensure the accuracy of the results, we first used X-ray imaging technology to observe the internal structure of the seeds to check for integrity. The results showed no significant difference in the degree of fullness within the seeds (Fig. A). Subsequently, the water content was tested, we found that the water content of the untreated seeds was around 7% overall, the lowest moisture content of the seeds stored at -18 °C was 7.95% among the treated seeds, and the highest was stored at 25 °C at 10.74%. Table S1 provides detailed data. Finally, we determined the germination rate of safflower seeds stored for one year under different conditions. The results revealed that seeds treated with vacuum and stored at -18 °C exhibited the highest germination percentage at 53%, whereas those naturally aged at 25 °C had the lowest germination percentage at only 15% (Fig. B). Detailed results are presented in Table S2. Additionally, the experiment also found that safflower seeds treated with vacuum generally had lower moisture content and slightly higher germination percentages compared to untreated seeds. To delve deeper into seed viability under varied storage environments, triphenyl tetrazolium chloride (TTC) staining was utilized. The outcomes elucidated a significantly enhanced viability of seeds subjected to a one-year ageing process at -18℃, when juxtaposed with seeds aged at 25℃ for an equivalent period. Moreover, the staining outcomes at 25℃ demonstrated that vacuum preservation markedly influenced seed viability. Remarkably, safflower seeds conserved under vacuum conditions manifested considerably elevated vitality in contrast to their non-vacuum preserved counterparts, as visually represented in Fig. C. Significant differences in seed viability can be viewed after the safflower seeds have undergone an aging process at -18 °C and 25 °C over the course of one year. To conduct an exhaustive exploration of seed alterations post one-year preservation across varying environmental conditions, Scanning Electron Microscopy (SEM) was employed to scrutinize the external surface morphology of the safflower seed cotyledon. Preservation at -18℃ seemingly conserved an unblemished cotyledon structure, with a scarcely discernible intercellular space (Fig. A). Moreover, vacuum preservation manifested an even more impeccable structural integrity (Fig. B). Conversely, seeds subjected to non-vacuum storage at 25℃ over a year exhibited significant cotyledon surface corrugation and conspicuous intercellular spacing (Fig. C). However, vacuum preservation somewhat attenuated this predicament, diminishing the intercellular space within the seed cotyledon (Fig. D). On the whole, the surface morphology of safflower seeds aged over a year under subzero temperatures in conjunction with vacuum preservation exhibited considerable structural preservation. Conversely, the cotyledon structure of seeds conserved at 25℃ without vacuum treatment incurred significant damage observable through disrupted cell spaces and potential annihilation of the seed cell membrane.
Catalase (CAT) is ubiquitously present in plant tissues, acting as a pivotal protective enzyme whose role encompasses efficacious eradication of hydrogen peroxide generated during cellular metabolic processes, thus thwarting detrimental effects of excessive peroxide on cellular health . Malondialdehyde (MDA), the terminal result of unsaturated fatty acid oxidation in seeds, can inflict profound damage on the seed membrane system ; ), serving as a reflection of the degree of membrane lipid peroxidation and membrane system structural integrity . Therefore, we evaluated CAT activity and MDA content in safflower seeds following a one-year preservation period under varying storage conditions. Our results illustrated that, in the absence of vacuum, the CAT activity exhibited thermosensitivity with an overall trend of initial surge followed by a subsequent reduction. While seeds preserved at lower temperatures illustrated diminished CAT activity, a progressive incline trailed by a decline was noted with augmenting temperatures. Conversely, under vacuum storage, seeds conserved at lower temperatures demonstrated consistently high CAT activity. Interestingly, aside from 25℃, CAT activity of seeds preserved at all other temperatures under vacuum exceeded that under nonvacuum storage conditions (Fig. A and Table ). The assessment of MDA content unveiled a discernible discrepancy in the MDA levels of seeds preserved under disparate conditions. Notably, seeds conserved at -18℃ in a non-vacuum setting displayed substantially reduced MDA concentrations in comparison to those maintained at -4℃ or higher temperatures. In a contrasting manner, a insight was gleaned as we discerned that the MDA content in the majority of safflower seeds subjected to vacuum preservation for one year surpassed the levels observed in seeds conserved without vacuum (Fig. B and Table ).
Safflower seeds, characterized by their high oil content, exhibited significant variations in seed viability under different storage conditions, particularly at -18 °C and 25 °C. We aimed to conduct an exhaustive analysis of the lipid profile at these temperatures. Lipidomic analysis was performed utilizing the high throughput Liquid Chromatography-Mass Spectrometry (LC–MS) technique. We identified a total of 1148 lipid compounds in positive ion mode and 863 compounds in negative ion mode, distributed across all 24 samples, thereby giving rise to 24 distinct species. Remarkably, triacylglycerol (TAG; 113 species subclass), phosphatidylcholine (PC, 254 species), phosphatidylethanolamine (PE, 152 species), and fatty acids (FA, 89 species) featured high abundance, primarily in the positive ionization mode (Fig. A, Tables S3 and S4). A differential analysis was subsequently conducted, and the outcomes of the Principal Component Analysis (PCA) delineated substantial variations between L1VL2 constituents, facilitating their clear segregation into independent clusters. Moreover, intra-cluster aggregation instances were high. Additionally, a clear distinction could be observed between L3VL4 and L2VL4 clusters. Despite partial overlaps, these two clusters could still be effectively differentiated. Such discernible separations were perceivable for both positive and negative ion modes. Nonetheless, the difference between L1VL3 was marginal, with a high degree of overlap and insufficient differentiation observed (Fig. B). Differential lipid compounds were analyzed to compare the lipidomic profiles of safflower seeds under various storage conditions, including positive ion mode (Fig. S1A) and negative ion mode (Fig. S1B). Venn diagrams highlighted significant differences in lipid composition, particularly between seeds stored at -18 °C and 25 °C, with vacuum storage at -18 °C further influencing lipid metabolites. Cluster analysis was performed to explore lipid metabolic patterns, revealing that lipid compounds with similar profiles may share functions or pathways. Hierarchical clustering analysis (HCA) of normalized data (Fig. S2) demonstrated distinct lipid expression patterns under different storage conditions in both positive (A) and negative ion modes (B). The heatmap showed upregulation (red), downregulation (blue), or no change (white/light) in lipid abundance. Significant differences in expression patterns were observed among storage conditions, with certain metabolites notably upregulated or downregulated in specific groups (L1–L4). These variations reflect the impact of storage conditions on lipid metabolism and indicate distinct biological functions or regulatory mechanisms. Both ion modes revealed substantial shifts in lipid abundance, emphasizing the complexity of lipid metabolism under different storage environments, particularly at low temperatures and vacuum conditions. These findings align with previous results obtained through TTC staining, which revealed a significant difference in seed vitality after one year of storage at -18 °C and 25 °C. However, vacuum storage at -18 °C showed no substantial variation in seed vitality. In both L1VL3 and L2VL4 clusters, the triacylglycerols (TAGs) exhibited a decline during natural seed aging. The L1VL3 cluster showed a significant reduction in all four TAGs, while the L2VL4 cluster displayed a substantial decrease in 18 out of 19 TAGs. Within the L1VL3 cluster, all three phosphatidic acid (PA) species experienced a sharp decrease, whereas within the L2VL4 cluster, only a third of the TAG species displayed a significant reduction (Table ). The lipid content of specific membrane lipids, including phosphatidylcholine (PC), phosphatidylethanolamine (PE), and PA, as well as storage lipids such as TAG and diacylglycerol (DAG), showed considerable variability. There were divergent trends observed in PA content under different conditions: non-vacuum storage at -18℃ resulted in reduced PA content, whereas a significant increase was observed following storage at 25℃. This underscores the substantial impact of temperature on the evolution of PA content, as higher temperatures amplify PA levels. Additionally, subsequent examinations revealed that the content of PC, PE, phosphatidylserine (PS), and phosphatidylglycerol (PG) species increased after one year of storage at -18℃, whereas they significantly decreased after a year-long storage at 25℃ (Fig. ).
We utilized TMT-based quantitative proteomics, successfully identifying 5004 proteins across 12 samples, of which 4991 proteins were quantified within a 95% confidence interval (Table S5). The differentially expressed proteins (DEPs) were divided into four subgroups (P1/P2, P3/P4, P1/P3, P2/P4) and subjected to principal component analysis (PCA) and clustering analysis. However, as shown in Fig. S3 and Fig. S4, no significant differences were observed between groups, and the within-group cohesion was poor. A total of 274 significantly differentially expressed proteins were identified between the four groups (fold change ≥ 1.2 or ≤ 0.83, P < 0.05) (Table S6). During safflower seed storage, respiration progressively consume soluble sugars and eventually lead to seed deterioration ; . In addition, a reduction in structural lipids, such as phospholipids and galactoselipids, can lead to a loss of seed viability . We focused on investigating the metabolic pathways related to sugar and lipid metabolism. These pathways include starch and sucrose metabolism (map00500), carbon metabolism (map01200), glycolysis/gluconeogenesis (map00010), fatty acid metabolism (map01212), glycerolipid metabolism (map00561), fatty acid degradation (map00071), glycosphingolipid biosynthesis – globo and isoglobo series (map00603) and sphingolipid metabolism (map00600). KEGG analysis identified 35 DEPs in group P1/P2, 21 in P3/P4, 21 in P1/P3, and 27 in P2/P4 (Table S7-S10). Table summarizes the total DEPs, along with up- and down-regulated proteins. Most DEPs were localized to five key pathways: glycerolipid metabolism, fatty acid metabolism, carbon metabolism, glycolysis/gluconeogenesis, and fatty acid degradation, though intergroup variation was minimal. Five specific gene-encoded proteins (HH-002899-RA, HH-002382-RA, HH-034570-RA, HH-013791-RA, HH-017308-RA) showed differential abundance (Fig. ). Notably, HH-013791-RA and HH-017308-RA exhibited increased expression with rising storage temperatures under non-vacuum conditions. Conversely, HH-002899-RA, HH-002382-RA, and HH-034570-RA displayed higher expression levels after one year of low-temperature storage (-18 °C) compared to high-temperature storage (25 °C), indicating that lipase genes are involved in lipid degradation. Statistical analysis of lipase-coding protein abundance (HH-0087250-RA) revealed its expression level was significantly lower at -18 °C compared to 25 °C after one year (Fig. ). These findings highlight that low-temperature storage effectively mitigates membrane lipid degradation during seed aging. Integration of lipidomic and proteomic data revealed significant differences in lipid profiles across storage conditions, while proteomic differences were less pronounced.Two plausible hypotheses were taken into consideration. Firstly, alternative modifying factors may have impacted enzyme activity, leading to insignificant differences in protein expression. However, the preceding activity experiment illustrated the evident influence of the preservation environment on enzyme activity. Secondly, there could be fragmented genome annotations, resulting in incomplete identification of several enzymes engaged in safflower lipid degradation. Additional investigations are necessary to investigate these specific factors.
Seed aging is a complex process influenced by various factors, primarily involving genetic factors (internal causes) and environmental factors (external causes). Our previous experimental results showed that with the extension of the aging period, catalase (CAT) activity significantly decreased, while malondialdehyde (MDA) content significantly increased. The aging process caused significant changes in both the external and internal structures of the cotyledons. Over time, the external structure of the cotyledons gradually deteriorated from its original plump state, and the oil bodies within the cotyledons transitioned from a compact to a more relaxed state. Additionally, the total content of diacylglycerol (DAG) and phosphatidic acid (PA) increased with aging, while the content of triacylglycerol (TAG) and phospholipids (such as , and phosphatidylglycerol (PG)) significantly decreased. Studies have shown that the levels of PA, PC, PE, PI, and PS significantly decrease during seed aging, while the level of PG increases . This suggests that the phospholipid metabolism changes related to seed aging may vary depending on the specific crop species and handling conditions. These changes reveal that during the natural aging process of safflower seeds, the cell membrane undergoes degradation, leading to significant changes in physiological indicators and a decline in seed vitality, which is also reflected in the changes in the proteome and lipid composition , . We hypothesize that during seed aging, enzymatic metabolism induces structural changes in the cell membrane, leading to the influx of reactive oxygen species (ROS) into the cells, which ultimately reduces safflower seed vitality. To determine the optimal preservation method for safflower seeds, we treated them under different temperature and sealing conditions and evaluated their vitality, structure, physiology, biochemistry, lipid composition, and proteome changes after one year under different environmental conditions. When the CAT (catalase) activity of safflower seeds stored in different environments for one year was measured, it was observed that the overall trend of CAT activity in seeds stored under non-vacuum conditions exhibited an initial increase followed by a decrease. This phenomenon can potentially be attributed to the activation of other enzymes present in the seeds that facilitate the dismutation of O 2 - into H 2 O 2 . The presence of increased H 2 O 2 subsequently enhanced the CAT activity, which aids in the elimination of excess H 2 O 2 . As the H 2 O 2 content decreased, the CAT activity gradually declined. Additionally, SOD (superoxide dismutase) was found to participate in the generation of H 2 O 2 . Our subsequent experiment aims to further evaluate the variations in SOD activity in seeds following preservation under different conditions. MDA (malondialdehyde) content is an important indicator of seed aging, and lower MDA content means lower membrane damage. Conversely, greater membrane damage results in higher MDA content . For example, the MDA content of pine seeds increases dramatically after 25 days of accelerated aging at 45 °C and increases to 21-fold after 35 days of control . In this paper, the effects of accelerated aging on MDA content in wheat seeds, and the effects of storage temperature and vacuum storage on MDA accumulation in safflower seeds were introduced. The results suggest that the accumulation of MDA during aging may vary depending on the plant species and the preservation environment, and further research is needed to determine whether lipid composition assays can better assess lipid peroxidation in seeds . The studies cited in the article suggest that the accumulation of MDA does not necessarily exacerbate membrane lipid peroxidation in seeds with extended storage time, and that the accumulation of MDA during aging may be species-related. By integrating lipidomics and proteomics analysis results, significant differences were observed between the lipid groups and the proteome in different preservation environments, while the disparities within the proteome were relatively inconspicuous. We considered the following four possibilities: First, low temperature and vacuum treatment slow down seed metabolic activity: low temperature and vacuum treatment can significantly slow down seed respiration and metabolic activity, thus delaying the natural aging process ; . Although this treatment extends the storage period of seeds, it does not completely stop the aging process. Seeds stored under low temperature and vacuum conditions still undergo gradual physiological and structural changes, so the differences in proteomics might be relatively small and not easily distinguishable from naturally stored seeds. Second, the stability of proteins: Some proteins in seeds, such as storage proteins and structural proteins, have strong stability and are less likely to degrade under low temperature and vacuum conditions. Since low temperature and vacuum reduce oxidative reactions and moisture influences, these proteins remain stable, meaning that changes in the proteome under these conditions may not be as significant as those observed in naturally stored seeds . Third, the gradual occurrence of the aging process: low temperature and vacuum treatment slow down metabolism, but they do not completely stop the aging process in seeds, particularly in terms of protein degradation and synthesis. This means that although the aging process is slower under these conditions, some subtle physiological changes will still occur over time, and these changes might not manifest as significant proteomic differences in the short term . Fourth, experimental conditions and detection sensitivity: In proteomic analysis, some minor changes might not be detected, especially when the preservation method effectively slows down the aging process. These subtle changes may not be significant enough in terms of protein expression or function to show noticeable differences in proteomic data . Lipases play a crucial role in the natural aging process , and earlier studies have found that two specific lipases (HH-026818-RA and HH-025320-RA) increase in expression during seed storage Moreover, lipases are also involved in glucose metabolism and fatty acid degradation, leading to the breakdown of oil bodies (triacylglycerols—TAG) and membrane lipids (PC, PE, PS, PI, PG). These processes result in the disruption of seed structure and a decline in seed vitality during natural aging. It can be speculated that during seed storage, the oxidation of seeds gradually increases with the extension of storage time, which may be related to lipid degradation. Lipid degradation leads to changes in both membrane structure and oil body structure, making it easier for ROS to penetrate the seeds. However, in the current experiment, it was observed that seeds stored under low-temperature vacuum conditions had a tighter surface structure compared to those stored under high-temperature non-vacuum conditions, with no collapse or cracking. This phenomenon may be due to the different seed treatment conditions used in this study compared to previous experiments. Specifically, low temperature and vacuum treatment were found to reduce seed respiration and effectively inhibit seed metabolism ; Our study revealed that the differences observed between the groups were consistent with the outcomes of previous experiments based on lipid analysis. Furthermore, we observed that the contents of PC, PE, PS, and PG were highest when stored at low temperature (-18℃), and gradually decreased with an increase in storage temperature. However, the contents of PA and PI showed an opposite trend, increasing with an increase in temperature, while the change in contents after vacuum storage was not significant. These observations suggest that changes in membrane lipid metabolism during seed aging vary by crop species and treatment conditions. We hypothesize that enzymes related to fat degradation are involved in the aging process under different preservation conditions, leading to the deterioration of membrane lipid degradation and impaired activity. During proteomic analysis, only five metabolic pathways were detected, and the five gene-coding proteins selected from them had not been identified in previous experiments. We speculate that the differences may be attributed to the selection of experimental materials. In the previous experiment, we used seeds that grew at different times of natural aging, while in this experiment, we used seeds that had been stored in different environments for one year. The different treatment methods of experimental materials may have led to differences in the proteome. In the forthcoming research, we will persistently investigate the detectability of a specific protein gene under distinct environmental conditions, following a one-year preservation period. Additionally, we will persist in scrutinizing the impact of the environment on the lipid composition and proteomic profile of seeds while concurrently striving to minimize preservation duration through deliberate artificial aging procedures. Expounding the identification of genes associated with seed aging, we will undertake a comprehensive exploration of the precise mechanisms underlying seed aging. Ultimately, the outcomes of this study present a novel theoretical framework for the scientific preservation of safflower seeds. Our research findings highlight the pivotal role of low-temperature storage in enhancing the viability of safflower seeds, recommending that seed practitioners should preserve seeds in an environment close to or below the freezing point. This not only helps to slow down the aging process but also effectively extends the seeds’ lifespan. Additionally, vacuum packaging technology has shown significant effectiveness in improving seed preservation quality by reducing moisture content and minimizing oxidation reactions, thereby extending the storage duration of seeds. To ensure seed quality, regular measurements of CAT and MDA levels are essential, aiding in monitoring the physiological status of the seeds and allowing for timely interventions when necessary. Control of humidity and temperature in the seed storage environment is equally important, with a recommendation to utilize storage facilities that maintain a constant temperature and humidity, along with regular checks to ensure optimal storage conditions. Implementing a seed rotation system is also an effective strategy to maintain the optimal vitality of seeds in storage, updating stock periodically to prevent a decline in vigor that could result from prolonged storage. Education and training are crucial for enhancing the understanding of seed storage personnel regarding the mechanisms of aging and storage techniques, ensuring they can properly execute and manage seed storage. Lastly, it is encouraged that seed storage practitioners stay informed and adopt the latest advancements in seed science, leveraging new technologies and methodologies to enhance the efficiency and effectiveness of seed storage. Through these comprehensive measures, there can be a marked improvement in the storage outcomes for safflower seeds, ensuring they exhibit high vitality and germination rates when applied in agricultural and medicinal contexSupplementary Information 13. Supplementary Information 14.
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Effect of size of capsulorhexis on the outcome of cataract surgery: a protocol for systematic review and individual participant data meta-analysis | 701f4d8a-1ef4-4081-be90-0b599c36e9d6 | 11667307 | Ophthalmologic Surgical Procedures[mh] | Approximately 20 million cataract surgeries are performed annually worldwide. Surgeons are focusing on improving preoperative measurements and calculations, standardising intraoperative manipulations and optimising postoperative care to achieve satisfactory visual outcomes. Capsulorhexis is one of the key steps during cataract surgery. A complete continuous curvilinear capsulorhexis (CCC) is crucial for successful phacoemulsification : (a) CCC aids containing ultrasonic turbulence and protects the endothelium against high-velocity lens fragmentation and cavitation. (b) CCC prevents asymmetrical contracture of the rim, which can lead to uneven tension on the capsule and zonules and helps maintain the stability of the capsular bag and intraocular lens (IOL), which in turn enables more accurate prediction of postoperative refraction. (c) A regular capsulorhexis rim helps prevent or reduce the occurrence of posterior capsule opacification. In addition to a contact CCC, the size of capsulorhexis has long been a concern in clinical practice. Usually, a smaller capsulorhexis (less than 5 mm in diameter or less than the diameter of the optical area of IOL) is correlated to less damage and is accepted by most surgeons. Moreover, the size of capsulorhexis may influence the stability of IOLs via its effect on postoperative capsule opacification. The capsule opacification, namely, the fibrotic response of the posterior and anterior capsule, can lead to capsular shrinkage, which in turn affects IOL stability. After cataract surgery, lens epithelial cells located in the remaining peripheral anterior capsule can transform into myofibroblasts, resulting in fibrotic anterior/posterior capsule opacification (ACO and PCO) and shrinkage of the capsular bag. It is supposed that a smaller capsulorhexis size and less polishing are associated with better stability IOL because the ACO, to some extent, could increase the adhesion between the anterior capsule and IOL and restrict IOL movement. Some researchers have argued that even a slight alteration in the diameter of the capsulorhexis can significantly affect the grade of ACO. However, in Grade 4 ACO, the capsular bag undergoes excessive constriction; the haptics of the IOL curls forward and the stability of IOL is impaired. In addition, in patients with high myopia, a large capsulorhexis may improve the long-term stability of IOL. Highly myopic eye is characterised by an axial length of ≥26 mm, with weak zonules and a large capsular bag. Usually, the likelihood of postoperative capsule shrinkage is greater in such patients, and the conventional capsulorhexis size is no longer appropriate. Studies have demonstrated that a longer axial length is associated with a higher risk of PCO. A relatively larger capsulorhexis (6 mm for an IOL with a diameter of 7 mm) may provide better stability for the special consideration of patients with highly myopia eyes. However, the proper size of the capsulorhexis and the extent of anterior capsule coverage in these patients remain unclear. The size of capsulorhexis is also specifically concerned in patients with cataract with diabetes mellitus (DM). These patients are at risk of developing retinopathy and may require intensified fundus examinations. Theoretically, patients with DM need to maintain good visual acuity while achieving the largest possible capsulorhexis size. Reduction of the anterior capsule aperture may hinder fundus visibility in pseudophakic eyes. In particular, DM is a risk factor for anterior capsule contraction, severe intraoperative corneal endothelial damage and slow postoperative recovery. Thus, three aspects should be considered when deciding the size of capsulorhexis: postoperative corneal recovery, IOL stability and fundus visualisation. At present, there are very few studies concerning the optimal size of capsulorhexis in patients with DM. As described above, the exact capsulorhexis size is important considering the rotational stability of the IOL and in patients with high myopia or DM. The number of relevant studies is very limited to date and there is no literature directly addressing this question, so we used raw data from individual relevant clinical trials to address this issue, that is, the individual patient data meta-analysis (IPD-MA), as employed by similar researches. The goal is to determine the optimal size of capsulorhexis to minimise the refractive changes caused by capsular contraction and maximise the benefits for patients with cataract with regular corneal astigmatism, high myopia or DM.
Before outlining this protocol, we strictly adhere to predetermined search methods and thoroughly review the text of relevant studies. The planned start date for this study is 4 July 2024, with an anticipated end date of 1 June 2025. Registration Types of studies Information sources and search strategy Selection, management and collection process of data included in IPD study Data items collected in IPD study Data integrity and outcomes and prioritisation Risk of bias and confidence in cumulative evidence Data synthesis and (statistical) analysis Amendments to protocol Patient and public involvement None. Ethics and dissemination This study inumber CRD42023459903). We follow the Preferred Reporting Items for Systematic Review and Meta-Analysis Protocols statement when reporting this article. Any protocol changes that may have occurred will be described in the final report.
This meta-analysis aims to identify eligible clinical trials, including observational studies and randomised controlled trials, involving adults undergoing phacoemulsification and IOL implantation with CCC and recorded capsulorhexis size. There are no restrictions on the year when the study is conducted, language of publication, date of publication or publication status. To be included in the IPD-MA, the study must provide the following information: Capsulorhexis size: diameter or area of the capsulorhexis, anterior capsule cover, centration and circularity of capsulorhexis. Primary outcomes: IOL stability or postoperative visual acuity. Secondary outcomes: capsule shrinkage and all the other outcomes.
A systematic search is conducted from inception until 4 July 2024 in the following databases: PubMed, Embase, Cochrane Library, Web of Science, SinoMed, China National Knowledge Infrastructure, Wanfang Data Knowledge Service Platform and China Science and Technology Journal Database. The structured search algorithm is: (((((((((continuous curvilinear capsulorhexis) OR (capsulorhexis size)) OR (diameter of capsulorhexis)) OR (area of capsulorhexis)) OR (anterior capsule opening)) OR (capsulorhexis position)) OR (shapes of capsulorrhexis)) OR (anterior capsule cover*)) OR (centration of capsulorrhexis)) OR (circularity of capsulorrhexis). The strategies of the search plan to be applied to all databases are detailed in ). The reference lists of the selected studies are reviewed to identify additional relevant studies. A final search will be conducted immediately before submission to ensure that no recent publications have been overlooked.
Selection process Data management Data collection process IPD can be obtained from data-sharing databases, such as Yale University Open Data Access, Clinical Study Data Request and Vivli ( https://vivli.org/ ). In cases where data are not available in data-sharing databases, we will directly obtain data from the authors of the respective study. Once it is established that the study meets the eligibility criteria, the authors of the study will be contacted via a secure university-registered email address. To collect as much individual patient (raw) data as possible from the included studies, we will use a multistep contact protocol that has been proven effective in previous studies. The steps for obtaining raw data from the authors of the included studies are summarised in . We will obtain the contact information of the corresponding author by (a) gathering it from relevant publications, (b) conducting a web search and (c) reaching other researchers. An email will be sent to the corresponding author, clearly stating the study’s objectives and providing clinical and methodological justifications for IPD. We will also convey our intentions to the authors through in-person meetings, as this may increase their willingness to share raw data. The corresponding author is given 9 weeks to respond to our email. If no response is received after 3 weeks, a second email will be sent. Similarly, if there is no response after another 3 weeks, a third email will be sent. If the corresponding author does not respond within these 9 weeks, we will repeat the aforementioned steps with other authors in the following order: first, last, second, third, fourth, etc. When the above steps are unsuccessful, the corresponding author will be contacted via letter and telephone to obtain the raw data. Letters will be sent at 3-week intervals. We will contact other authors in the same manner only when the corresponding author cannot be reached. Finally, if there is still no response, we will attempt to establish communication with colleagues who may be acquainted with the authors. Study data will be considered unavailable only if all the methods have failed or if authors indicate that the raw data have not been retained or that they refuse to share these data.
A dedicated team of five persons is formed with two key members responsible for developing the process and standards. The two assessors conduct the processes of study screening, selection, data extraction and bias risk assessment independently. The final plan will be determined through a group discussion. All team members should receive training before data extraction and collection. The titles and abstracts of potential studies are compared with the eligibility criteria and thoroughly assessed by two appraisers to determine their suitability. Subsequently, these two appraisers will carefully review the full text of the potential studies. Any discrepancies between the two appraisers are to be resolved through a consensus meeting involving the entire study team. During the screening process, we first use software to remove duplicate references, followed by manual deletion. After removing duplicates, the two assessors independently perform manual screening, excluding case series, conference reports, grey literature, news articles and literature reviews from the analysis. After screening, a total of 94 articles meeting the inclusion criteria have been identified, including five articles focusing highly on patients with myopia and six articles focusing on patients with diabetics (see for specific flow diagram). Readers who possess knowledge in this field and are aware of studies that have not yet been included are encouraged to contact us.
The data collection protocol employed at the group consensus meeting will be continuously adjusted by two key members based on various studies. When possible, we will contact the corresponding author of the study via email to acquire, if any, original data that were not mentioned in their article. The data collection process will be divided between two members with respective roles. All data will be sent to the data manager in the most convenient format and stored in an Access database. The data will be uniformly organised by two team members responsible for this task. All information will be securely maintained and treated as strictly confidential. The data will not be allowed to be used in any publication without permission from original trialists.
ll prognostic indicators accessible in a sufficient number of included studies that may affect the outcome of cataract surgery are included. The elements included in the achieved studies are summarised in . The information provided in the text and tables will be combined into a systematic narrative synthesis that will explain and summarise the characteristics and conclusions of the included investigations. Box 1 Information and baseline characteristics of the included clinical studies Information about the included trials: Baseline characteristics of the included studies: Information about the included studies Baseline characteristics of the included studies Information to be explained Capsule shrinkage: the percentage reduction in anterior capsule opening at follow-up is calculated as follows: (previous anterior capsule opening–anterior capsule opening at this follow-up) × 100/anterior capsule opening at the last follow-up. Types of IOLs: capsule contraction is affected by IOL optic material, haptic design, and size. Anterior chamber depth: the distance between the anterior surface of the IOL and central corneal endothelium is known as ACD. Circularity: 4π (area/perimeter 2 ). Calculation of the capsulorhexis diameter. Actual diameter = (maximum diameter+minimum diameter)/2 Relative diameter=actual diameter/expected diameter Stability of the IOL: Tilt: an IOL tilt of 7° or more is considered clinically significant. Decentration: clinically significant decentration of the IOL is defined as 0.4 mm or greater. Rotation (suitable for toric IOL): this parameter refers to the angle between the IOL axial position at the postoperative follow-up and the expected position of the IOL axis before the operation. An angle greater than 1° indicates axial rotation. Anteroposterior IOL shift Visual acuity: for analysis, all measurements of visual acuity are transformed into logMAR units.
Study (primary author, published date) Eyes/patients per study (N) Country Special populations: high myopic eyes, patients with diabetes mellitus Types of capsulorhexis size included diameter, anterior capsule opening area, anterior capsule cover, centration and circularity Refraction: uncorrected distant visual acuity, best-corrected distant visual acuity Capsule shrinkage Stability of IOL: tilt, decentration, rotation and anteroposterior IOL shift Types of IOL Postoperative time points (months)
Participant-level data items: Age Gender Axial length Anterior chamber depth Grade of the lens nucleus or grade of lens opacity Capsular bag diameter IOL power Predicted refraction Capsulorhexis area and/or anterior capsule opening area Capsulotomy or CCC circularity Visual acuity, logMAR Intraocular pressure Corneal edema Postoperative aqueous flare intensity Corneal endothelial cell density Central corneal thickness Anterior capsule opacification Posterior capsule opacification Surgically induced astigmatism Rate of anterior capsule contraction Surgical-level data items: Surgical time Cumulative ultrasound energy Abbreviations: CCC, continuous curvilinear capsulorhexis; IOL, intraocular lens.
For the included studies, we will collect basic information (author, publication date, country, number of eyes/patient per study and special populations), intervention description (diameter of the capsulorhexis, area of anterior capsule opening, anterior capsule cover, centration and circularity of capsulorhexis), outcome description (refraction, capsule shrinkage, stability of IOL intraocular pressure) and follow-up time, as summarised in .
Participant-level data items: we request that investigators provide the following original data, to the extent possible, for the subjects in the included studies: age, sex, axial length, anterior chamber depth (ACD), grade of the lens nucleus or lens opacity, capsular bag diameter, IOL power, predicted refraction and preoperative visual acuity. Data collected at the follow-up visit include the area of capsulorhexis and/or anterior capsule opening, circularity of capsulotomy or CCC, visual acuity, intraocular pressure, corneal oedema, postoperative aqueous flare intensity, corneal endothelial cell density (ECD), central corneal thickness (CCT), ACO, PCO, surgically induced astigmatism and the rate of anterior capsule contraction. Surgical-level data: the surgical time and cumulative ultrasound energy used during the operation are recorded.
The primary analysis of each article will be replicated, and the endpoint values will be compared with those of the original publication. In cases of missing data, investigators from the included studies will be contacted to determine the reasons for loss to follow-up and to request any incomplete data if available. The primary outcomes of this study are IOL stability and visual parameters during follow-up. The main objective is to evaluate the impact of different capsulorhexis sizes on IOL status and visual acuity. Secondary outcomes include capsule shrinkage during follow-up and any other outcomes (eg, ACD, corneal oedema, aqueous flare intensity, corneal ECD, CCT, ACO or PCO) that will require assessment in at least two studies.
Risk of bias in individual studies Confidence in cumulative evidence Publication or data-sharing bias The standard risk of bias assessments is not applicable to these studies because we will use data from clinical trials for purposes unrelated to their original research topic. Previous studies have identified the refined Quality in Prognosis Studies (QUIPS) tool as suitable for assessing the risk of bias in studies of prognostic factors. Six domains are evaluated:The outcomes are classified as yes, partial, no or uncertain, based on the prompting items within each domain. The answers will be then summed, and each domain will be rated as having a high, moderate or low risk of bias. Two independent appraisers will use the QUIPS tool to assess the risk of bias in the included studies. The entire research team will convene a consensus meeting to resolve any disagreements among appraisers. A study will be classified as having a low risk of bias when all six domains are rated as low or moderate, with at least four items rated as low, including the outcome measurement domain. Studies that receive high scores in two or more domains will be classified as having a high risk of bias. The remaining studies will receive a moderate rating.
The overall certainty of the evidence will be assessed by two independent reviewers using a modified Grading of Recommendation, Assessment, Development and Evaluation (GRADE) approach. This modified approach is specifically designed to evaluate prognostic evidence, including study design, risk of bias, inconsistency, imprecision, indirectness and publication bias. Effect sizes and trends will also be considered. In the event of any disagreement, consensus meetings will be held with the group members to resolve them. We will take the following steps to implement the modified GRADE framework: Determine the type of study: we will first classify the outcomes based on the study design (eg, randomised controlled trials and observational studies). This classification will assist us in establishing a baseline for assessing the quality of evidence. Assess the risk of bias: we will use standardised tools, such as the Cochrane Bias Risk Tool, to evaluate the risk of bias for each study. Studies identified as having a high risk of bias will be considered for downgrading. Assessment of consistency: we will compare the consistency of results across studies. In cases where significant differences are observed, we will consider downgrading the quality of evidence. Indirectness of evidence: we will conduct an indirect assessment of whether the results apply to our study population or the intervention. Downgrading will be considered if the intervention or population in the study is deemed distant from our own. Precision assessment: we will evaluate the precision of the results, including the width of the confidence intervals. If the confidence intervals are wide, indicating insufficient precision, we will consider downgrading. Publication bias: we will check for signs of publication bias, for example, by employing a funnel plot. Should evidence of bias be identified, a downgrade will be considered. Upgrade or downgrade: on completion of the assessments, we will synthesise all factors to determine whether to upgrade or downgrade the quality of evidence. For instance, if a particular study demonstrates strong performance in terms of risk of bias and consistency but exhibits issues with precision, we may opt to maintain the current quality of evidence. Transparent recording: we will meticulously document each step of the evaluation process and the final grading of evidence quality to ensure transparency and reproducibility.
To assess the concern regarding publication or data-sharing bias, we will implement several strategies: Literature review: we will undertake an extensive literature review to identify all pertinent studies, including smaller ones that might be excluded from IPD analyses. Tool: for analyses involving 10 or more trials, we assess the potential publication bias by examining asymmetry in funnel plots using Egger’s test. Funnel plots will also be generated to visually inspect for potential bias in smaller studies. Sensitivity analysis: we will perform sensitivity analyses to evaluate the impact of including or excluding smaller studies on the overall results. Meta-biases: In 2016, Smith et al compared IPD-MA outcomes with aggregate data meta-analysis outcomes and reported inconsistent results. To assess potential bias in data availability, we will perform t-tests for continuous variables and χ2 tests for categorical variables to compare characteristics across studies that received IPD and those that did not. The effect size information obtained from the publications will be used to calculate Cohen’s d effect sizes. To compare effect sizes between studies with and without IPD, the effect sizes are pooled and analysed across studies using the traditional ‘subgroup analysis’ approach of meta-analysis.
High statistical power indicates a high feasibility of the project. However, due to the inclusion of various study types and the fact that some studies did not primarily focus on the outcomes of interest, there is a lack of standardised power calculation method in this study. Meanwhile, the assessment of bias risk and overall certainty of evidence can, to some extent, validate the reliability of our findings. Appropriate tabular and graphical summaries can be used to present the descriptive statistics. Quantitative variables are summarised using means and SD, whereas qualitative variables are summarised using frequencies and percentages. Strategy for data synthesis Sensitivity analysis Subgroup analysis Prespecified subgroup analysis will be conducted based on: Presence or absence of high myopic eyes Patients with or without DM Types of IOL Types of capsulorhexis size Types of study If sufficient data are available, the ocular axial length and the type of astigmatism will also be considered as factors in the grouping. By collecting individual patient data, it is possible to estimate potential differences between these subgroups. Analysing the reasons for these differences is more effective than relying solely on individual trial reports. The analyses will be conducted using Review Manager C.5.3.
A one-stage IPD-MA using multilevel regression model will be chosen based on previous experience. The effect measures will be converted to a standardised scale (z-scores) to facilitate visual comparisons. Notably, most clinical trials included in the IPD-MA did not assess the primary endpoint. A multilevel regression model will be established based on all available data, with the size of the capsulorhexis, preoperative visual acuity and other prognostic indicators as independent variables, and postoperative visual acuity as a dependent variable. A similar multilevel regression model will be used to examine the impact of capsulorhexis size on the rotational stability of the IOL or other outcome variables. Multilevel linear regression models will be used for continuous outcomes, while multilevel logistic regression models will be used for categorical outcomes. To evaluate their moderating effect on treatment outcomes, the model will incorporate the interaction between the intervention group and the moderators of interest. If heterogeneity is permitted, raw data will be combined into a single set. To address data clustering, a study identification variable will be added as a random effect variable. When quantitative synthesis is not feasible, we will perform a narrative analysis. Another analysis will investigate the short-term effects of capsulorhexis size on IOL rotation of less than 15° within 1-week period.
To conduct a sensitivity analysis, studies with an overall high risk of bias will be excluded. Further sensitivity analysis will use interaction terms to explore whether pairs of predictors show nonlinear effects on the primary outcome.
To ensure transparency and maintain the integrity of our research, the following plan will be implemented in case of documenting any amendments to the study protocol. This plan will include the following key components: Documentation: all amendments will be recorded in a designated section of the study protocol, detailing the nature of the change, the rationale behind it and the date of the amendment. Reporting: any amendments are to be explicitly reported in future publications. This will include a summary of the changes made, along with an explanation of their significance in relation to the study’s objectives. Justification: for each amendment, a point-by-point justification is to be provided which outlines the reasons for the changes, whether they are based on emerging evidence, feedback from peer reviewers or unforeseen circumstances encountered during the study.
None.
Ethical approval is not required because the study does not involve individual patients. The study results are to be disseminated via professional journals as well as academic media.
10.1136/bmjopen-2024-092002 online supplemental file 1 10.1136/bmjopen-2024-092002 online supplemental file 2
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Helpful or harmful? Navigating the impact of social media influencers’ health advice: insights from health expert content creators | 6f572995-b8ee-4e86-b62c-91e78e5b1425 | 11657387 | Health Communication[mh] | Harff and colleagues describe SMIs as “regular people who became well-known on social media due to their efficient self-presentation on these platforms” (p. 831). In other words, SMIs achieve popularity through their proactive, self-managed presence and personal branding on social media platforms . This factor distinguishes SMIs from traditional celebrities, who usually achieve recognition through industry gatekeepers, such as editors or producers . Nevertheless, the influence of SMIs can surpass traditional celebrities . Additionally, SMIs are often categorized based on the number of followers they have, with classifications ranging from micro-influencers to mega-influencers, depending on the size of their audience. The cut-off points for these classifications can vary across the literature, with different studies applying different follower thresholds . The number of followers SMI has can also influence the effects of their messaging, with micro-influencers, with smaller audiences, being often perceived as more authentic and relatable , while SMIs with a high number of followers tend to be seen as more likable and popular . Moreover, SMIs also vary in their focus areas, with some dedicated solely to specific health-related content, such as fitness or nutrition, while others focus on broader lifestyle topics or unrelated issues, yet still occasionally share health-related information . SMIs tend to use a conversational style of communication, which enables them to build close connections with their followers , who might perceive them as friends . This phenomenon is attributed to parasocial relationships (PSRs) - one-sided relationships where audience members feel a bond with a media persona, despite no direct interaction. Originally described by Horton and Wohl , PSRs allow individuals to feel familiar with and emotionally close to a media figure, while experiencing the relationship as evolving . While the influence of SMIs has been extensively studied in the context of advertising and marketing , their content is not always driven by marketing objectives but might also often include personal insights and everyday life experiences , motivated by SMIs’ desire to build a community and help their followers . The dissemination of health-related information by SMIs is of high significance due to its capacity to shape health perceptions and practices of their audience, thus influencing public views on a broad spectrum of health subjects, such as fitness , vaccination , or cancer screening . SMIs have been shown to have a positive impact on the exercise intentions of their audience , encourage healthy diet or promote positive attitudes toward preventative screenings and vaccinations . However, SMIs can also motivate followers to consume unhealthy food or spread health-related misinformation . Health experts as social media content creatorsMisleading health information on social mediaDespite concerns regarding the dynamics of patient-physician interactions on social media , health experts are increasingly becoming active on these platforms, recognizing the significant potential of their active engagement for public health purposes . In this context, health experts are understood as professionals with advanced knowledge and training in medical or health-related fields, evidenced by education from accredited institutions; examples include medical doctors, nutritionists, or psychotherapists. Health professionals nowadays engage with users on social media to address their personal health concerns, spread knowledge, and assist with lifestyle decisions . Many health experts from a wide array of fields - including neurosurgery , plastic surgery , or cardiology - have even been recognized as SMIs, due to their success in attracting large followings and exerting substantial influence within their communities. These experts embrace the dual role of being SMIs and health experts in their self-presentation on social media , thereby enhancing the public dissemination of health information. Active social media engagement of health experts appears to be beneficial as evidence suggests that they are perceived as more credible and authentic compared to non-expert SMIs . While existing research has explored the motivations behind SMIs’ content creation, ranging from intrinsic motivations like personal enjoyment, to extrinsic motivations such as financial incentives , how these motivations translate to other content creators such as health experts, who are increasingly more active on social media, remains underexplored. To address this gap, we ask: RQ1:RQ2:What motivates HECCs to create and post health-related content on social media? Despite the evidence of SMIs’ potential significant influence on the health-related attitudes and behavior of their audience, research suggests that many often disseminate health-related messaging without possessing relevant professional qualifications and give preference to their commercial rather than public health interests . This trend is concerning because as users increasingly turn to social media for health information , it raises significant concerns regarding the reliability and accuracy of the health information available there. Given their dual expertise in medical knowledge and social media dynamics, HECCs are uniquely positioned to understand the real-world implications and potential threats posed by misleading health advice disseminated by non-expert SMIs. This leads to our second research question: What are the attitudes of HECCs toward SMIs disseminating health-related information without professional qualification? In recent years, the proliferation of misleading health information on social media platforms has garnered significant scholarly interest . Studies from this field usually use the term health misinformation , which can be defined as “information that is contrary to the epistemic consensus of the scientific community regarding a phenomenon” [ , p. 434], or as “a claim that contradicts or distorts common understandings of verifiable facts” [ , p. 10]. It differs from disinformation due to the absence of clear intent to deceive its recipients . While the prevalent focus of research is on health misinformation as information that is factually incorrect , we argue that this might not be the only form of misleading health messaging potentially leading to adverse outcomes. We base our argument on the fact that human health is an extremely complex concept with no one-size-fits-all solutions, and individual assessment of one’s health condition by a professional is always warranted . Communication of SMIs is a form of mass communication since they typically spread generic messaging to large undifferentiated audiences . However, mass dissemination of identical health messaging to large groups of recipients naturally fails to consider the unique factors influencing individual health-related choices and behaviors of a person . Therefore, we introduce the term overgeneralized health messaging , which we define as health-related information that, while factually correct and accurate for the source, possesses the potential to mislead or confuse recipients due to its limited applicability or relevance to a broader audience. This phenomenon can occur when individuals share their personal health experiences, symptoms, or advice, which may lead others to draw inaccurate conclusions about their health conditions or situations. The concept of overgeneralized health messaging might be exceptionally relevant in the context of health communication of SMIs as these media figures can have a significant impact on their audience’s health-related behaviors . Moreover, on the account of PSR, the generic character of the mass-communicated messaging of a SMI might not be immediately evident to the recipients, who tend to identify with the influencer, viewing them as someone close and similar . As highlighted in broader studies on science communication, generalized messaging can be effective in promoting health-related behavioral intentions across populations despite individual differences , which might result in unintended negative consequences in the context of SMI communication. Research shows that social media influencers (SMIs) routinely share their experiences with both physical and mental health conditions, describing their symptoms, treatment, and personal advice . While this practice can provide valuable insights, it can be also be seen as potentially problematic due to the personal and individual nature of the topic. For example, when SMIs publicly discuss their mental health symptoms, it could lead viewers to self-(mis)diagnose as a result of self-identification with the content creator . Harris and colleagues described that SMIs often generalize their mental health-related personal experiences, which might result in viewers’ self-misdiagnosis and an overreliance on solutions that may not be suitable for everyone. While such messaging is not necessarily incorrect, it can still result in negative consequences like worsening mental health or incorrect treatment . Furthermore, non-expert health advice in general can exacerbate confusion and worsen health conditions . Current research has yet to fully explore the impact of overgeneralized health messaging by SMIs. However, it is crucial first to consult health experts to avoid drawing parallels between two potentially distinct concepts - misinformation and overgeneralized messaging. Therefore, in this study, we aim to examine the outcomes of the two concepts: RQ3:RQ4: What measures should be taken to counter the spread of misleading information by SMIs from the perspective of HECCs? Are the potentially harmful effects of overgeneralized health messaging by SMIs comparable to those of health misinformation spread by the same individuals? Furthermore, health experts are on the front lines, directly confronting the consequences of misleading information disseminated by SMIs. Therefore, their experience dealing with the repercussions of such misleading messaging on patient outcomes and public health provides them with unique insights. We argue that this perspective positions them as valuable sources of knowledge on effective strategies to counteract misleading health information. Consequently, this leads to the research question: To investigate the posed research questions, we conducted semi-structured qualitative interviews with experts on both physical and mental health, who are also active social media content creators. The applied method allowed us to gain in-depth understanding of the perceptions, attitudes, and experiences of HECCs regarding potentially misleading health information shared by non-expert SMIs, as well as their recommendations for countering practices and motivations for social media engagement. The research design was approved by the Institutional Review Board of the Faculty of Social Sciences, University of Vienna (approval ID: 20230522_023) and adhered to the ethical guidelines of the American Psychological Association (APA). The research process was reported following the Consolidated criteria for reporting qualitative research (COREQ) (see Supplementary file 1) . SampleData collectionData analysisTo find potential participants, we conducted an Instagram search of various profession-related keywords (e.g., “doctor”, “nutritionist”, “psychotherapist”). All terms were searched in both male and female variations to account for the gendered nature of the Czech language. The selection of search terms was based on their relevance to health-related professions and the ongoing identification of key terms throughout the research process, ensuring coverage across various specializations, including both physical and mental health. Based on the results, we compiled a list of potential participants, who were both health professionals and active creators of social media health-related content from Czechia. To qualify as a health professional, individuals had to possess a recognized qualification in a health field, such as a degree in medicine or nutrition, from an accredited institution. For active content creation, individuals needed to have published at least one health-related post on their social media profile every month for the last three months. Suitable candidates were contacted via Instagram messages and, where available, also via email. Altogether, 45 HECCs were contacted. Participants were provided with a consent sheet that included detailed information about the study, including their rights, the voluntary nature of their participation, and how the data would be used. They were asked to review the sheet and provide their signed informed consent prior to participating. The final sample comprised 12 health experts from various fields to enhance the diversity of perspectives in the findings and ensure a broad representation of expertise, including three psychotherapists, three nutritionists, three medical doctors , two pharmacists, and a physiotherapist. Their professional qualification was verified during the interviews. Despite the diversity in their professional backgrounds, saturation was reached with this sample as no new themes or insights emerged from the data. Given the relatively small population size of Czechia, the study’s sample size can be considered proportionate. Participants were 24 to 42 years old ( M age = 29.67; SD = 5.03); nine of them identified as female and three as male. The sample was diverse also in terms of number of followers ranging from 484 to 64,800 (M = 14,774.83; SD = 20,776.87), thus encompassing HECCs across different scales of social media engagement and further enhancing generalizability. For an overview of the sample, see Table . The interviews lasted between 26 and 55 min ( M = 34.83; SD = 9.55) and were conducted via Zoom video calls . In one case (#4), the interview was conducted via telephone call per interviewee’s preference. Data collection took place between August 7th and December 18th, 2023. The interviews were conducted by the first author (JK; female), who was a PhD candidate at the time of data collection. JK had received training in both quantitative and qualitative research methods and was the sole member of the research team present during the interviews. Before starting the discussion, JK requested participant consent to digitally record the session. Upon receiving approval, she then obtained oral consent for participation and proceeded with a welcome and personal introduction. JK provided an overview of the study and its objectives, clarifying that it was part of her dissertation research. There was no prior relationship between participants and JK; the only interaction before the interview occurred during the recruitment process. Following this introduction, JK posed the questions outlined in the interview guide, which was developed by the authors in line with the research questions. The conversations were conducted in Czech, which was the native language of both the participants and the interviewer. The interviews were subsequently transcribed verbatim and translated into English for analysis and reporting. The transcripts were not returned to participants for comments or corrections. The English version of the interview guide is available online: https://osf.io/zbma8/?view_only=36d50595d0be40b88f6c111e6dd83e52 . The material was analyzed using MAXQDA following the approach to thematic qualitative text analysis as outlined by Kuckartz . This method involves a structured, multi-step process designed to systematically categorize and interpret qualitative data while remaining flexible enough to capture emerging themes. Kuckartz’s approach consists of several stages: familiarization with the data, theme identification, coding, categorization, interpretation, verification, and reporting. This ensures a comprehensive analysis, blending inductive and deductive methods to allow for both predefined research questions and the content of the data itself to inform the analysis. Initially, JK familiarized herself with the interview transcripts by reading through the material multiple times to immerse herself in the data and identify preliminary overarching themes relevant to the research questions. Following this initial familiarization, JK proceeded to code the material on a sentence-by-sentence basis, assigning segments of text to relevant categories that emerged from the data. As new overarching themes emerged throughout the analysis, JK revisited the entire dataset to recode the material, ensuring that all relevant data was consistently categorized and aligned with the emerging themes. Once the coding process was completed, JK interpreted the results by identifying patterns and relationships between the themes. To enhance the reliability and validity of the analysis, JK consulted with co-author (AB) during the verification phase, sharing the transcripts and coding process. AB reviewed the data, provided feedback on the coding, and ensured that the interpretation of the findings was sound and accurately reflected the content. Finally, the results were synthesized and reported in line with the overarching themes. Participants did not provide additional feedback on the findings after the analysis phase and no repeat interviews were conducted. Motivation for content creation (RQ1)Health experts’ attitudes toward health communication of non-expert SMIs (RQ2)Misleading health-related information shared by SMIs (RQ3)Countering measures (RQ4) Participants provided suggestions that could, from their point of view, help combat the spread of misleading health-related information by SMIs. Overall, five overarching themes were identified, described in Table with corresponding sample quotes. Firstly, there was a strong emphasis on the need to enhance both health and social media literacy to empower individuals to critically assess the health information they encounter online. Alongside this, the importance of educating SMIs on the accurate and responsible dissemination of health information was underscored. Furthermore, participants advocated for the implementation of regulatory interventions. These could take the form of legal measures or policies enforced by social media platforms, designed to limit the spread of misleading health content. In addition, the value of health professionals maintaining an active presence on social media was recognized as a key factor in improving the situation. By directly engaging with the public, experts can offer accurate information and effectively counteract misleading information. In relation to this, health experts suggested an official social media verification system based on qualifications. Such a mechanism would help users easily identify genuine health experts, thereby enhancing the credibility of the health information shared and making it easier for the public to distinguish between qualified and unqualified sources. Our first research question was concerned with HECCs’ motivation for creating and sharing content on social media. Based on the responses of the interviewees, four main motivations were identified, as presented in Table . First, HECCs aim to educate the general public about health-related topics through social media content. The interviewees mentioned focusing on various topics based on their expertise, such as medicine in general (#5), their specific work and qualification (#10), mental health and eating disorders (#2), or addressing the “knowledge gap” (#7) between experts and laypeople regarding nutrition. In the case of mental health, experts emphasized their goal of destigmatizing the topic. One of the interviewed psychotherapists highlighted their approach by stating, “I have chosen the strategy to focus on showing the public that psychotherapy, and generally mental health care, does not have to be a taboo subject and it is a completely normal thing.” (#9). The second identified motivation highlights HECCs’ recognition of social media as a strategic tool for enhancing public visibility, which, in turn, could potentially expand their client or patient base. This perspective was echoed by most of the interviewees, indicating a widespread acknowledgment of social media’s professional benefits within the health community. For instance, one interviewee articulated the direct advantage by stating, “It is of course also beneficial for me regarding my business because it allows me to reach people who might become my clients because they know me from social media.” (#12). Similarly, another expert noted, “I would be lying if I said it does not have the potential to help me professionally.” (#6). Furthermore, HECCs acknowledged that their activity on social media is driven by their desire to interact with people and foster a sense of community. Interviewee #2, a psychotherapist specializing in eating disorders, expressed her intention to “build a community of people who maybe can pass on their experiences” (#2), emphasizing the aim to create a supportive space for sharing experiences. Moreover, a gynecologist and sexologist noted that social media acts as a platform for discussing sensitive topics, benefiting from the anonymity and openness of the online community. This professional observed that “people are not shy” and are more willing to discuss matters they might hesitate to bring up in person (#4). Lastly, HECCs highlighted their motivation to engage actively on social media was to counteract the spread of false health information. For instance, interviewee #3 shared his rationale for starting on social media, stating, “I thought that it would be nice to start fighting with the myths that were actually pouring in on me from all sides on social media and start saying sort of my opinion about it.” (#3). The second research question focused on understanding HECCs’ perceptions of non-expert social media content creators disseminating health-related messaging. The consensus among the interviewees was rather negative, perceiving this trend as both problematic and irritating. One expert mentioned, “I try not to follow many influencers because it makes me really mad when they give health advice” (#8). This sentiment was echoed by others, who stated, “I guess we, as doctors, have a fundamentally united view on this, and that is it bothers us very much, it makes us angry.” (#5), and “I perceive it very negatively.” (#1). HECCs observed that in many instances, non-expert SMIs tend to offer health-related advice based on their personal experiences while positioning themselves as experts. Women who have experienced childbirth offering advice on women’s health and pediatrics or individuals advising on nutrition following personal weight loss exemplify this trend. One expert highlighted the broader issue of assumed expertise: “This is kind of an epidemic of inexpertise, where the non-experts act as if they were experts. For example, I am a psychotherapist who really likes coffee. So, I could go and tell people how to make coffee correctly because I am the expert. Well, that is just nonsense, because experience does not equal expertise.” (#9). Adding to this sentiment, another comment was made: “Everyone who was overweight, then lost some weight and posted about it on social media is now a self-proclaimed expert on nutrition and fitness.” (#1). Despite the overwhelmingly negative stance toward SMIs providing health-related advice without proper qualifications, some interviewees recognized a positive shift. They noted a trend where SMIs include disclaimers in their posts, stating they are not health professionals and advising their audience to treat their advice accordingly. One expert appreciated this development, saying, “I feel like there’s a bit of an effort right now when an influencer is talking about mental health, they are talking about their story. That’s where I feel like it’s opening up. I also notice that there’s an effort to add some disclaimer in there that they’re not experts, that it’s totally not their place to pass on some advice, to pass on some support and stuff, which I appreciate.” (#2), while another added, “I must say I started noticing lately that influencers try to emphasize at least that this is what helped them specifically or was recommended to them by a doctor.” (#8). Despite the prevalence of health misinformation, i.e., factually incorrect information, in discussions about misleading health content on social media, most interviewed HECCs emphasized that information does not necessarily have to be factually incorrect to be misleading and potentially harmful. They underscored the importance of considering the individual needs and conditions of each person. As one expert put it, “I think that misinformation is not the only type of information that could be harmful to human health. There can also be accurate information that is potentially harmful because it really heavily depends on overall medical condition of the person concerned. It needs to be assessed whether it is applicable to and safe for this specific person.” (#10). To illustrate the point, interviewees provided numerous examples of what could be described as overgeneralized messaging. This refers to information that, while not factually incorrect, could still be potentially harmful to human health due to its lack of specificity and failure to account for individual health conditions and needs. For instance, a nutritionist expressed her concern over various diets, noting, “In this case, for example, it really depends on who the person is. For example, if it’s an adolescent, who’s still growing up, I would be very careful with veganism. […] I think that if someone wants to inspire their followers to adopt some alternative diet, such as veganism, they should always also point out all the potential downsides and say that while it might be suitable for one person, it might not be for everyone.” (#7). Similarly, two interviewees mentioned the popular concept of “what I eat in a day” videos as an example of overgeneralized messaging. These videos, typically shared by SMIs, showcase everything the influencer eats in a day, often as a form of inspiration for their followers. One interviewee explained: “I think a good example would be the ‘what I eat in a day’ posts. When a nutritionist does that, they always point out that this is based on their own individual needs and goals, and it would look different for other people. So, they bring attention to individual differences, and it serves more as an inspiration. […] However, many profiles do not point these differences out. It’s also common when people share recipes and their portion sizes. Sometimes I see portions and think that for me as an active person, the portion would need to be much larger. I don’t think the majority of the audience realizes this, though. Instead, they may think something is wrong with them if they’re still hungry after eating the meal.” (#7). Another interviewee added: “I believe people who don’t know much about nutrition and are desperate to lose weight might take these ‘what I eat in a day’ videos literally - like ‘eat exactly like this if you want to look like me’ - and that can naturally be a big problem.” (#1). Discussing childbirth experiences shared on social media, an expert pointed out the dangers of oversimplifying complex medical decisions based on personal experience, stating, “If someone says, ‘I gave birth at home, it was an amazing experience and everything went well,’ that is not misinformation because it might actually be true for them. However, it does not change the fact that home births are extremely dangerous and if somebody decides to give birth at home based on this good experience of an influencer, this person and their baby do not have to be that lucky and come out of it both healthy and alive.” (#1). Another expert criticized exercise advice given by SMIs, saying, “For example, somebody posts a video with ‘the best five exercises for back pain,’ but it is clear that there are no five best exercises for everyone. This influencer says in the video that these are the absolute best five exercises that everyone should be doing, but in reality, while these exercises might be great for some people, they are not going to be suitable and helpful for everyone. It is also very possible that the back pain of some people will get much worse if they do these exercises.” (#11). Furthermore, all interviewed mental health experts raised concerns about the public disclosure of mental health condition symptoms by SMIs, particularly the risk of self-(mis)diagnosis this may encourage. Interviewee #2 observed, “I think that people would self-diagnose based on what influencer says because of their need to belong and identify with someone. So, for example, for adolescents, these young people are trying to find out who they are and where they belong, and if they see that everyone around me who I consider important had some mental illness, then they will want to have one too and probably self-diagnose with one.” (#2). This sentiment was reinforced by another expert, who remarked, “People try to self-diagnose. For example, if you have this and that, you need to have this diagnosis. Most commonly I feel like people self-diagnose themselves with ADHD for some reason, and usually completely incorrectly.” (#12). The third mental health expert also acknowledged this trend, noting that it is something he encounters also in his practice, and saying that “it is the price for the fact that mental health is now increasingly more discussed.” (#9). Therefore, the public discussion of mental health symptoms can be seen as a form of overgeneralized health messaging, according to the definition used in this study. Answering RQ3, the interviewees argued that the health-related consequences of misinformation and accurate but inapplicable health advice are generally comparable. One expert elaborated on this point by stating, “I would say that I don’t really see a difference between misinformation and some kind of unsuitable advice in terms of their consequences because in the end, both can lead to very similar results.” (#9). Nevertheless, some experts observed that the two types of messaging may differ in terms of the immediacy with which undesirable consequences manifest. While the adverse health effects of following misinformation can be immediate, the consequences of adhering to overgeneralized advice could emerge over a longer period. One expert explained, “I’d say the consequences of these two types of information might differ in the speed in which they appear. So, for misinformation it might be quicker and acute, whereas when a person does something like a long-term lifestyle change then the consequences can be damaging to health, but I’d say there’s higher probability somebody will notice and do something about it.” (#7). To avoid the undesirable consequences of overgeneralized health messaging, HECCs advocated for SMIs to include disclaimers highlighting their lack of professional expertise and the personalized nature of health messaging. They emphasized the importance of recognizing that health conditions vary significantly from person to person. One expert noted, “It’s just extremely important to keep emphasizing that everyone’s journey is different and what worked for one person does not have to work for others.” (#2). Another emphasized the potential risks of overgeneralized advice, suggesting, “I think it should always be stated there that while these exercises might help some people, they can also be harmful for others so you should always consult a medical professional, who can evaluate their individual case. I think a disclaimer like this would be really helpful because it really can potentially be dangerous and harmful.” (#11). This study aimed to provide in-depth insights into the HECCs’ perspectives on non-expert SMIs disseminating health information, especially concerning potentially harmful and misleading messaging. First, we identified four primary motivators for health experts’ active engagement with social media: (a) they are driven by a commitment to public health education, aiming to share accurate health information and advice with the broader public; (b) they are interested in building and engaging with communities on social media, fostering spaces for dialogue and support around health topics; (c) they proactively combat the spread of misleading health information; and (d) they recognize the potential personal benefits of public visibility and effective self-promotion, acknowledging that an active social media presence can enhance their professional reputation and opportunities. These motivations help explain the growing numbers of health experts turning to social media to extend their influence beyond traditional medical settings, as evidenced by recent research . Since social media users search for health-related information on these platforms , it is desirable to increase the presence and active engagement of health experts on social media . By understanding the motivators for health experts’ participation on social media, agencies can develop targeted strategies and support systems to facilitate and enhance the online activity of health professionals. Next, our study revealed a general skepticism among HECCs toward non-expert SMIs. This skepticism arises from concerns that personal health-related experience, frequently shared by non-expert SMIs, might often be mistakenly equated with expertise, potentially leading to the spread of misleading information and harmful advice. These apprehensions resonate with the broader discourse on the destabilization of traditional expertise in the digital era, where lived experiences and peer-to-peer advice on social media platforms are increasingly valued over formal qualifications and medical training . These findings align with concerns highlighted by previous studies, which noted that SMIs often share health advice on social media without the necessary expertise, and might prioritize commercial interests (e.g., promoting unhealthy food products) over public health concerns . While the observation that some non-expert SMIs increasingly include disclaimers represents a positive shift - indicating a growing awareness of the limitations of personal experience in offering health advice and the potential impact of SMIs’ influence on their followers’ decision-making and lifestyle choices - research in the context of back pain-related TikTok content found that creators sparingly encouraged viewers to seek professional help . In this context, collaborations between SMIs and health professionals, such as the cooperation of British SMI Zoe Sugg with Jo’s Cervical Cancer Trust , could present a valuable opportunity to contribute to the spread of accurate and beneficial health information , and should be explored by further research. Furthermore, in this study, we introduced the term overgeneralized health messaging to describe information that while technically accurate, could be potentially misleading and result in undesirable health outcomes, paralleling the effects of health misinformation. HECCs emphasized that health information does not need to be factually incorrect to be detrimental, especially when disseminated by SMIs, with whom audiences often develop close, friend-like relationships . This underscores the importance of context and individual health needs in evaluating the accuracy and helpfulness of health information, challenging the binary of true/false in assessing the value and impact of health content on social media. As highlighted by the HECCs, this potentially harmful messaging can manifest in various ways, such as sharing personal dietary or exercise routines, or discussing individual experiences with health conditions by detailing symptoms and treatments. The latter seemed to be particularly relevant to mental health, where all interviewed mental health experts expressed concern over the potential for such disclosures to lead to self-(mis)diagnosis among followers. This aligns with previous news reports that public discussions of mental health symptoms could lead to confusion, self-(mis)diagnosis, and inappropriate treatment choices. Nevertheless, this concern also extends beyond mental health to physical health, as previously explored by journalists as well . In contrast to SMI messaging, overgeneralized health messaging also appears in other contexts, such as peer support groups. However, in these communities, the advice is typically solicited, and members share actual similarities, seeking experiential advice from individuals with similar conditions . Sillence further highlights, that advice in peer support groups often includes recommendations to seek professional medical guidance, with members specifically requesting advice from individuals whose health circumstances closely mirror their own. Therefore, while advice in peer support groups may still suffer from inaccuracies, especially when it lacks professional medical guidance, it is likely more relevant to an individual’s specific health situation. Conversely, the perceived closeness and similarity with SMIs is often illusory, based on PSRs . The risk here parallels findings from broader research on politicized scientific communication, where generalized messages can have widespread effects across diverse audiences due to their broad, untargeted nature, which may however overlook individual needs and context . As a result, overgeneralized health messaging from SMIs may pose a greater risk of harm due to its broader reach, lack of tailored relevance, and potential to mislead audiences. Moreover, the interviews highlight that overgeneralized messaging by SMIs can manifest in various forms, not limited to direct health advice, such as symptom disclosures, showcasing personal exercise or dietary regimens, or sharing personal experiences with health conditions. These different types of messaging warrant further exploration in future research. Our study is the first empirical work to describe the phenomenon of overgeneralized health messaging by SMIs and validate its significance through the perspectives of HECCs. Moreover, this study revealed that, according to the HECCs, the adverse consequences of health misinformation and overgeneralized health messaging can be considered essentially comparable in severity, despite the latter being factually accurate. Nevertheless, it was observed that in some cases, the repercussions of overgeneralized health messaging might not manifest as immediately as those resulting from misinformation. The conceptualization of overgeneralized health messaging highlights a critical pathway for future research to probe into the intricacies of overgeneralized health messaging disseminated by SMIs. Such inquiries should aim to delve deeper into the specific characteristics, consequences, and comparability of overgeneralized health messaging to health misinformation. We argue this exploration is vital for developing effective communication strategies and interventions to counteract the nuanced challenges posed by the dissemination of misleading health information on social media. The consensus among HECCs emphasized the critical need for educating both audiences and content creators, augmented by regulatory measures to ensure the accuracy of health information shared online, echoing recommendations from research . In addition, the active engagement of health experts on social media platforms was proposed as a key strategy for mitigating the spread of misleading health information. Their visible presence is not only desirable but also potentially effective in counteracting misleading content, given that health professionals are regarded as more credible and authentic sources of information compared to non-expert SMIs . Moreover, the suggestion to implement a verification process for health experts’ social media accounts presents a practical tool to enhance the visibility and influence of credible health information sources. By verifying professional credentials and marking their profiles accordingly, social media platforms could significantly aid in distinguishing qualified health experts from non-experts. In line with this, platforms like YouTube have introduced initiatives such as YouTube Health , which fosters cooperation between the platform and health professionals and increases the visibility of authoritative health content by featuring verified health professionals and institutions. While the effectiveness of such interventions in the context of health experts on social media is yet to be examined, existing research presents mixed findings. On one hand, Edgerly & Vraga found that the credibility of a user is not significantly influenced by the presence of a verification badge. On the other hand, recent studies, such as that by Liao et al. , suggest that verification badges enhance trust in posts. Therefore, we encourage future research to delve into this issue as understanding the nuanced effects of verification on the perceived credibility and trustworthiness of health experts’ social media accounts is crucial for developing effective strategies to combat misleading health information spread on the platforms. Limitations First, the interviews were conducted exclusively online or via phone calls, precluding the ability to observe non-verbal cues such as body language, which could offer additional insights into participants’ responses. Second, the research focused solely on health experts active on Instagram, potentially overlooking the perspectives of health professionals who utilize other platforms, such as TikTok , which might exhibit different dynamics and audience engagements. Next, the country of data collection needs to be considered when interpreting the results. Specifically, the interviews were conducted in Czechia, an EU member state with social health insurance system with universal membership, ensuring widely accessible health services at no direct cost to citizens . The country’s healthcare system could potentially influence both the health information seeking behaviors on social media and the motivations behind HECC’s social media engagement. Additionally, the study likely encountered self-selection bias, as potentially only those experts who had an existing interest in the topic agreed to participate, possibly skewing the sample. Finally, while most of the HECCs described their social media engagement as an altruistic effort, the possible social desirability bias needs to be taken into account when interpreting the results. This exploratory study identified primary motivators for health experts’ active engagement with social media and revealed a general skepticism among HECCs toward non-expert SMIs and their health-related communication. There exists a critical need to broaden the scope of health communication research to include overgeneralized health messaging, which can be just as detrimental as outright misinformation. HECCs recognized the importance of context and individual health needs in evaluating health information and validated concerns over the potential for overgeneralized messaging to lead to self-(mis)diagnosis and inappropriate health decisions, emphasizing its comparability in severity of its consequences to misinformation. They highlighted the necessity of educating both audiences and content creators, alongside regulatory measures, and verification of health experts’ social media accounts to ensure the accuracy of online health i |
Incidence and factors associated with postoperative headache among adult elective surgical patients at the university of gondar comprehensive specialized hospital, northwest Ethiopia, 2022: a prospective follow-up study | 1d634400-e327-4921-9d13-1b4fa359bb6a | 11776306 | Surgical Procedures, Operative[mh] | Headache is conceptualized as a somatic and chronic disorder with acute episodes of pain, resulting in varying levels of disability, emotional distress and disruption of daily life . The International Classification of Headache Disorders (ICHD-3) classified headaches in to three different groups: Primary headaches, secondary headaches and neuropathies and facial pains and other headaches. Primary headaches includes ; migraine, tension-type headache (TTH), trigeminal autonomic cephalalgias (TACs) and other primary headache disorders. Secondary headache includes ; headache attributed to trauma or injury to the head and/or neck, headache attributed to cranial or cervical vascular disorder, headache attributed to nonvascular intracranial disorder, headache attributed to a substance or its withdrawal, headache attributed to infection, headache attributed to homeostasis disorder, headache or facial pain attributed to facial or cervical structures and headache attributed to psychiatric disorder. Neuropathies and facial pains and other headaches includes; painful lesions of the cranial nerves and other facial pain and other headache disorders . In different studies, it has been reported that postoperative headache (POH) is distressing complication of anesthesia and surgery and, reach 54% up to 84% . It is the most commonly reported neurologic condition and can be severely disabling with several underlying causes, including primary headache disorders and secondary causes. It is a public health problem of major concern in all countries and it represents a drain on a country’s productivity. The economic costs involved (direct and indirect) as well as the psychosocial and human costs are enormous burden on society in general . Postoperative headache is a complication that occurs shortly after anesthesia. It is associated with increased morbidity, prolonged hospital stays, poor quality of life and additional economic burdens and constitutes a health problem . It has also been shown that POH increases the requirements for pain medications and affects the overall patient satisfaction and even delays patients’ discharge from hospital. In the postoperative period, many patients develop postoperative symptoms, most of which are attributable to anesthesia and surgery. These symptoms persist for more than 24 h, and implicated as the main reason for decreased mobilization after surgery and anesthesia . In a study conducted in USA suggested that the risk of post-surgical hospital readmission due to pain was reduced by 35% in patients who received painkiller in the perioperative period . Different studies reported that the risk of POH and its complications is elevated specifically in patients who undergo ambulatory surgery and it also prolongs hospital stay . In patients who underwent ambulatory surgical procedures, postoperative headache was identified as a major risk factor for 30 day hospital readmission. Patients present to hospital with varied symptoms. However, they usually present with headache and abdominal pain. Surgical patients with a history of headache are at an elevated risk of perioperative ischemic stroke. It is therefore, recommended that chronic headache be considered in the risk assessment for perioperative ischemic stroke . Treatment of headache requires multidisciplinary approaches includes; obtaining history, description of the headache(quality, severity and location) following this, narcotics and gabapentin can be used to treat severe headache in the postsurgical period . Caffeine supplementation of surgical patients is part of the enhanced recovery after surgery program(ERAS).Adding caffeine both intravenous and oral caffeine in the perioperative period in chronic caffeinators can reduce the incidence of caffeine withdrawal headache in the perioperative period . Although, there are different studies on the incidence of postoperative headache and associated factors in the abroad, as it is described below in conceptual frame work (Fig. ), there are limited studies both in the study area and all over the country, Ethiopia. There is no published data in the study area and in the country. So, we hope the study will provide a base line data regarding the incidence and associated factors of postoperative headache. Objective The objective of this study was to assess the incidence and factors associated with postoperative headache among adult elective surgical patients at University of Gondar Comprehensive Specialized Hospital Northwest Ethiopia.
Study design and period An institution-based prospective follow-up study was conducted from 9 April to June 20, 2022. Source and study population Eligibility criteria Study variables Operational definition Sample size and sampling procedure Data collection procedures Data quality assurance Data management and statistical analysis Ethics committee approval Before the start of actual data collection, ethical clearance was obtained from Ethical clearance was obtained from University of Gondar College of Medicine and Health Science, School of Medicine, Ethical Review Committee with date and reference number of 25/3/2022 and Ref.No SOM/1404/2022, respectively. The objective of the study was explained to each study participant explicitly. Both written and verbal informed consent was obtained from each study participant after a clear explanation of the merits of the study. Study participants were informed that they had free will to abandon the study at any time. The data was kept secret in a way that only the researcher could get access to it. The privacy of patients during data collection was kept confidential. Confidentiality was maintained at all levels of this study by avoiding identifiers, using codes to identify each participant, keeping the privacy of each study participant during data collection and locking questionnaires securely.
An institution-based prospective follow-up study was conducted from 9 April to June 20, 2022.
Source population Study population All adult elective surgical patients who underwent surgery during the study period.
All surgical patients who were admitted to University of Gondar Comprehensive Specialized Hospital for operation were our source populations.
All adult elective surgical patients who underwent surgery during the study period.
Inclusion criteria All adult elective surgical patients who were operated during the study period were included. Exclusion criteria Patients with cognitive dysfunction and communication problem, pregnant mothers, patients operated under local anesthesia and patients who were directly transferred to intensive care unit were excluded.
All adult elective surgical patients who were operated during the study period were included.
Dependent variable Postoperative headache. Independent variables .
Age, gender, daily caffeine consumption, duration of preoperative fasting, prior history of headache, general anesthesia technique, intraoperative-hypotension and hypertension, smoking, alcohol consumption, low albumin level, increased white blood cell count, type of surgery, intraoperative hypercarbia, intraoperative hypoxemia.
Postoperative headache The presence of headache mostly within 3 days of postoperative period. Smoking history A record of an individual’s background in regard to smoking tobacco. Prolonged fasting A fasting time or nothing per os of ≥ 16 h. Hypertension Hypotension Alcohol history Previous or prior history of headache Intraoperative hypoxemia Intraoperative hypercarbia History of caffeine consumption If someone has a habit of taking caffeine of 400mgs per day.
The presence of headache mostly within 3 days of postoperative period.
A record of an individual’s background in regard to smoking tobacco.
A fasting time or nothing per os of ≥ 16 h.
If there is greater than 20% of systolic blood pressure from the base line it will be considered as hypertension.
A decrement of systolic blood pressure greater than 20% from the base line it will be considered as intraoperative hypotension.
was rated as (non-drinkers) individuals who never drink any kind of alcohol, frequent- drinkers’ individuals who drink any kind of alcohol once or more days per week and ex-drinkers individuals who do not drink any kind of alcohol currently but who used to drink alcohol in the past. Generally if a person drinks one or more bottle of alcohol per day frequently so that we can say the patient has alcoholic history.
Defined as self-reported or recorded migraine or any other kind of recurrent headache before surgical exposure.
If the intraoperative oxygen saturation of the patient decreases below 90% which is measured by pulse-oximetry.
If there is any intraoperative increment of end tidal carbon dioxide (> 50 mmhg as measured by capno-meter).
If someone has a habit of taking caffeine of 400mgs per day.
Sampling procedure Sample size determination For this particular study, all consecutive patients who underwent elective surgery in the study period were selected based on inclusion and exclusion criteria. Data was collected till the calculated sample size was reached.
A single population proportion was used to estimate the sample size. A 50% incidence of postoperative headache with a margin of error (d) of 5% at a confidence level of 95% was assumed and a 10% non-response rate was added and the final minimum estimated sample size was n = 424.
Data were collected by two data collectors after being given training about the data collection procedure and they were supervised by a postgraduate anesthesia student and the principal investigator. The data collection procedure included an observational checklist and an interview based on questionnaire. The data collectors were requested to select voluntary participants to interview and record anesthetic and surgical data. A structured questionnaire obtained from a previous study conducted to assess postoperative headache was used for data collection . Information regarding demographic variables like, Age, Sex, Previous history of headache, History of smoking, History of alcohol, Caffeine consumption, Family history of headache and NPO-status was obtained by interviewing patients preoperatively. Data regarding, American society of anesthesiologist’s physical status of the patient (ASA), intraoperative hypotension, intraoperative hypertension, intraoperative hypoxemia and the respective measures taken, intraoperative blood transfusion, type of anesthesia, anesthetic drugs used intraoperatively, both for induction and maintenance, type of surgery, position of the patient during surgery, duration of anesthesia and surgery were taken from the anesthesia recording sheet and patients chart. Headache severity can be reliably measured in self-reporting population with one dimensional tools and the visual analogue scale is considered appropriate (VAS). It ranges from no headache (0) to the worst possible headache. Patients were grouped into having no headache (score of 0), having mild headache (score of 1–3), having moderate headache (score of 4–6) and having severe headache (score of 7–10). So, visual analogue scale was used to assess severity of postoperative headache. Patients were interviewed with questionnaires six hours after surgery and were followed up to the 72th postoperative hours. Since the common occurrence of Postoperative headache is during the first 48 h after surgery where the maximum incidence occurs during the first 12 h after surgery we followed patients for three postoperative days . Patients who developed POH during the data collection were advised to seek medical care and the responsible heath care providers were informed about the headache each patient had.
Training was given for two data collectors before the start of data collection. The training was basically about how they should approach study subjects, how they could use the data collection tool and keeping the privacy of study subjects. The data collectors were final year anesthesia BSC students and they were supervised by one Anesthesia post graduate student and other activities were covered by the principal investigator. To ensure the quality of data, a pretest was carried out using 5% (22 patients) of the sample population and these patients were excluded from the main study. The pretest was conducted in the UOGCSH two weeks prior to the start of the actual data collection. The purpose of the pretest was to check the questionnaire for completeness and comprehensibility. The actual data collection was started by patient interviews and by extracting data from patient medical records. The consent form was translated into Amharic using an online Google English to Amharic translator.
After ensuring that the data was complete and consistent, it was cleaned, coded and entered to Epi data version 4.6.0 before being exported to stata software version 14.01 for analysis. Tables and texts were used to present the data, which included both descriptive and analytic statistics. Categorical variables are presented with frequencies and percentages and corresponding cross-tabulation values are presented in tables. The association between categorical variables was tested using chi- square test. Binary logistic regression was conducted to identify factors associated with postoperative headache. All independent variables were identified using bivariate logistic regression and Factors with P -value < 0.2 during the bivariate analysis were further entered into multivariate logistic regression analysis to identify independent risk factors. Multi-Co-linearity of covariates was assessed. To check for their association with the dependent variable (POH) p -value < 0.05 was considered statistically significant. The strength of association was presented using both crude and adjusted odds ratios with their respective confidence intervals. Hosmer Lemeshow test of goodness of fit was used to check the appropriateness of the model for analysis and the model was found to be appropriate.
In this study, a total of 424 patients who met our inclusion criteria were included and the response rate was 99%. Four patients were excluded from analysis, because of incomplete data as they were discharged earlier than 24 h after surgery. Socio -demographic and clinical characteristics of study participants Personal habits and previous history of headache Clinical data of study participants The overall incidence and severity of postoperative headache Factors associated with postoperative headache During the bivariate logistic analysis variables like, intraoperative hypertension, prolonged preoperative fasting, intraoperative hypotension, general anesthesia, female gender, both self and family history of prior headache were significantly associated with postoperative headache. However, in multivariate logistic analysis, female gender (AOR = 1.62, 95%CI = 1.03–2.53), intraoperative hypotension (AOR = 1.74, 95%CI = 1.06–2.88), general anesthesia (AOR = 1.96, 95%CI = 1.25–3.18), prior history of headache (AOR = 4.84, 95%CI = 2.40–9.74) and prolonged preoperative fasting (AOR = 2.28, 95%CI = 1.45–3.60) and caffeine consumption (AOR: 1.60, 95%CI = 1.03–2.54) were significantly associated with the postoperative headache (Table ). Accordingly, being female increases the risk of postoperative headache by 1.6 times as compared to being male (AOR = 1.62 95% CI = 1.03–2.53). Patients operated under general anesthesia were 1.96 times more likely to develop postoperative headache as compared to counterparts (AOR = 1.96, 95%CI = 1.25–3.18). The risk of developing postoperative headache in patients who consume caffeine was increased by 1.6 times as compared to non-consumers (AOR = 1.60, 95%CI = 1.03–2.54). Intraoperative hypotension increases the likelihood of postoperative headache by 1.7 times as compared to patients who had normal blood pressure during surgery (AOR = 1.74, 95%CI = 1.06–2.88). Patients who had prolonged preoperative fasting time had a two-fold increased risk of POH (AOR = 2.28, 95%CI = 1.45–3.60) and patients who had prior history of headache had a four-fold increased risk of developing postoperative headache (AOR = 4.84, 95%CI = 2.40–9.74) (Table ).
More than half of our study populations were males (233) and majority of study participants were in the age between18 and 29 years (42.2%). From the total study participants 60.5% of them were ASA class I. More than one third of our study participants (37.9%) had a preoperative fasting time of ≥ 16 h and 30.2% of participants had a baseline serum albumin investigation that revealed 6.7% of them had low albumin level. (Table ).
This study showed that 20.2% (85) of participants had prior history of headache and 18.8% (79) of participants had family history of headache (Table ).
The majority of our study participants were operated under general anesthesia and majority of them were induced with combined ketamine and propofol. Isoflurane was the only inhalational anesthetic agent used for maintenance in all cases. More than half of type of surgery was categorized under general surgery ( n = 223) which incorporates; genitourinary surgery ( n = 25), Neurosurgery ( n = 30), ENT surgery ( n = 88), gastrointestinal surgery ( n = 60) and hepatobiliary surgery ( n = 20) and most of participants were operated in supine position. The duration of surgery in majority of the cases was from 1 to 3 h. (Table )
The overall incidence of postoperative headache in the current study was found to be 54.3% (95% CI = 49.5–59.0). In patients who developed postoperative headache, the highest incidence 80.7% (184) was observed at day one of postoperative period. At day one about 60% and 20.6% patients developed postoperative headache at 12 and 24 h of postoperative period, respectively. The incidence of severity of mild, moderate and severe postoperative headache was 14.3%, 21.2% and 18.2%, respectively (Table ).
Postoperative headache is a complication that occurs shortly after anesthesia . According to our findings, more than half of our study participants experienced postoperative headache 228 (54.3%) (95% CI = 49.5–59.0).This finding is in line with a study which reported a 54% incidence of headache in postoperative patients . However, a prospective cohort study conducted in Greece reported a lower incidence of postoperative headache (28.3%) . The possible reason for this may be due to intraoperative anesthesia maintenance difference, they maintained with intravenous propofol but our maintenance was inhalational anesthetic drugs. The use of propofol reduces the incidence of postoperative headache as compared to conventional type of anesthesia . Furthermore, clinical practice difference, demographic distribution of headache, differences in postoperative care, follow up and the access to symptomatic treatments might be the other possible reason for the different incidence of postoperative headache. Similarly, a study done in China reported a lower incidence of postoperative headache(27.5%) as compared with our study . This might be explained by clinical practice difference, differences in postoperative care, follow up and the access to symptomatic treatments and a difference in study design. In another follow-up study done in Brazil showed that high incidence of postoperative headache (91%) . The difference may be due to the fact that their study was done on patients who are risky for developing postoperative headache since the procedure was craniotomy as compared to our study participants and also there may be a difference in age and gender, type of surgeries performed, endemic conditions, and even different prevalence of primary headache disorders in their area. Another study carried out to compare the effect of anesthesia technique on the incidence of postoperative headache among orthopedic surgical patients found a 44% incidence of POH . This is also low as compared to our findings. The difference might be explained by the type of surgical procedure differences, differences in postoperative care, follow up and the access to symptomatic treatments. In our study, the majority of patients (60%) developed postoperative headache during the first 12 h of postoperative period. This finding is consistent with the findings of a research which reported a maximum incidence of postoperative headache during the first 12 h after surgery . This might be explained by active residual effect of anesthetic drugs exposes the patient to headache because the drugs need some times to be metabolized. In our study, type of anesthesia, gender, intraoperative hypotension, previous history of headache, prolonged preoperative fasting and caffeine consumption were identified as independent risk factors for the development of postoperative headache. The result of our study showed that patients operated under general anesthesia had an increased risk of postoperative headache when compared with patients operated under regional anesthesia. Our finding is supported by a study conducted to compare the incidence of postoperative headache using general anesthesia versus spinal versus combined general and regional anesthesia. It showed that higher incidence of postoperative headache in patients operated under general anesthesia . This might be because of the physiologic disruption of central nervous system by general anesthesia medications. Similarly, a study conducted on ambulatory surgical patients to evaluate the incidence of postoperative complications and their relation with anesthetic techniques, found that the use of inhalational anesthetic agents were significantly associated with postoperative headache . Our finding is also supported by existing evidences. Inhalational anesthetic agents including halothane and isoflurane are known causes of postoperative headache because of cerebral vasodilation . So, patients who undergo surgery under general anesthesia may need regular perioperative assessment for headache and possible intervention. Correspondingly, in the current study, we found a significant association between prolonged preoperative fasting hours and postoperative headache. Patients with prolonged preoperative fasting hours were more likely to develop postoperative headache. This finding is also supported by other studies . The reason behind might be explained by prolonged starvation may end up with intravascular volume depletion and hypoglycemia in turn blood flow to the brain also decreases and pressure sensitive brain vasculature began to develop pain. So, shortening the preoperative fasting may decrease fasting related headache disorders in the perioperative period. Furthermore, the finding of our study showed that intraoperative hypotension is associated with postoperative headache. Our finding is supported by a study conducted in Greece . Existing evidences also showed an association between headache and hypotension. The possible pathophysiology as explained by existing evidences is that, hypotension induces a reduction in cerebral blood flow and affects cerebral perfusion. finally, results in a significant mismatch of cerebral oxygen delivery and demand . So, improving perioperative cardiac function may help to decrease the occurrence of postoperative headache . Furthermore, in our study, female gender was strongly associated with postoperative headache. This is also supported by many comparative studies conducted to compare the occurrence of postoperative complications between men and women . The possible explanation may be, since women have fluctuating hormones(estrogen, serotonin and progesterone) during their menstrual cycle they may experience headache and they are more likely to be affected by psychosocial factors. In addition, pain expression is more socially acceptable in women than men. So, women are more likely to report headache attacks . The other most important risk factor identified as a cause of postoperative headache was prior history of headache. In this study, patients with prior history of headache are more likely to develop postoperative headache. This finding is consistent with other research findings . The association may be explained by endogenously formed substances in prior history of headache that are assumed to interact with intracranial nociception during disruption of brain physiology because of anesthesia. Among such substances, which may be elevated in patients’ plasma during headaches or which can induce headaches, are neuropeptides, in particular calcitonin gene-related peptide (CGRP), nitric oxide (NO) and its metabolites, and prostaglandin E 2 (PGE2) these cause post operative headache easily on the other hand Headache attributable to disorders of homeostasis represents a diverse diagnostic category in which external (i.e., high-altitude) or internal (i.e., fasting) demands exceed homeostatic capacity, resulting in symptomatic headaches. Metabolic headaches, especially fasting headache, have been observed to be common in the general population. In many cases, a history of migraine is a risk factor, consistent with the hypothesis of an adaptive, evolutionary role of headache to warn against environmental and physiological threats. Because many exposures may also be interpreted as migraine triggers, some authors have considered certain disorders in this category to more accurately represent primary headache disorders. Nonetheless, these disorders not only represent important clinical entities, recognition of which may prevent medical morbidity, but also interesting exposure-response models to further our understanding regarding headache pathophysiology . Our study has revealed that the risk of postoperative headache in coffee consumers is high in comparison with non-consumers. Cessation of use of caffeine containing drinks after chronic exposure leads to withdrawal syndrome with headache as its dominant symptom . The interruption of daily consumption of caffeine-containing beverages can cause headache and other symptoms within 8 h . In contrast to our finding, a study found no association between postoperative headache and caffeine consumption . The difference with our result may be due to the fact that this study was conducted on day case surgical patients with short follow up time. Caffeine is a commonly used Neuro stimulant that produces cerebral vasoconstriction by antagonizing adenosine receptors and its withdrawal results in cerebral vasodilation and the end result will be headache (61). In addition, the enhanced recovery after surgery(ERAS) protocol recommends caffeine supplementation in chronic caffeine consumers to mitigate the development of postoperative headache and it is already part of a clinical practice at many institutions . Although, history of smoking, history of alcohol, intraoperative hypertension, intraoperative hypercarbia, white blood cell count and albumin level are well known causes of postoperative headache, we did not find significant association with the outcome variable . The difference may be due to the fact that we included few smokers and most patients had no baseline albumin level as well as less incidence of intraoperative hypertension was documented and we did not analyze the association between hypercarbia and postoperative headache because most patients were not monitored with Capnometry due to limited access to it.
This study is the first of its kind bothWe have also used adequate sample size and included diverse groups of surgical patients. However, this study is not without limitations. First, it is a single-centered study which might make generalizability difficult. Secondly, our primary end point was to determine the incidence of postoperative headache and also there might be a recall bias of headache since we started patient follow up at 6 h of post operative time. So, we did not evaluate the type of headache each patients had and some patients were asked for headache retrospectively, this may result in recall bias as well we did not evaluate the quality of pain to specific etiologies.
According to the findings of our study, more than half of our study participants suffered from postoperative headache. Use of general anesthesia techniques, intraoperative hypotension, being female, consuming caffeine, prior history of headache and having prolonged preoperative fasting hours were significantly associated with postoperative headache and finally, based on our findings, we would like to recommend strict adherence of fasting guideline since it decreases the incidence of postoperative headache and preventing and managing intraoperative hypotension may also help to reduce the risk of postoperative headache. Patients who undergo surgery under general anesthesia, caffeine consumers, patients with prior history of headache and women may need regular perioperative assessment. These risk factors and the presence of headache need regular assessment in the perioperative period in order to offer analgesics and risk reduction strategies. We also recommend future researchers to conduct a research on the impact of postoperative headache on patient’s postoperative outcome. Since the problem is wide spread and understudied, more high level studies are recommended we hope there is a lot to discover.
Below is the link to the electronic supplementary material. Supplementary Material 1
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The future of AI clinicians: assessing the modern standard of chatbots and their approach to diagnostic uncertainty | 491cf254-0c53-4ac0-90da-dd4112711f1e | 11470580 | Family Medicine[mh] | In recent years, the potential benefits of artificial intelligence (AI) in healthcare have been extensively explored . Among the barriers faced by outpatients at specialist care centers, more than half experience issues related to information availability and healthcare communication . The advent of rapidly developing chatbots, such as ChatGPT, has highlighted the utility of AI in medical information dissemination and early patient education. These chatbots, with their advanced fluency and technical linguistic capabilities, offer the general patient population a wealth of easily accessible and accurate information . They deliver context with careful consideration, potentially mitigating the occasionally alarming nature of highlighted internet search results . AI has already demonstrated benefits in triage, providing diagnostic results comparable to those of clinicians and offering safer recommendations on average . Furthermore, the rise of telemedicine as a medium for patient management presents an additional dimension suitable for language models . Nonetheless, the intricacies of real-world medical practice go beyond static knowledge and involve domains fraught with diagnostic uncertainty. Diagnostic uncertainty arises when, differential diagnosis, and often, iterative patient evaluation . This aspect of medical practice poses challenges even for seasoned clinicians, demanding a synthesis of experience, intuition, and continuous learning . Previous studies have demonstrated that ChatGPT performs well on structured medical knowledge assessments, including the United States Medical Licensing Exam (USMLE) . However, there is a paucity of research evaluating the performance of AI chatbots in scenarios involving diagnostic uncertainty. In addition, it is crucial to consider the distinct ethical frameworks and training methodologies that different AI chatbots employ, as these factors can significantly influence their responses. For instance, ChatGPT is programmed with several moral principles, including privacy, non-maleficence, non-discrimination, and transparency, while Claude is trained within a virtue ethics framework, which emphasizes honesty and a context-sensitive approach . This latter framework could potentially allow for more nuanced and empathetic responses, particularly in complex scenarios such as those involving diagnostic uncertainty. This study aims to assess the efficacy of AI chatbots in addressing medical scenarios characterized by diagnostic uncertainty and to compare the responses of chatbots trained on different ethical frameworks. Understanding the constraints and capabilities of AI chatbots in managing diagnostic uncertainty is crucial for their effective integration into clinical practice. Study designData collectionStatistical analysisThe Progress Test, conducted by the Department of Family and Community Medicine (DFCM) at the University of Toronto functions as a formative tool to evaluate the development of residents towards becoming Family Medicine Experts and supports their preparation for Board Certification. This biannual examination is structured as a closed, four-hour multiple-choice test, curated by subject matter experts in Family Medicine. Each item on the test presents four response options, labeled A through D. For this study, all questions from four Progress Tests administered between 2022 and 2023 to a cohort of 320 Family Medicine residents in their first (PGY-1) and second (PGY-2) postgraduate years that were tagged with the “diagnostic uncertainty” assessment objective, as highlighted by The College of Family Physicians in Canada, were extracted . Diagnostic uncertainty questions were defined as those presenting clinical scenarios where symptoms, clinical findings, and patient histories do not converge on a definitive diagnosis, necessitating nuanced interpretation and differential diagnosis. The performance of medical residents ( N = 320) on these questions was then compared against the performance of AI models GPT-4o and Claude-3 pro. Ethical approval for this study was granted by the University of Toronto Research Ethics Board. To maintain study integrity, each question was input into both GPT-4o and Claude-3 in the same format as presented in the official examination, with multiple-choice answers labeled A through D, without any alterations or additional cues. Prior to entering each question, the chatbots’ conversation history was reset, and memory cleared to avoid any influence from previous interactions. The chatbots’ responses were reviewed by two independent reviewers (R.S.H., A.B.) to identify the chosen multiple-choice options. Each LLM was queried with the same question three times to assess for variability. Collected data included the date of question input, response length in characters, response time in seconds, the presence of a rationale for excluding other options, and the root cause of any incorrect responses. If the AI chatbot selected “all of the above” or “none of the above,” the answer was marked incorrect since these were not valid choices. For each question, it was documented whether the response provided reasons for excluding incorrect options. Incorrect responses were classified into three mutually exclusive types by the reviewers (R.S.H., A.B.): statistical errors, information errors, and logical errors. Statistical errors were defined as mistakes in arithmetic calculations. Information errors occurred when the chatbot gathered incorrect information either from the question itself or external sources, resulting in an incorrect answer. Logical errors were identified when the AI chatbot had access to the correct information but failed to apply it accurately to arrive at the correct answer. The primary outcome of this study was to compare the performance of AI chatbots and PGY-1 and PGY-2 residents in answering questions involving diagnostic uncertainty. Secondary outcomes included comparing GPT-4o and Claude-3 performance, response length, response time, and the proportion of questions. Resident performance was calculated as an aggregate of the performance statistics on diagnostic uncertainty questions from the Family Medicine Progress Tests administered between 2022 and 2023, with 95% confidence intervals (CIs) derived using a binomial generalized estimating equation model. Chatbot performance was calculated based on the percentage of correct responses to the extracted questions. Analyses were stratified across each of the nine priority question areas. Paired t-tests were employed to compare means, and chi-squared tests were applied to compare proportions. We applied the Bonferroni correction method to control the family-wise error rate, ensuring that the significance level was appropriately maintained across the multiple comparisons . A p-value threshold of 0.05 was set to determine statistical significance. Statistical analyses were conducted using Stata version 17.0 (StataCorp LLC, College Station, Texas). A total of ninety questions involving diagnostic uncertainty across nine categories within Family Medicine were included in the study selected from a total of 440 questions across four Progress Tests administered between 2022 and 2023 (Table ). Overall, Claude-3 correctly answered 57.7% ( n = 52/90) of the questions, while GPT-4o correctly answered 53.3% ( n = 48/90) (Fig. ). Both chatbots provided the same multiple-choice answer across all three trials for each question. The performance difference between the two chatbots was not statistically significant ( p = 0.55). When comparing the performance of GPT-4o and Claude-3 to Family Medicine residents on diagnostic uncertainty questions, both chatbots underperformed relative to the residents. PGY-1 residents achieved an average correctness rate of 61.1% (95% CI: 58.4–63.7), and PGY-2 residents scored 63.3% (95% CI: 60.7–66.1), both significantly higher than the chatbots ( p < 0.01). In specific categories, GPT-4o outperformed the residents in cardiovascular and gastrointestinal questions, with scores of 80% and 70%, respectively, compared to 64.5% and 65.7% among PGY-1 and PGY-2 residents ( p < 0.01). Claude-3 excelled in geriatric care, mental health, and women’s health, scoring 70%, 80%, and 70%, respectively, outperforming the residents’ scores of 59.6%, 52.4%, and 56.4% ( p < 0.01). Conversely, residents outperformed both chatbots in the endocrine, musculoskeletal, pediatric, and respiratory categories ( p < 0.01). Claude-3 had a longer mean response time of 24.0 s (95% CI: 21.0-32.5) compared to GPT-4o, which had a mean response time of 12.4 s (95% CI: 9.3–15.3) ( p < 0.01) (Table ). In terms of response length, Claude-3 also produced longer answers, with a mean of 2001 characters (95% CI: 1845–2212) compared to GPT-4o’s 1596 characters (95% CI: 1395–1705) ( p < 0.01). Both chatbots frequently provided rationales for other answer options, with Claude-3 doing so slightly more often than GPT-4o, although not statistically significant (86.7% vs. 78.9%; p = 0.17). Regarding the types of errors made, GPT-4o predominantly made logical errors, accounting for 62.5% of its mistakes, followed by information errors (18.8%) and statistical errors (18.8%). In contrast, Claude-3 had a lower proportion of logical errors at 44.7%, but higher rates of information errors (31.6%). An example of the output from GPT-4o and Claude-3 is provided in Table and examples of errors are provided in Table . Our study compared the performance of AI chatbots, GPT-4o and Claude-3, against Family Medicine residents in addressing diagnostic uncertainty using questions from official progress examinations at the University of Toronto DFCM. Overall, both chatbots underperformed relative to the residents. Although Claude-3 generated longer and more rationale-rich responses, it was more prone to information errors compared to GPT-4o. In a previous study examining chatbot performance on a Family Medicine Progress Test, ChatGPT demonstrated superior performance compared to the best-performing resident, highlighting its capability in handling well-defined medical knowledge assessments . However, the results from our novel study, focusing solely on questions involving diagnostic uncertainty, reveal a significant shift in performance dynamics. Both GPT-4o and Claude-3 performed worse than first-year Family Medicine residents. This discrepancy underscores the heightened complexity and nuanced judgment required in scenarios characterized by diagnostic uncertainty, which current AI systems struggle to navigate effectively . There are several plausible explanations for why AI systems struggle with this dimension of healthcare provision. Primarily, AI systems lack the contextual understanding required to appreciate the intricacies of modern medicine . Their algorithms, trained on statistical patterns within limited data sets, are ill-suited to handle rare disease presentations, compounding illnesses, and conflicting clinical data . This bias towards trained data leads AI systems to fill gaps in information with assumptions, resulting in incomplete and incorrect diagnoses . For instance, AI systems like GPT-4o have been found to prefer clinical diagnoses over pathological causes, such as selecting frontotemporal dementia over frontotemporal lobar degeneration, possibly influenced by the available training data . The authenticity and quality of the training data used by these systems are of great consequence . The validity, diversity, and representativeness of the datasets included reinforce the decision-making capacity of the system when approaching rare and complex cases. Conversely, human physicians possess a wealth of experience regarding disease presentation, allowing them to consider individual circumstances, history, prevalence, and additional investigations to make a holistic diagnostic process . This level of nuanced understanding is challenging to encode into an AI system. Another critical consideration is that ChatGPT has been found incapable of recognizing and expressing uncertainty . A cornerstone of modern medical practice and the training of medical practitioners is the risk assessment process, which involves calculating the probabilities of failure or complications while considering the patient’s comorbidities and leveraging these against the potential benefits of the intervention . Salihu et al. (2024) describe seven cases where AI selected invasive treatments, whereas human physicians determined that medication would suffice . These decisions were based on a complex array of considerations involving frailty, comorbidities, and life expectancy . A similar finding emerged in our study, with ChatGPT recommending investigations when none were required. AI systems tend to answer decisively and confidently, often overestimating their confidence level regardless of the validity of their responses. ChatGPT was also found to be incapable of using low confidence levels to increase the number of unanswered questions in a sample exam designed to challenge its strategic capabilities . This overconfidence may be considered a linguistic trait essential to the marketability of the system, but it underscores a significant concern regarding its integration into healthcare delivery. The observed performance differences between GPT-4o and Claude-3 in specific medical domains can potentially be attributed to the distinct ethical frameworks and training methodologies employed for each AI system. GPT-4o performed better in areas such as cardiovascular and gastrointestinal health, possibly due to its programming with a predetermined set of moral principles, including privacy, non-maleficence, non-discrimination, and transparency . These principles may guide GPT-4o towards clear, decisive answers in well-defined medical scenarios where established protocols and concrete data are available, as is often the case in cardiovascular and gastrointestinal health. Conversely, Claude excelled in mental health, women’s care, and geriatric care, which may be attributed to its training based on virtue ethics, emphasizing honesty and intention within a flexible, context-sensitive framework . The nuanced and individualized nature of these domains likely benefits from the virtue ethics approach, which allows for more empathetic and contextually appropriate responses. Mental health, women’s care, and geriatric care often involve complex, subjective factors and require a deep understanding of the patient’s unique circumstances. Claude’s ethical framework may better equip it to navigate these complexities, providing more thoughtful and tailored responses. Consistent with the literature, the majority of ChatGPT’s errors were also in logical reasoning . Given that diagnostic uncertainty questions often arise from incomplete or highly nuanced information that escapes common medical databases, ChatGPT may simply overlook steps in logical reasoning . Claude-3, in contrast, committed fewer logical errors. These findings suggest that the ethical training heuristics embedded in AI systems may influence their performance across different medical domains, especially in scenarios involving diagnostic uncertainty. In addition to comparing the accuracy of each chatbot, Claude-3 responded to the prompts more slowly than ChatGPT, but its answers were generally longer on average. Longer response times may suggest that the LLMs are engaging in more detailed analysis, which could correlate with higher accuracy in scenarios requiring nuanced decision-making. This is partially supported by our findings where Claude-3, with longer response times, performed slightly better than GPT-4o, although the difference was not statistically significant. However, it is important to recognize that response times are also subject to server latency and other external factors, which could introduce variability unrelated to the LLM’s cognitive processing. Therefore, while response time provides some insight into the LLM’s functioning, its interpretation should be approached with caution. Our investigation is subject to several limitations. Given that ChatGPT is updated regularly, incorporating user feedback, its responses to identical queries might vary over time. We attempted to control for these variations by having the models respond to all multiple-choice questions on the same day, and we confirmed the consistency of responses across two different web browsers and three trials per question. It is essential to consider that the findings of this study are relevant to the specific period when they were collected, as the capabilities of both GPT-4o and Claude-3 are expected to evolve. Moreover, these models depend on cookies for optimal functionality and their responses can be affected by prior inputs. To counteract this, we regularly cleared conversation histories and memory before entering new prompts. Another consideration is that our questions were multiple-choice; the models’ performance might differ with open-ended questions or tasks requiring prioritization. In conclusion, while AI chatbots like GPT-4o and Claude-3 show promise in handling structured medical knowledge, their performance in scenarios involving diagnostic uncertainty remains suboptimal compared to human residents. The influence of ethical rule sets on AI performance warrants further investigation, as a virtue ethics framework may offer some advantages in managing complex clinical decisions. Future studies should focus on exploring the capabilities of AI in authentic healthcare contexts, particularly in its role as a clinical decision support tool intended to augment, not replace, physician clinical reasoning. |
Plasma GlycA, a Glycoprotein Marker of Chronic Inflammation, and All-Cause Mortality in Cirrhotic Patients and Liver Transplant Recipients | ff44e630-621b-414a-9de6-988f814c899d | 11765328 | Surgical Procedures, Operative[mh] | Cirrhosis represents the final phase of chronic liver disorders, where liver function deteriorates irreversibly, leading to severe complications and high mortality . The primary treatment for severe cirrhosis is liver transplantation (LT), which offers a chance for a cure . However, despite the success of LT in prolonging life expectancy, recipients continue to face major health risks, including increased mortality . These persistent challenges are partly attributed to cirrhosis-associated immune dysfunction, which is increasingly recognized as a critical factor influencing clinical outcomes in patients with liver diseases . This dysfunction involves two different immune phenotypes: low-grade systemic inflammation and high-grade systemic inflammation, with recent research increasingly focusing on the low-grade systemic inflammatory response . To date, various biomarkers of enhanced low-grade inflammation have been utilized to measure inflammation and predict outcomes in liver disease. High-sensitivity C-reactive protein (hs-CRP) is a widely recognized systemic inflammation marker. Elevated levels of hs-CRP have been associated with worse outcomes in patients with liver disease , reflecting ongoing inflammation and cellular injury. Although hs-CRP is a useful indicator, it may not capture the full scope of low-grade inflammatory activities, especially in chronic conditions like cirrhosis or in assessing the health of transplant recipients. This highlights a crucial need for more comprehensive biomarkers that can better represent the extensive inflammatory activities in these patients. Recently, GlycA has emerged as a novel marker of low-grade systemic inflammation . GlycA is determined using nuclear magnetic resonance (NMR) spectroscopy. The signal comes from N-acetyl methyl groups mostly bound to acute-phase proteins, mainly α1-acid glycoprotein, haptoglobin, α1-antitrypsin, α1-antichymotrypsin, and transferrin . Although GlycA and hs-CRP are found to be interrelated , GlycA seems to be a less variable biomarker of low-grade systemic inflammation . This stability is likely due to its reflection of the acute-phase protein glycosylation process, providing a broader assessment of the inflammatory state. Previously, GlycA has been proven relevant in diseases such as cardiovascular disorders and diabetes, where it correlates with long-term inflammation and predicts negative health outcomes . However, so far, the potential role of GlycA in patients with cirrhosis and among liver transplant recipients (LTRs) remains unexplored. We therefore initiated the present study on plasma concentrations of GlycA, along with hs-CRP, in patients with cirrhosis on the waiting list for LT and those who have received LT. We aimed to investigate: (1) to which extent plasma GlycA and hs-CRP are altered in cirrhosis and post-transplantation, (2) the determinants of plasma GlycA and hs-CRP in cirrhosis patients and LTRs, and (3) the associations of GlycA and hs-CRP with all-cause mortality in these patient groups.
2.1. Comparison of Baseline Clinical Characteristics Between Patients with Cirrhosis, Liver Transplant Recipients, and PREVEND Participants 2.2. Associations of GlycA and Hs-CRP with Clinical and Laboratory Variables in Patients with Cirrhosis and Liver Transplant Recipients 2.3. Longitudinal Analysis of GlycA and Hs-CRP with All-Cause Mortality in Patients with Cirrhosis and Liver Transplant Recipients re were 129 patients with cirrhosis and 367 LTRs who participated in this study. Their clinical and laboratory characteristics were compared with 4837 participants from the Prevention of Renal and Vascular End-stage Disease (PREVEND) study . The median age was higher in the cirrhotic and LTR groups compared to the PREVEND group ( p < 0.001), with no significant difference between the cirrhotic and LTR groups ( p = 0.65). Male predominance was noted in both the cirrhotic and LTR groups compared to the PREVEND cohort ( p < 0.001), while differences between the cirrhotic patients and LTRs were not significant ( p = 0.19). Cirrhotic patients had the highest body mass index (BMI) ( p < 0.001). Lifestyle factors varied, with only 12.4% of cirrhotic patients and 9% of LTRs currently smoking compared to 27.3% in PREVEND ( p < 0.001); alcohol consumption was also lower in cirrhotic patients. Diabetes prevalence was substantially higher in both cirrhotic and LTR groups vs. PREVEND ( p < 0.001). Use of glucose-lowering and antihypertensive drugs was higher in cirrhotic and LTRs ( p < 0.001). Total cholesterol, low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, and triglyceride levels were all significantly different among the groups ( p < 0.001), with cirrhotic patients typically showing lower levels than LTRs and PREVEND participants. hs-CRP was elevated in cirrhotic patients and to a lesser extent in LTRs compared to PREVEND participants ( ; p < 0.001). Remarkably, GlycA was lower in cirrhotic patients than in the other groups ( p < 0.001). Applying propensity score matching for age, sex, BMI, blood pressure, smoking status, history of cardiovascular disease (CVD), diabetes, and usage of glucose-lowering, lipid-lowering, and antihypertensive drugs, 111 and 338 participants from the PREVEND cohort were, respectively, matched for subsidiary analyses with the cirrhotic and LTR groups. This analysis confirmed that hs-CRP levels were elevated in both the cirrhotic patients and LTRs compared to their matched PREVEND participants (10 vs. 1.9 mg/L and 2 vs. 1.2 mg/L, respectively; p < 0.001 for both comparisons). Again, GlycA was lower in cirrhotic patients than in LTRs and PREVEND participants, whereas GlycA was elevated in LTRs compared to PREVEND participants (317 vs. 346 µmol/L and 371 vs. 347.5 µmol/L, respectively; p < 0.01 for both comparisons). A,B show plasma GlycA and hs-CRP levels in cirrhotic patients and in LTRs. GlycA measurements, both before and after the transplant, were available for 30 patients, and hs-CRP levels for 23 patients. For these individuals, the GlycA concentration increased ( C; p < 0.001), contrasting the decrease in hs-CRP ( D; p < 0.01). shows baseline characteristics of cirrhotic patients and LTRs, categorized by tertiles of plasma GlycA levels. In cirrhotic patients, those in the highest GlycA tertile had significantly lower BMI and reduced HDL cholesterol, as well as elevated triglycerides and hs-CRP compared to those in lower tertiles . For LTRs, higher GlycA tertiles were associated with older age; higher BMI; total cholesterol, LDL cholesterol, and triglycerides; and lower estimated glomerular filtration rate (eGFR). Additionally, significant differences were observed in the use of sirolimus, azathioprine, and liver function markers, such as total bilirubin, gamma-glutamyl transferase (GGT), and alkaline phosphatase (ALP), with increasing GlycA levels .
A strong positive correlation was observed between plasma GlycA and hs-CRP levels in LTRs ( R = 0.679) and PREVEND participants ( R = 0.669). In cirrhotic patients, this relationship was weaker ( R = 0.459) . To disclose whether the relationship between GlycA and hs-CRP was different in cirrhotic patients than in LTRs and in PREVEND participants, we compared the regression lines between these groups. The results indicated that the interaction between hs-CRP and the group was statistically significant ( p < 0.001), suggesting that the slopes of the regression lines differ significantly among the three groups. The main group effect was significant ( p < 0.001), indicating differences in the intercepts. Univariable and multivariable linear regression analyses in cirrhotic patients and LTRs identified significant associations between GlycA, hs-CRP, and various clinical and laboratory parameters . In cirrhotic patients, GlycA was inversely associated with BMI and HDL cholesterol and positively with total cholesterol, LDL cholesterol, triglycerides, GGT, ALP, and hs-CRP in univariable analysis. Multivariable analysis confirmed positive associations with LDL cholesterol, triglycerides, and hs-CRP . For hs-CRP, univariable analysis showed an inverse association with HDL cholesterol and positive associations with Child–Turcotte–Pugh (CTP) stage, Model for End-stage Liver Disease (MELD) score, and GlycA, with multivariable analysis confirming positive associations with MELD score and GlycA . In LTRs, GlycA was related to age, BMI, lipid-lowering and glucose-lowering drugs, antihypertensives, metabolic dysfunction-associated steatotic liver disease (MASLD), use of mycophenolate, prednisone, sirolimus, tacrolimus (inverse), azathioprine (inverse), total cholesterol, LDL cholesterol, triglycerides, alanine aminotransferase (ALT), aspartate aminotransferase (AST), GGT, ALP, fasting glucose, glycated hemoglobin (HbA1c), and hs-CRP in univariable analysis. Multivariable analysis confirmed positive associations with age, MASLD, sirolimus use, LDL cholesterol, ALT, ALP, HbA1c, and hs-CRP . For hs-CRP, univariable analysis revealed positive associations with BMI, glucose-lowering drugs, prednisone use, sirolimus use, total cholesterol, ALT, AST, GGT, ALP, HbA1c, and GlycA, with GlycA remaining the strongest predictor in multivariable analysis .
Of the 129 cirrhotic patients, 29 (22.5%) died during follow-up of 140 (IQR 52–381) days. Of the 367 LTRs, 30 (8.2%) died during follow-up of 9 (IQR 3–17) years. presents the Kaplan–Meier curves for all-cause mortality of cirrhotic patients and LTRs stratified by tertiles of GlycA and hs-CRP levels. In cirrhotic patients, higher GlycA ( A) and hs-CRP ( B) tertiles were associated with lower survival probabilities. Specifically, in GlycA tertiles, the survival probability was significantly lower in T3 compared to T1 (log-rank test, p = 0.033). Similarly, for hs-CRP, T3 showed a lower survival probability compared to T1 (log-rank test, p = 0.003). In Cox proportional hazards regression analyses, both GlycA and hs-CRP were associated with increased all-cause mortality risk in cirrhotic patients ( , Model 1, HR per 1-SD increment in GlycA: 1.73 [95% CI: 1.15–2.60], p = 0.009; HR per 1-SD increment in hs-CRP: 1.74 [95% CI: 1.23–2.47], p = 0.002). After adjusting for age and sex ( , Model 2) and MELD scores ( , Model 3), GlycA remained significantly associated with mortality, while the significance of hs-CRP diminished. With further adjustments for LDL cholesterol and triglycerides, the association with GlycA remained ( , Model 4, HR per 1-SD increment: 2.76 [95% CI: 1.53–4.99], p = 0.001), while hs-CRP became non-significant. Even after adjusting for hs-CRP, GlycA remained significantly associated with mortality ( , Model 5, HR per 1-SD increment: 2.34 [95% CI: 1.07–5.13], p = 0.033), whereas hs-CRP showed no significant association when adjusted for GlycA ( p = 0.38). In LTRs, higher GlycA ( C) and hs-CRP ( D) tertiles were associated with compromised post-transplantation survival. For GlycA, T3 showed significantly lower survival compared to T1 (log-rank test, p = 0.005). Similarly, for hs-CRP, both T2 and T3 demonstrated a higher risk compared to T1 (log-rank test, p = 0.003 and p = 0.024, respectively). Cox regression analyses in LTRs revealed significant associations of GlycA and hs-CRP with increased all-cause mortality in the crude model ( , Model 1, HR per 1-SD increment in GlycA: 1.69 [95% CI: 1.29–2.21], p < 0.001; HR per 1-SD increment in hs-CRP: 1.25 [95% CI: 1.09–1.44], p = 0.002) and after adjusting for age, sex, and MASLD ( , Models 2 and 3). However, when adjusted GlycA for hs-CRP or vice versa, neither GlycA nor hs-CRP remained significantly associated with mortality ( , Model 4). In a sensitivity analysis, excluding participants with a hs-CRP level >10 mg/L, point estimates of GlycA and hs-CRP with mortality in cirrhotic patients ( n = 43; 6 deaths) were comparable to those in the whole group but did not reach significance . Also, in LTRs, point estimates of GlycA and hs-CRP with mortality were comparable to those found in the whole group ( n = 305; 25 deaths). However, in LTRs, the association of GlycA with mortality was significant in the crude model (HR per 1-SD increment: 1.75 [95% CI: 1.06–2.90], p = 0.029). In LTRs, the association of hs-CRP with mortality was non-significant in all models.
The present study has demonstrated that plasma GlycA concentrations were unexpectedly lower, whereas hs-CRP levels were considerably higher in patients with cirrhosis compared to the levels found in the general population. Following liver transplantation, GlycA levels increased by 38%, while hs-CRP levels decreased by 74%. In LTRs, both GlycA and hs-CRP levels were elevated compared to the control cohort. Furthermore, both GlycA and hs-CRP were associated with all-cause mortality in both cirrhotic patients and LTRs, with the association of GlycA with mortality being stronger than that of hs-CRP in cirrhotic patients. Collectively, these findings may suggest that the extent to which GlycA is associated with all-cause mortality in patients with cirrhosis and LTRs is at least as strong as that for hs-CRP. We found an independent association of GlycA with LDL cholesterol and triglycerides, which are known to progressively decline with worsening of liver function , whereas hs-CRP was associated with the MELD score in multivariable analysis. Combined, these findings align with an effect of progressive liver failure on GlycA and hs-CRP. However, despite the positive relationship between GlycA and hs-CRP , as now also found in cirrhosis, we observed a notable discrepancy between these inflammation markers in cirrhotic patients, with hs-CRP levels being higher, while GlycA levels are lower compared to the general population. In fact, for a given hs-CRP, GlycA levels were lower in cirrhotic patients than in LTRs and PREVEND participants. hs-CRP, an acute-phase protein mainly produced by the liver in response to inflammatory cytokines , is known to increase in patients with severe liver cirrhosis due to heightened inflammatory responses . However, GlycA is derived from the glycosylation of acute-phase proteins, a process that is also primarily regulated by the liver . Although both hs-CRP and GlycA are markers of acute-phase response, differences exist in their regulatory mechanisms and metabolic pathways. The synthesis of acute-phase glycoproteins, such as α1-acid glycoprotein and α1-antitrypsin, is more complex than that of hs-CRP, especially during the glycosylation step. Glycosylation requires a well-functioning liver with sufficient sugar supply, energy metabolism, and normal enzyme activity . When liver function is severely compromised, the glycosylation process may be disrupted or less efficient, resulting in impaired acute-phase glycoprotein synthesis. Conceivably, ZIP8, a manganese transporter encoded by the SLC39A8 gene that influences N-glycan branching, could be involved in lower circulating levels of GlycA in patients with cirrhosis. The SLC39A8 locus plays a crucial role in regulating the metabolism and transport of essential metal ions, particularly manganese and zinc, thereby affecting the N-glycosylation process . ZIP8 is involved in the cellular uptake of these metal ions, both of which are critical for numerous biological processes, including immune function, antioxidant defense, and inflammation regulation . Of further relevance, the SLC39A8 locus may also confer protection against the progression of hepatic steatosis to steatohepatitis and fibrosis . Evidently, further study is needed to delineate more precisely the mechanisms responsible for the lower GlycA levels in cirrhosis and the role of ZIP8/ SLC39A8 therein. Following liver transplantation, we observed a significant increase in GlycA levels and a decrease in hs-CRP levels. As the new liver gradually regains normal function, it better regulates inflammatory responses, leading to reduced hs-CRP production. Even an intra-operative decrease in hs-CRP levels has also been shown to have the potential to predict overall post-transplant outcomes in LTRs . However, hs-CRP may be less sensitive to chronic low-grade inflammation and could fail to detect ongoing systemic inflammation. Conversely, GlycA seems to be a more sensitive and less variable biomarker of low-grade systemic inflammation in non-cirrhotic individuals . Interestingly, our study found elevated GlycA levels after LT, indicating the persistence of enhanced chronic low-grade inflammation. Recent research has highlighted the gut microbiota, specifically the composition and metabolites produced by gut bacteria, as a significant contributor to low-grade inflammation . A key biomarker in this context is the gut-derived metabolite trimethylamine N-oxide (TMAO), which has been shown to be elevated in LTRs , potentially contributing to persistent low-grade inflammation. Another possible factor is the use of immunosuppressive agents. Our multivariable regression analysis found a positive association between GlycA levels and the use of sirolimus in LTRs. Immunosuppressive agents can impair gut barrier function, increasing intestinal permeability. When the gut barrier is compromised, bacteria, toxins, and inflammatory factors can more easily enter the bloodstream, exacerbating systemic inflammation . Other factors, including age, diet, and pharmacological treatments, can significantly impact GlycA and hs-CRP levels and must be considered. These factors may interact with underlying inflammatory processes, contributing to the variability in these biomarkers. Future research that includes comprehensive data on these variables will be essential for fully understanding their roles in influencing GlycA and hs-CRP levels in LTRs. In this study, we evaluated the association of hs-CRP and GlycA for all-cause mortality in cirrhotic patients and LTRs. While hs-CRP has been extensively studied and established as a biomarker for various diseases, GlycA is emerging as a novel marker, with most of the research focusing on its role in diabetes and CVD and its value to predict all-cause mortality in the general population and to confer reduced life expectancy . To our knowledge, our study is the first to investigate GlycA specifically in a population of patients with cirrhosis and LTRs. Notably, GlycA could exhibit superior predictive capability for all-cause mortality in cirrhotic patients. After adjusting for hs-CRP, the association between GlycA and mortality remained significant, whereas the relationship between hs-CRP and mortality disappeared after adjusting for GlycA in the cirrhotic group, although in LTRs, the associations of GlycA and hs-CRP with mortality were similar. This suggests that GlycA might be a more robust predictor in the context of cirrhosis despite lower circulating levels. Moreover, it is important to highlight that GlycA is influenced by cardiovascular risk factors, suggesting that its levels may be modifiable through lifestyle interventions. In our study, we confirmed a relationship between GlycA and cardiometabolic risk factors, such as BMI, hyperglycemia, and total cholesterol, in univariable analysis, as found in non-liver disease populations . Previous studies have demonstrated that lifestyle interventions can significantly lower GlycA levels. For instance, GlycA was markedly lowered in obese, prediabetic adolescent Latinos following lifestyle changes , and surgical weight loss was shown to lower GlycA levels as well . Controlling modifiable risk factors can thus lead to a reduction in GlycA levels. This presents a promising opportunity for future therapeutic strategies aimed at lowering GlycA to improve patient outcomes. Further research is warranted to elucidate the underlying mechanisms linking GlycA with disease progression and to explore its potential as a predictive biomarker for clinical applications or perhaps even as a therapeutic target. Notably, however, the association of GlycA with mortality was independent of age and sex, suggesting that GlycA retains its prognostic value independent of age and gender, indicating its robustness as a biomarker. Strengths of this study include combined GlycA and hs-CRP measurements in a considerable group of cirrhotic patients and LTRs with a detailed and standardized assessment of clinical and laboratory characteristics consequent to the TransplantLines Biobank and Cohort study set-up in comparison with data from the PREVEND study, which served as a large community-dwelling control cohort from the same region in the Netherlands. We included LTRs only who had received an LT at least one year before the collection of clinical and biochemical characterization and GlycA blood sampling to obviate confounding due to short-term LT-related complications. Moreover, a sensitivity analysis among cirrhotic patients and LTRs with an hs-CRP <10 mg/L, thereby essentially excluding participants with potential latent infections, defined by hs-CRP levels exceeding 10 mg/L, yielded similar hazard point estimates regarding associations of GlycA and hs-CRP with mortality, though not significant in most models due to lack of power. Our study’s limitations should also be acknowledged. As this was a single-center study, the external validity of the results may be restricted, reducing the ability to generalize the findings to broader or more diverse populations. Furthermore, the observational nature of our study and the retrospective setup of the TransplantLines Biobank and Cohort study may have induced insufficiently identified bias, precluding inference to causality. Additionally, our cohorts were predominantly composed of Western European ancestry, which may limit the generalizability of our findings to other ethnic groups.
4.1. Study Population 4.2. Data Collection and Clinical Measurements 4.3. Laboratory Measurements 4.4. Statistical Analysis This study adhered to the STROBE (Strengthening the Reporting of Observational Studies in Epidemiology) guidelines . Patients with cirrhosis and LTRs were recruited from the TransplantLines cohort, a prospective observational study based at the University Medical Center Groningen (UMCG), The Netherlands (NCT03272841) . The TransplantLines study was approved by the UMCG Medical Ethics Committee (METc 2014/077). All participants provided written informed consent, and this study was conducted in compliance with the Declaration of Helsinki . Exclusion criteria included inability to understand the Dutch language, cognitive impairment preventing comprehension of questionnaires or physical tests, prior retransplantation, and absence of GlycA measurements. LTRs who had received a liver transplant shorter than one year were excluded to obviate bias due to short-term complications and infections after transplantation. For the control cohort, data were drawn from the PREVEND study , a population-dwelling cohort in the city of Groningen, The Netherlands. The PREVEND study, initially started between 1997 and 1998, has been described in detail elsewhere . In brief, Groningen residents aged 28 to 75 years were invited to submit a morning urine sample and complete a demographic and cardiovascular history questionnaire. Pregnant women and those with insulin-dependent diabetes were excluded. Participants with a urinary albumin concentration of ≥10 mg/L, along with a randomly selected control group with lower concentrations, were invited for further evaluation. For this analysis, we focused on those who completed the second screening round between 2001 and 2003 and were confirmed to be free of liver disease (based on a questionnaire and medical records obtained from primary care physicians) and had available plasma GlycA measurements. The PREVEND study was approved by the UMCG Medical Ethics Committee (MEC96/01/022) and conducted according to the Declaration of Helsinki . All participants from the TransplantLines and the PREVEND cohorts gave written informed consent. shows a CONSORT flow chart of study participants from the TransplantLines and the PREVEND cohorts.
The TransplantLines study is continuously collecting data from June 2015 onwards. For the current study, samples were collected up to June 2021. During outpatient visits, questionnaires and blood samples were obtained from all patients with standardized procedures . At the time of blood collection, participants continued their regular medication. A standardized protocol was used to obtain anthropometric measurements. Patient information, including weight, height, BMI (calculated as weight divided by height squared), blood pressure, smoking status, alcohol consumption (standardized to 10 g per drink in the Netherlands), medication use (glucose and lipid-lowering drugs, antihypertensive medication, and immunosuppressants), and medical histories such as CVD and diabetes (defined as fasting plasma glucose >7.0 mmol/L, non-fasting plasma glucose >11.1 mmol/L, a self-reported diagnosis, or the use of glucose-lowering drugs), was extracted from the TransplantLines Biobank. Blood pressure was measured using an automatic device with multiple readings to ensure accuracy. The eGFR was calculated using the 2012 CKD Epidemiology Collaboration creatinine-based formula . Additional review of electronic patient records of study participants was performed to obtain additional data concerning the etiology of liver disease. For cirrhotic patients, cirrhotic evaluations (based on imaging, histology, or transient elastography) and biochemical and clinical variables were used to compute the MELD and CTP scores to assess the severity of cirrhosis. The MELD score was calculated by serum total bilirubin, creatinine, and the international normalized ratio (INR) . The CTP score was calculated by total bilirubin, serum albumin, INR, the presence of ascites, and hepatic encephalopathy . For the TransplantLines cohort, data on mortality were obtained from electronic patient records and verified by the Dutch Central Bureau of Statistics. In the PREVEND cohort, data were collected on demographics, lifestyle factors, anthropometric measurements, medical history, and medication use, which was combined with information from a pharmacy-dispensing registry as previously described .
Venous blood samples for both TransplantLines and PREVEND cohorts were collected after an overnight fast. Laboratory methods for PREVEND are reported as described in detail previously . A panel of standardized laboratory assays, including serum ALT, AST, GGT, ALP, total bilirubin, albumin (only available in the TransplantLines cohort), serum creatinine, hemoglobin, thrombocytes, and leucocytes (only available in the TransplantLines cohort), HbA1c (only available in the TransplantLines cohort), and plasma glucose, were analyzed with standardized laboratory measurements and quality assessment control at the department of Laboratory Medicine of the University Medical Center Groningen, The Netherlands. hs-CRP was measured using nephelometry with a threshold of 0.18 mg/L (BNII; Dade Behring Diagnostics, Marburg, Germany). Ethylenediaminetetraacetic acid (EDTA)-anticoagulated plasma samples were centrifuged at 1400× g for 15 min at 4 °C and then stored at −80 °C. Plasma samples were sent frozen at −80 °C to Labcorp (Morrisville, NC, USA) for analysis on the Vantera ® Clinical Analyzer. For lipoprotein profiles, plasma samples were prepared on board the instrument and automatically delivered to the flow probe in the NMR spectrometer’s magnetic field. Total cholesterol, HDL cholesterol, and triglycerides were measured employing the LP4 algorithm . LDL cholesterol was calculated using the Friedewald equation. GlycA levels were determined as previously described . In short, the GlycA NMR signal comes from the N-acetyl methyl group protons of the N-acetylglucosamine moieties located on the bi-, tri-, and tetra-antennary branches of plasma glyco. The coefficients of variation (CVs) for the GlycA assay range from 1.3% to 2.3%, and the GlycA signal remains stable in EDTA-plasma samples after prolonged storage at −80 °C.
Statistical analyses were performed using IBM SPSS software (version 25.0, IBM Corp, Armonk, NY, USA) and R software (version 4.2.1, Vienna, Austria). A two-sided p -value of less than 0.05 was considered statistically significant. Continuous variables were presented as mean ± standard deviation (SD) for normally distributed data or as medians (interquartile range, IQR) for non-normally distributed data, while categorical variables were reported as frequencies and percentages. The GlycA concentrations were divided into tertiles separately among patients with cirrhosis and LTRs. Baseline characteristics were compared between two groups (cirrhotic patients and LTRs) and across three groups (cirrhotic patients, LTRs, and PREVEND participants) or across tertiles. For normally distributed continuous variables, an independent t -test was used for two-group comparisons and one-way analysis of variance (ANOVA) for three-group comparisons. For non-normally distributed continuous variables, the Mann–Whitney U test was applied for two-group comparisons, while the Kruskal–Wallis test was used for three-group comparisons. Categorical variables between two groups were compared using the chi-square test or Fisher’s exact test, as appropriate, while three-group comparisons were assessed using the chi-square test. Paired data for GlycA and hs-CRP were analyzed using the Wilcoxon signed-rank test. For the propensity score matching analysis, covariates included age, gender, BMI, systolic and diastolic blood pressure (SBP and DBP), current smoking, history of CVD, diabetes, glucose-lowering drugs, lipid-lowering drugs, and antihypertensive medication. A match tolerance of less than or equal to 0.2 was applied to enhance the precision of the matching. The relationship between GlycA and hs-CRP (Ln-transformed) levels among the cirrhotic group, LTRs, and PREVEND participants was assessed using Pearson correlation coefficients, with 95% confidence intervals (CIs). The generalized linear model was conducted to evaluate whether the relationship between GlycA and hs-CRP differs among cirrhotic patients, LTRs, and PREVEND participants. The analysis included both main effects and interaction terms to compare the slopes and intercepts of the regression lines for each group. Univariable and multivariable linear regression analyses were conducted to examine the associations between clinical or laboratory variables and GlycA or hs-CRP levels. Variables with significant associations in univariable analysis ( p < 0.05) were included in the multivariable models. The variables identified as independently associated with GlycA were subsequently included in the Cox regression model to adjust for potential confounding effects. To assess the survival distributions across tertiles of GlycA and hs-CRP levels, Kaplan–Meier curves were generated, and comparisons were made using the log-rank test. Survival time was defined from baseline until the date of the last examination that participants attended, the date of their death, or June 2021 (the final month of follow-up). Univariable and multivariable Cox proportional hazards regression analyses were conducted to explore the associations between GlycA, hs-CRP, and the risk of all-cause mortality. Multivariable Cox models were constructed using the enter method to include potential confounding factors simultaneously. Results were expressed as hazard ratios (HRs) with corresponding 95% confidence intervals (CIs). HRs of GlycA and hs-CRP-associated risk were reported per 1-SD increment. The proportional hazards assumption was tested to ensure that it was not violated. Sensitivity analyses were conducted to exclude participants with potential latent infections, defined by hs-CRP levels exceeding 10 mg/L.
In conclusion, GlycA increases, but hs-CRP decreases after live transplantation. We suggest that both inflammation markers may be associated with all-cause mortality in patients with cirrhosis and LTRs. The association of GlycA with mortality may be at least as strong as that with hs-CRP. These findings underscore the potential of GlycA, an NMR-based inflammation biomarker, for predicting all-cause mortality in cirrhotic patients and LTRs. GlycA may provide additional insights into risk assessment and long-term outcomes, emphasizing its role in identifying high-risk individuals and improving mortality prediction.
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Epidemiological profiles and causes of sudden deaths of various ages in Ethiopia: an autopsy-based study | ff68f7bd-9e2e-4615-bcec-5a40ceee9a97 | 11829148 | Forensic Medicine[mh] | Sudden death is an important global public health issue, especially when it occurs in apparently healthy individuals due to the bearing these losses have on individuals, families, communities and wider society. The definition of sudden death varies according to authorities and conventions. According to the World Health Organization (WHO), death is considered a sudden death (SD) when a nonviolent and unexpected death occurs within 24 hours after the commencement of a fatal illness. The frequency and pattern of sudden death in different parts of the world vary greatly due toSudden unexpected natural death is often the initial clinical appearance of an underlying condition in people who had previously been asymptomatic and seemingly healthy. , Furthermore, since many of these deaths are unwitnessed and unattended by clinicians, the extent of the problem is difficult to determine. In this circumstance, an autopsy is an initial and sole opportunity to determine and document the precise cause of death. , , In Ethiopia, SD is regarded as a type of medicolegal death for which an autopsy is required to determine the cause. Public health authorities, therefore, depend heavily on vital statistics systems with a focus on the cause of death (COD) for formulating their programs, both curative and preventive specialist services. Mortality statistics are an essential component of the nation’s vital statistics system. They are the ingredients to measure population growth and provide a demographic perspective for the planning of development in social services. Cause-specific mortality rates are crucial measures of population health trends. There are several challenges in interpreting epidemiological information in resource-limited settings, including a lack of uniformity and low quality of death certificates as well as the utilization of verbal autopsies. – Thus, vital health information may not reach the national registry, which could prevent the implementation of crucial interventions and prevention measures. Accurate evidence on causes of death is necessary to recognize the general epidemiological profile of diseases in Ethiopia and to support decision-makers in prioritizing the public health agenda. An autopsy is an important source of epidemiological data, as the considerable causes of SD remain hermetic until postmortem examination. Therefore, epidemiological autopsy-based data on sudden death causes are vital to understand the characteristics of the affected population and customize public health programs. To our knowledge, this is the first study paper on SD in Ethiopia and is devoted to documenting the sociodemographic, behavioral, clinical and pathological characteristics of sudden death cases of various ages. This is an observational, prospective, descriptive study that included all sudden unexpected death with a known natural cause of death observed from July 1, 2019, to June 30, 2020 , in the Department of Forensic Medicine and Toxicology of St. Paul’s Hospital and Millennium Medical College (SPHMMC), Addis Ababa, Ethiopia. SPHMMC is a tertiary hospital and medical college governed by a board under the Federal Ministry of Health. SPHMMC in Addis Ababa and Aider Hospital in the Tigray region were the only two centers in the country providing forensic medicine and toxicology services during the study period. The Department of Forensic Medicine and Toxicology, SPHMMC, offers postmortem services to almost all regions of Ethiopia, except cases from the Tigray region served by Aider Hospital. This study included all SD cases (according to the WHO definition) brought for autopsy from July 1, 2019, to June 30, 2020. The WHO defines SD as death that occurs within 24 hours after the disease’s commencement in a person not known to have been diagnosed with a serious disease, accident, or poisoning. In consideration of this definition, two factors were considered: 1) the time of onset of terminal signs or symptoms until death and (2) the expectation of death at the time of occurrence. The definition of the onset of terminal signs or symptoms used in this study was the time when an individual had to change his activity because of the illness. The time of death was defined as the time an individual was pronounced dead. For unwitnessed deaths in which an individual was known to have been alive within 24 hours before the pronounced time of death, they were considered sudden death. The second factor in deciding whether or not the death was sudden was the unexpected nature of the occurrence. The degree of disability reported before death was used as a measure of the expectation of death at the time of the event. Individuals who died but were confined to their homes, health facilities, or other related institutions due to illness for more than 24 hours before death were not considered unexpected deaths and were not included. Principal investigators and five well-trained medical doctors collected the data. Information on the biodata of the cases, clinical data and circumstances of the death was collected from all potential sources, such as police files, medical records, and direct interviews with the eyewitnesses, relatives, and friends of the deceased. The duration of hospital stay before death was extracted from the hospital record, autopsy referral papers, and police requests. Full postmortem examinations were carried out in each instance using the Letulle evisceration procedure, systematically inspecting all cavities, including the cranial, cervical, thoracic, and abdominal cavities. The standard gross examination of the heart was conducted following several key steps. First, the pericardium was checked for any abnormalities and opened to explore the pericardial cavity. Second, the anatomy of the great arteries was checked, with the pulmonary artery opened in situ to identify any emboli, before being transected 3 cm above the respective valves. The pulmonary veins were then transected, and the superior vena cava was transected 2 cm above the junction of the right atrial appendage to preserve the sinus node. The inferior vena cava was transected near the diaphragm. When congenital heart disease or aortic dissection were suspected, the recommended examination procedure was followed. The coronary arteries were serially sectioned every 3-4 mm from their origin to their distal portion to check for any thrombosis or atherosclerosis. The heart chambers were opened and inspected in the direction of blood flow. Finally, the hearts were weighed after dissection and had their chambers washed to remove any blood clots. All organs were dissected, examined and checked for signs of gross pathological changes and violence. Autopsied specimens (whole organs or organ pieces) were fixed in 10% formalin for histopathological examination. Multiple sections with thicknesses of 4-5 mm were taken. The tissues were processed, subjected to paraffin sectioning at a thickness of 4 micrometers and then stained using hematoxylin and eosin staining. In addition, toxicological screening, biochemical tests, and microbiology were performed in selected cases. However, genetic testing was not part of the investigations due to the absence of appropriate facilities in our setting. A structured data collection form was used to obtain all relevant clinical, epidemiological and pathological data. The predesigned data collection form was pretested in 50 cases to maintain data quality, and the necessary amendment was made to the form before the actual data collection. Data entry was performed using Microsoft Excel 2016 and exported to the Statistical Package for Social Science (SPSS window version 26) for analysis. Descriptive summary measures were used to characterize sociodemographic, behavioral, clinical characteristics and circumstances of death. A chi-square test was used to compare categorical variables, and the level of significance was set at a p-value <0.05. Ethics approval was obtained from the SPHMMC Institutional Review Board (Ethical clearance reference no: PM 23/188). The study was conducted as per the Declaration of Helsinki. All information was treated anonymously and confidentially using unique identification codes rather than individual names and identifications. Epidemiological characteristicsClinical characteristics and habits of substance abuse dataCircumstances of sudden deathCauses of sudden unexpected deaths Cardiovascular system diseases (CVS) were the leading causes of SD, accounting for 36.1% of all SD cases. This was followed by respiratory and gastrointestinal system pathologies, accounting for 32.6% and 19.5%, respectively. Central nervous system (CNS) and genitourinary system (GUS) pathologies were the least prevalent causes of death, accounting for 10.6% and 1.2%, respectively. Male cases were affected more than female cases in each system, except in the GUS, in which female cases were prevalent. There was a statistically significant difference between the causes of death by organ system and sex, p<0.004. Comparisons of COD by organ system and sex are summarized in . Ischemic heart disease (IHD) and pneumonia are the most common underlying causes of SD. The underlying COD in each organ system is detailed in . Pneumonia was the most common cause of sudden death in the first, third, and fourth decades of life and the second leading COD after the fifth decade following IHD. Ischemic heart disease was the most common COD in general and in both sexes, was the leading cause after the fifth decade and was the second most common COD in the age group of 21-40 years. summarizes the common causes of SD by age group. The most common COD among CVS was ischemic heart disease, accounting for 69.8% of CVS deaths and 25.2% of all sudden deaths, that is, the leading cause of SD overall and in both sexes. Cardiomyopathy was the second most common COD in the CVS category and caused 5.3% of all sudden deaths . Only 4.4% of cases among these categories of sudden deaths had previously been diagnosed with cardiovascular disease. In the cardiovascular category of sudden death, the modifiable cardiovascular risk factors (CVRFs) identified were diabetes mellitus in 31.7% (65/205) and hypertension in 17.6% (36/205) of the cases. Furthermore, a history of alcohol, khat, and smoking was declared in 45 cases. Chronic alcohol use was the most common substance used and was reported in 38 (18.5%) of 205 cases. This was followed by the use of khat and cigarettes, each of which was reported in 14 cases (6.8%). The most common terminal symptoms reported among CVS category of sudden death were syncope (n=39 cases) and chest pain (n=25 cases). In 61 of 205 cases (29.8%), there were no previous symptoms, and SD was the first manifestation of sudden cardiac death. There was a statistically significant difference between the CVS causes and sex (p=0.019). Among SD cases attributed to CVS causes, 86.7% of SD from ischemic heart diseases, 96.7% of SD from cardiomyopathies, and 71.9% of SD from other CVS causes occurred in males . The most common respiratory pathology was pneumonia in 57.2% of respiratory cases and 18.7% of all sudden deaths, that is, the second most common underlying cause of SD in general and in both sexes. This was followed by tuberculosis in 19.5% of respiratory cases and 6.3% of all sudden deaths . A history of substance abuse was reported in 24.8% (n=46) of 185 cases of sudden death from respiratory pathology. Chronic alcohol use was the most common substance used and was reported in 37 (20%) of 185 cases. This was followed by smokers and chronic khat use, each reported in 13 cases. The most common terminal signs and symptoms reported among respiratory causes of sudden death were dyspnea (n=34 cases, 18.4%) and chest pain (n=20 cases, 10.8%). Gastrointestinal system (GIS) pathologies were the third leading cause of SD, occurring in 97 (19.5%) cases. Liver and pancreatic diseases were the two most common causes of sudden death among GIS pathologies, accounting for 53.2% (n=59 cases) and 37.8% (n=42 cases), respectively . A history of substance use was reported in 46.8% (n= 52) of 111 cases of sudden death from GIS pathology. Chronic alcohol use was the most common substance used and was reported in 45% (n=50/111) of victims, followed by khat and cigarettes in 19.8% and 4.5% of cases, respectively. The most common terminal signs and symptoms reported among the GIS causes of sudden death were abdominal pain (n=24 cases, 21.6%) and vomiting (n=7 cases, 6.3%). CNS pathologies were the fourth most common cause of SD, occurring in 10.6% (n=60 cases) of all sudden deaths. Subarachnoid hemorrhage and spontaneous intraparenchymal hemorrhage were the two common CNS pathologies, which occurred in 48.3% (29/60) and 31.7% (5/22) of cases, respectively . The least common cause of sudden death was related to GUS, which accounted for 1.2% of all sudden deaths. There was a statistically significant difference between the pathologies of GUS and sex, where p=0.48. There was a female preponderance of 71.4% and a mean age of 32.14±18.614 years. Pregnancy-related sudden deaths were the main cause of death in this group, occurring in five cases. Three of them died from obstetric bleeding (two cases of postpartum hemorrhage and one case of antepartum hemorrhage). A postpartum hemorrhage occurred after delivery at home in a rural area. Two cases of SD were due to rupture of ectopic pregnancy in 15- and 22-year-old single women, in which the family did not know the pregnancy status of the deceased. A total of 4,942 medicolegal autopsies were performed during the study period, of which 568 cases were due to SD, accounting for 11.5% (95% CI: 10.6-12.4) of the total autopsied cases. The youngest case was a 1-day-old male newborn, and the oldest was a 98-year-old man, with a mean age of 44.8±17.349 years. Males (n = 482/586) were predominant over females (n = 86/586) at a ratio of 5.6:1. The maximum number of sudden deaths (24.3%) was in the age group of 31-40 years, followed by 41-50 years, which represents 19.6% of all SD cases. SD was less prevalent in extreme age groups, less than 10 years (1.8%) and over 70 years (6.7%). Males outnumbered females in all age groups. The fourth decade was the most predominant age group seen in males and females, accounting for 23.9% and 26.7% of total sex-specific cases, respectively. The number of cases by age and sex is summarized in . More than two-thirds of the cases of SD (70%) belonged to urban areas, whereas 30% of cases were from rural areas. There was a statistically significant difference between the residence of the SD cases and sex (p<0.001). Among the cases of sudden death, 54.7% of females lived in rural areas and 45.3% lived in urban areas. 25.7% of males lived in rural areas and 74.3% lived in urban areas. Most of the cases were from Addis Ababa city, the capital city of Ethiopia, accounting for 59.2% (n=336) of cases, followed by the Oromia region (23.6%), and the remaining 17.2% were from six other regions of the country. summarizes the sociodemographic characteristics of the cases of sudden death. Substance use was reported in approximately 40.1% (n=228) of all SD cases. Chronic alcohol use was reported in 24.3% of cases, followed by khat (Catha edulis) and cigarettes in 9.3% and 6.5%, respectively. Moreover, 10% of the cases use two or more of these substances. There was a statistically significant relationship between substance abuse and sex (p<0.001) and residence (p=0.004). Specifically, substance abuse was reported in 32% of males, in contrast to just 4.7% of females. Among individuals with documented substance use, 77.8% resided in urban areas. A different distribution was observed for chronic alcohol use and sex (p<0.001). Chronic alcohol use was documented in 27.8% of males compared to only 4.7% of females. More than one-third of cases (38%) had a history of prior chronic medical illness, and diabetes mellitus (n=97 cases, 17.1%) was the most common disease. In 5.8% of cases, there is a history of SD in first-degree relatives. There was a statistically significant relationship between the presence of a prior chronic illness and sex (p=0.022). No prior chronic illness was reported in 39.5% of female cases and in 26% of male cases. summarizes the clinical characteristics and habits of substance use and SD in first-degree relatives of SD cases. More than three-quarters of the cases were found dead (68%, n=387/568 cases), followed by cases who died at the time of arrival or after a short period of arrival in health facilities (23.1%, n=131/568 cases). Approximately 6.3% (n=36 /568 cases) died instantaneously. Prodromal signs and symptoms were reported in 178 cases of SD. Of these 178 cases, 70.8% (n=126) sought medical help. The most common terminal signs and symptoms of SD were chest pain (n=45 cases), followed by dyspnea (n=43 cases) and syncope (n=42 cases). Of the 178 cases, 42.1% died within 1 hour of the onset of symptoms, while 36% and 21.9% of the cases died between 1-6 hours and 6-24 hours of the onset of symptoms, respectively. The majority of sudden death incidents occurred at home in 34.9% (n=198) of the cases, followed by public places and health facilities in 30.3% and 23.4%, respectively. This is the first study article on sudden deaths in Ethiopia and is devoted to evaluating the sociodemographic, behavioral, clinical and pathological characteristics of 568 sudden deaths of various ages in Ethiopiavarious genetic and environmental factors. , The present study reveals that sudden unexpected death with a known natural cause of death constitutes approximately 11.5% (95% CI: 10.6-12.4) of all autopsy cases. This finding matches those of other developing countries. This finding is inconsistent with that reported in developed countries. , It is challenging to compare the magnitude of SD in different parts of the globe because it varies mainly as a function of the diversity of prevalent diseases in various nations and due to diverse genetic and environmental factors. , Furthermore, various definitions of SD, inclusion criteria and age groups that were evaluated in the study all contribute to the variation in SD incidence described in different studies. The findings revealed a mean age of 44.8±17.349 years. This is consistent with the studies conducted in Nigeria and Libya. , , The maximum number of sudden death cases (24.3%) was in the age group of 31-40 years, followed by 41-50 years, representing 19.6% of all SD cases. This finding matches various studies. – All of these studies noted that middle age groups of 30 to 50 years are at high risk for SD. This finding appears to reflect behavioral and environmental factors that impact the health of society resulting from urbanization, the shift to a Western lifestyle, and the rapid nutritional transition and sedentary lifestyle with increased substance abuse habits. Of 568 sudden death cases, men (n = 482/586) were predominant over women (n = 86/586), with a ratio of 5.6:1. The observed male predominance aligns with other studies. , , , Several factors may contribute to this gender difference. The known cardioprotective effects of estrogen in premenopausal women, which can lower cardiovascular risk, likely play a role. Beyond biological factors, the higher prevalence of substance use among men, which was also evident in our study, is another potentially contributing psychosocial factor. Furthermore, the relatively high male-to-female ratio observed here can be partially attributed to the higher male-to-female ratio (3.7:1) of autopsy cases within the study period. In our study, the majority (70%) of SD cases were from urban areas, which is consistent with other similar studies. , This is due to the sedentary lifestyle and westernization with the increased smoking and alcohol consumption habits adopted by people in urban areas. In addition, the stress levels of urban and rural life are well known to differ. According to the current study, more than three-quarters of sudden deaths were unwitnessed. This could be because the shorter the survival period, the more likely it would go unnoticed, meaning that the likelihood of death being unwitnessed or unattended is directly correlated with the duration of survival. Our study found that chest pain, dyspnea, and syncope were the most common terminal symptoms reported in 68.6% of SD cases. These are the principal symptoms of cardiovascular and respiratory diseases, which were the two leading causes of SD. The finding of chest pain as the most common prodromal symptom coincided with a study in South Africa. In light of this, we advise emergency medical professionals to pay more attention to people who exhibit these prodromal symptoms. The fact that 86.6% of all sudden deaths occurred outside of a hospital setting shows that the majority of these cases may not have known about their underlying medical problems or may not have been able to pay for the necessary medical care. This shows that most cases of SD are silent without preexisting symptomsTwo additional findings from this study further support this view. The first is that 34% of the SD cases had visits to medical facilities in the weeks before death, followed by deaths at home, which could be an indication that the diagnosis was incorrect, that therapy had failed, or that the deceased or caretakers lacked sufficient financial power to complete further investigation or management. The other reason is the high frequency of SD cases from chronic illnesses diagnosed for the first time at autopsy. The occurrence of SD in males outnumbers females in all systems except GUS. Our findings coincided with other similar studies. , In the current study, men were predominantly affected by cardiovascular causes of sudden natural death, and a significant association was observed between CVS disease and sex. Our findings coincided with other similar studies. , , The fact that men are more frequently exposed to CVS pathologies can be attributed to the fact that estrogen hormone in women acts as a protective factor against most cardiovascular events, explaining the male preponderance that coexists with the high prevalence of CVS causes of sudden death. , Furthermore, it could also be the result of men’s higher rates of substance misuse compared to women, as well as higher levels of financial stress. Cardiovascular and respiratory diseases are the most common causes of SD. These results are consistent with those of various studies conducted in different parts of the world. , , , , , The fact that CVS pathologies are becoming the leading cause of SD could probably be the shift to the Western type of lifestyle that our societies are acquiring. The most common underlying cause of SD is ischemic heart disease. This finding is consistent with those of various studies conducted in different parts of the world. , , , , , Cardiomyopathy was the second most common COD in the cardiovascular system, accounting for 5.3% of all sudden deaths. This agrees with the result achieved in Libya. Cardiomyopathies can be inherited and therefore it is important to screen surviving blood relatives for these conditions. In many series, sudden cardiac death (SCD) is the initial manifestation of the condition in 20%–40% of cases. , In the current study, 29.8% of cases of cardiovascular death had no antecedent symptoms, and SD was the first manifestation of sudden cardiac death in 29.8% of cases, which is consistent with many similar studies. , Approximately 6.3% of all cases (n=36) experienced instantaneous death. Among these, a substantial majority, 88.9% (n=32), were attributed to cardiovascular (CVS) causes, specifically ischemic heart disease and cardiomyopathies. The remaining cases were due to spontaneous intracranial hemorrhage. Because SD is frequently the first sign of the disease, it is impossible to detect high-risk individuals, making the prevention of SCD considerably more challenging. Primary prevention is highly challenging because early identification of subjects in the community who are at a high risk of SCD is impossible owing to the significant percentage of SCD occurring in individuals with no previously known disease. However, sudden cardiac deaths have a relatively high prevalence of CVRFs. The risk of SCD in asymptomatic subjects with CVRFs is higher than that in the general population but lower than that of symptomatic patients with a diagnosed condition. As a result, early identification and management of modifiable CVRFs is one potential strategy to reduce the burden of sudden cardiac deaths in the community. In this study, significantly higher CVRF frequencies were observed in SCD cases, including diabetes mellitus (31.7%), hypertension (17.6%), and substance addiction (22%). Therefore, community education on preventive strategies, early detection, and control of CVRFs together with preventive strategies for substance abuse would be effective for the prevention of SD. Respiratory causes of SD accounted for 32.6% of cases in the current study. Pneumonia was the leading cause of death in the respiratory system, representing 18.7% of sudden death cases, making it the second most common cause of SD. This is closely followed by tuberculosis, comprising 6.3% of all sudden death cases. This is consistent with the results achieved in Libya and South Africa. , Gastrointestinal pathologies were the third leading cause of SD, occurring in 111 (19.5%) cases. Liver and pancreatic pathologies were the two most common causes of sudden death among GIS pathology, accounting for 53.2% (n=59 cases) and 37.8% (n=42 cases), respectively. Hepatic and pancreatic pathologies together contribute to 90.1% of GIS causes of sudden death and are generally the third and fourth underlying causes of death, respectively. This finding is inconsistent with various similar studies showing that CNS pathologies are the third most common cause of SD. , , , , A possible explanation could be the high frequency of substance abuse reported among cases of SD from GIS pathology. A history of substance ab50 (45%) of 111 cases, followed by khat and cigarettes in 19.8% and 4.5% of cases, respectively. Alcohol has a causative association with both liver and pancreatic diseases. , – Additionally, there is a growing body of evidence linking khat to the emergence of both acute and chronic liver disease. – Khat (Catha edulis), a plant that is chewed for its psychedelic effects, is widely used in the eastern and southern parts of Ethiopia, but less frequently in the northern region. Central nervous system pathologies accounted for 10.6% of SD cases in this study. Cerebrovascular accidents were the most common causes (81.7% of all CNS causes), followed by infectious causes (15% of all CNS causes). This is in agreement with a study from Türkiye. The number of male versus female cases in all organ systems was higher except for GUS. Our findings coincided with various studies. , , The reason is that in our study, a high frequency of maternal deaths was recorded. While this study provides valuable insights into the epidemiological data necessary for planning medical services, prevention and control activities, education, and further research, it is important to acknowledge its limitations. Notably, 6.2% of the total autopsy cases remained undetermined during the study period, indicating a need for additional examinations. Although samples were collected for histolopathology (mainly H&E), toxicological screening, biochemical tests and microbiology in selected cases, molecular autopsy facilities were not available in our context. As a result, cases in which the cause of death could not be established due to advanced putrefaction or incomplete ancillary examinations, such as molecular autopsy, were excluded from the study. This exclusion of undetermined cases may potentially affect or underestimate the prevalence estimates. Traditional autopsy techniques, though essential, may not always reveal the cause of death, particularly in cases of sudden unexplained death (SUDs). To overcome this limitation, molecular autopsy should be utilized. Molecular autopsy involves genetic analysis to investigate sudden death, and is particularly useful in cases where traditional autopsy findings are negative or only show non-diagnostic results. It is also instrumental in the diagnosis of inherited conditions, such as cardiomyopathies. SUDs are frequently due to underlying inherited arrhythmogenic cardiac diseases, and molecular autopsy helps identify these causes. Identifying the cause of death using molecular autopsy is vital for medico-legal inquiries and it also guides cascade genetic screening of the victim’s relatives. It is important to note that molecular autopsy aims at identifying the cause of death, which may be particularly relevant for risk prediction in family members, and the management of those at risk. – Studies conducted in resource-limited facilities often share similar limitations, particularly concerning genetic testing. – Considering the future and to mitigate similar limitations, and with the likely expanding role of molecular autopsy, the preservation of DNA samples from all cases is critical to enable future investigations. Additionally, the weight of the deceased was not measured during the postmortem examination due to a malfunctioning body weight scale during the study period, leading to the omission of a key variable: body mass index. Therefore, it is crucial to consider these factors in future research. Despite these limitations, our study stands out as the first nationwide prospective autopsy-based study that comprehensively includes epidemiological, clinical, and pathological characteristics of all sudden unexpected natural deaths over a one-year period. In every case, we gathered information from all available sources and conducted complete postmortem autopsies. Sudden death is an important global public health issue. Cardiovascular, respiratory and gastrointestinal system pathologies were the most common causes. The two main underlying causes of sudden death were ischemic heart disease and pneumonia. Although the causes of SD observed in the current study were comparable to those previously reported elsewhere, the rate of occurrence of certain conditions was different, particularly the higher frequency of liver and pancreatic pathologies. The association of these diseases with chronic alcohol and khat (Catha Edulis) abuse was also documented. Most of the causes of SD in Ethiopia can be prevented and treated. The majorities of sudden deaths are silent without preexisting symptoms and occur outside a hospital setting. Therefore, it is vital to develop public health measures that will help educate the community about the importance of recognizing the manifestation of various clinical conditions and the need to seek immediate clinical help. Furthermore, efforts should be made to make healthcare facilities accessible and affordable with adequate diagnostic and management capacity. Documentation of autopsy-based data could provide important epidemiological information to guide medical services, prevention efforts, and control measures. |
Association Between Positive Results on the Primary Care–Posttraumatic Stress Disorder Screen and Suicide Mortality Among US Veterans | 7676a739-fdbb-4b82-9bc7-d965ddaee964 | 7489804 | Preventive Medicine[mh] | Posttraumatic stress disorder (PTSD) is one of the signature wounds of war among veterans who served in the conflicts in Afghanistan and Iraq. The estimated lifetime prevalence of PTSD among veterans is 11% to 12%, nearly double that of the general adult population in the US, which is 6% to 8%. , , Posttraumatic stress disorder is associated with an increased risk of morbidity , , and mortality. Among veterans, PTSD is associated with increased impairment in psychosocial functioning, decreased health-associated quality of life, and increased mortality. , , , , , , Veterans also have an increased risk of suicide. Suicide prevention is the first clinical priority of the US Department of Veterans Affairs. Posttraumatic stress disorder is associated with increases in suicidal ideation and suicide attempts. , Findings regarding the association between PTSD and suicide mortality among veterans have differed across studies, in part depending on the covariates included in the analyses. In general, analyses without adjustments for mental health comorbidities have reported positive associations between a diagnosis of PTSD and suicide risk, , , , while analyses with adjustments for mental health comorbidities have observed negative associations. , , , , Previous studies have typically relied on PTSD diagnoses reported in medical records; however, patients may experience clinically important PTSD symptoms without receiving a diagnosis, and PTSD symptoms may fluctuate over time. Therefore, it may be informative to evaluate the suicide mortality risk associated with self-reported PTSD symptoms among veterans without an established PTSD diagnosis. Recent studies have identified positive associations between PTSD symptoms and both suicidal ideation and suicidal behaviors , , , , , , ; however, to our knowledge, no study has examined whether veteran-reported PTSD symptoms are associated with the risk of suicide mortality. The US Veterans Affairs/Department of Defense Clinical Practice Guidelines recommend periodic PTSD screening, including annual assessments during the first 5 years after military separation and every 5 years thereafter. The US Veterans Health Administration (VHA) primarily uses the Primary Care–Posttraumatic Stress Disorder Screen (PC-PTSD) for its annual PTSD screening. This screen was based on criteria from the Diagnostic and Statistical Manual of Mental Disorders (Fourth Edition) and was developed to quickly identify patients with PTSD who receive primary care in the VHA heath system. The PC-PTSD includes only 4 items, facilitating its administration during routine care, which most often occurs in primary care settings. To our knowledge, no study has examined the association between PTSD symptoms and suicide mortality. Information regarding suicide mortality risk after the administration of the VHA PC-PTSD may inform veteran suicide prevention activities and provide new information regarding the association between PTSD symptoms and suicide. We hypothesized that positive PC-PTSD results (ie, total scores of 3 or 4) would be associated with suicide mortality. Because PTSD symptoms may fluctuate over time, we further hypothesized that associations between reports of symptoms and suicide mortality risk would decrease over time. Using the VHA Corporate Data Warehouse, we identified 1 837 114 completed PC-PTSD screens in 2014. Data regarding vital status and cause of death were obtained from the joint US Veterans Affairs/Department of Defense Mortality Data Repository. Screens were excluded if data on vital status and cause of death were unavailable or if the recorded date of death was before or on the date of the PC-PTSD. Additional exclusion criteria included having no record of a VHA inpatient or outpatient encounter on the screening date, administration of the PC-PTSD at a facility outside of the 50 US states and the District of Columbia, documented death in Puerto Rico or the Virgin Islands, missing or invalid data on patient age (ie, ages <18 years or >115 years), and nonveteran status as indicated by VHA patient records and Department of Defense personnel records. The analyses were conducted as part of ongoing VHA operations and program evaluation and were approved by the Veterans Affairs Office of Mental Health and Suicide Prevention with a waiver of informed consent. This reportA total of 4 items are included in the PC-PTSD. Each item is a response to the question, “In your life, have you ever had any experience that was so frightening, horrible, or upsetting that”: (1) “in the past month, you have had nightmares about it or thought about it when you did not want to?”; (2) “tried hard not to think about it or went out of your way to avoid situations that reminded you of it?”; (3) “were constantly on guard, watchful, or easily startled?”; and (4) “felt numb or detached from others, activities, or your surroundings?” Patients respond to each item with an answer of yes (considered a positive response) or no (considered a negative response). The PC-PTSD has been reported to have high diagnostic validity in primary care and mental health care settings. , , For the main analysis, we limited the cohort of screens to those administered to veterans without a PTSD diagnosis ( International Classification of Diseases, Ninth Revision, Clinical Modification [ ICD-9-CM ] code 309.81) associated with a VHA inpatient stay or outpatient encounter in the 365 days before each PC-PTSD was administered. These criteria identified 1 693 449 total screens administered to 1 552 581 individual patients. Suicide mortality was measured from 1 day after each PC-PTSD administration date through December 31, 2016. Data on patient mortality and cause of death were obtained from the Mortality Data Repository, and suicide death was defined bys U03, X60 to X84, and Y87.0. Most covariate data were collected from the Corporate Data Warehouse and the VHA National Patient Care Database. Patient age and sex were obtained from the Mortality Data Repository and, if missing, from medical records. Data on suicide attempts were collected from the Veterans Affairs Suicide Prevention Applications Network database as well as medical records. At the patient level, risk factor data collected were age, sex, service-connected disability status for PTSD, marital status, race, and mental health diagnoses. Age was assessed on January 1, 2014. The service-connected disability compensation program provides tax-free monthly benefits to veterans for disabilities that were incurred or aggravated during military service. Service-connected disability status for PTSD was defined as an effective date of disability status that occurred before the date of the patient’s initial PC-PTSD in 2014. The presence of other mental health diagnoses was assessed in the 365 days before and including the initial PC-PTSD administration date in 2014. Specific ICD-9-CM codes for identifying the presence of mental health diagnoses are available in eTable 1 in the . At the screening level, 6 indicator variables were created to categorize the types of care patients received on each PC-PTSD administration date: primary care services through the Primary Care–Mental Health Integration program, primary care services outside of the Primary Care–Mental Health Integration program, specialty mental health care services, other outpatient services, inpatient mental health stay, and other inpatient stay. Variables were created to capture emergency department or inpatient mental health stays as well as the number of emergency department visit days and inpatient mental health bed-days in the 365 days before the screening date. An indicator variable was created for the presence of a documented suicide attempt in the 365 days before and including the screening date when the patient had an ICD-9-CM diagnosis code of E95.0-E95.8 in the medical records or when a suicide attempt was documented in the Suicide Prevention Applications Network database. Indicator variables were also created to record prescription fills for antipsychotic, antidepressant, benzodiazepine, mood stabilizer, and anxiolytic or sedative medications in the 365 days before and including the screening date. Responses for each PC-PTSD were prepared for analysis in 3 ways. First, an indicator variable was created to record whether each PC-PTSD had a positive (total score of 3 or 4) or negative (total score of 0, 1, or 2) result. Second, each of the 4 items was treated as an individual indicator based on whether that item received a positive or negative response. Third, each screen was assigned a score (ranging from 0-4) for the total number of items with positive responses. Statistical AnalysisFor descriptive analyses, frequencies were calculated, both overall and by suicide mortality status, for all variables among 1 552 581 patients. In bivariate analyses, the responses on the patient’s initial PC-PTSD in 2014 were examined by suicide mortality status through December 31, 2016. Fisher exact tests and χ 2 tests were used to assess the differences in proportions, and Kruskal-Wallis tests were used to evaluate differences in means. Suicide rates per 100 000 person-years were calculated at the patient level by multiplying the number of observed suicides by 100 000, then dividing that value by the number of person-years at risk from the day after each patient’s initial PC-PTSD in 2014 until death or December 31, 2016, whichever occurred first. In the proportional hazards regression analyses, each PC-PTSD screen was used as the unit of analysis. The patient’s time at risk began the day after the PC-PTSD was administered and ended at death or on December 31, 2016, whichever occurred first. We used partially conditional proportional hazards regression models and controlled for nonsuicide mortality as a competing risk. By treating each screen as the unit of analysis, the models were conditioned on the baseline covariates (age, sex, PTSD service connection, marital status, race, and mental health diagnoses) but not on the time-varying covariates (PC-PTSD responses, types of care received, and presence of a previous suicide attempt). Five main proportional hazards regression models were used to examine suicide mortality risk by comparing positive (total score of 3 or 4) vs negative (total score of 0-2) PC-PTSD results, with adjustment for varying levels of covariates. An additional set of models were used, in which each item on the PC-PTSD was included as the main effect. Because we hypothesized that the suicide mortality risk among those with positive PC-PTSD results would be higher during the time closer to the screening date, we included an interaction term between the main effect and time in all models. Covariates were included in the models to adjust for potential confounding variables in the association between PC-PTSD responses and suicide mortality. , We adjusted for covariates using a stepped process to better understand the associations between the covariates, the focal indicator, and the outcome. Covariance sandwich estimators were used to adjust for the nested nature of the data, with screens nested within individuals and screens of individual patients nested within VHA facilities. The proportional hazards assumption was evaluated for all models. A supplemental post hoc trend analysis was performed in which PC-PTSD scores were treated as a continuous variable. Three sensitivity analyses were conducted for both sets of models. First, to examine how screening results were associated with the risk of suicide mortality regardless of a previous diagnosis of PTSD, we analyzed all models by using a larger cohort of 1 790 355 patients, in which we retained screens that were associated with a PTSD diagnoses in the previous 365 days. Second, to eliminate overlapping time at risk, we analyzed all models by censoring time at risk at each patient’s subsequent PC-PTSD administration. Third, to assess whether the elimination of repeated PC-PTSD administrations would alter the observed associations, we restricted the analyses to 1 552 581 initial PC-PTSD screens in 2014. None of the sensitivity analyses produced results that substantially differed from those observed in the main analysis. All tests were 2-sided and unpaired, with a significance threshold of α = .05. Data collection and analyses were conducted from November 13, 2018, to June 18, 2019. Data analyses were performed using SAS software, version 9.4 and the SAS Enterprise Guide, version 7.1 (SAS Institute Inc). Among 1 552 581 patients included in the analysis, 73.9% were White, 52.2% were married, 91.1% were male, and 62.5% were 55 years or older . Most patients (92.1%) in the cohort received only 1 PC-PTSD in 2014, and the maximum number of screens received per person was 23. The number of days at risk ranged from 1 to 1095 (ie, 3 years), with a median time at risk of 904 days (interquartile range, 809-997 days) for all screens. A total of 1423 patients (0.1%) died by suicide within the follow-up period. The unadjusted suicide mortality rate was 37.6 deaths per 100 000 person-years. A total of 46 759 PC-PTSD screens were excluded from the analysis. Excluded screens were less likely to have had positive results compared with included screens (9.1% vs 13.8%, respectively; χ 2 P < .001). Among those excluded, 42 948 screens were able to be linked to a patient’s vital status. An analysis of those patients indicated that they were more likely to die by suicide through December 31, 2016, compared with patients whose screens were included (0.3% vs 0.1%, respectively; P < .001). Among 166 685 patients with positive initial PC-PTSD results in 2014, 88.5% were male, 64.7% were White, and 34.8% were 55 years or older. Among 1 385 896 patients with negative initial PC-PTSD results, 91.5% were male, 75.1% were White, and 65.9% were 55 years or older. In the bivariate analysis, among patients with positive initial PC-PTSD results, 0.13% died by suicide compared with 0.09% of patients with negative initial PC-PTSD results ( P < .001). In the analysis unadjusted for covariates, a positive PC-PTSD result was associated with a 90% increase in the risk of suicide mortality at 1 day after screening (hazard ratio [HR], 1.90; 95% CI, 1.44-2.52) compared with a negative result (model 1 in ). This risk decreased throughout the follow-up period (β for interaction = −0.0006; P = .04), which was up to 1095 days. The unadjusted suicide-specific survival probabilities are shown in the . In the analysis adjusted for all covariates, a positive PC-PTSD result was associated with a 58% increase in the risk of suicide mortality at 1 day after screening (HR, 1.58; 95% CI, 1.19-2.10), and this risk decreased over time (β for interaction = −0.0006; P = .03) (model 5 in ). At 30 days after screening, the HR for a positive PC-PTSD result was 1.55 (95% CI, 1.18- 2.04). At 365 days after screening, the HR was 1.26 (95% CI, 1.07-1.48), which represented a 26% increase. The HR at 475 days after screening was nonsignificant (HR, 1.17; 95% CI, 0.99-1.38). Covariate results are available in eTable 2 in the . In the unadjusted analysis examining each PC-PTSD item separately, a positive (yes) response to item 4 (“felt numb or detached from others, activities, or your surroundings”) was associated with a 117% increase in the risk of suicide mortality at 1 day after screening (HR, 2.17; 95% CI, 1.61-2.92), and this risk decreased over time (β for interaction = −0.0007; P = .009) (model 1 in ). In the analysis adjusted for all covariates, a positive response to item 4 was associated with a 70% increase in the risk of suicide mortality at 1 day after screening (HR, 1.70; 95% CI, 1.27-2.28), and this risk also decreased over time (β for interaction = −0.0007; P = .007) (model 5 in ). At 30 days after administration of the PC-PTSD, the HR for suicide mortality associated with a positive response to item 4 was 1.67 (95% CI, 1.26-2.21). At 365 days after screening, the HR was 1.31 (95% CI, 1.07-1.59), and the HR at 460 days after screening was nonsignificant (HR, 1.22; 95% CI, 0.99-1.48). Covariate results are available in eTable 3 in the . A post hoc test for trend revealed a 14% increase in suicide mortality risk for each unit increase in the PC-PTSD score (baseline HR, 1.14; 95% CI, 1.05-1.23; β for interaction = −0.0002; P = .01) (model 5 in ). Among patients who received PC-PTSD screens through the VHA health system in 2014, positive PC-PTSD results were associated with an increased risk of suicide mortality through 1 year after screening. We observed a significant dose-response pattern in the association between PC-PTSD score and suicide mortality, with greater risk observed as the number of positive responses to PC-PTSD items increased. We found a positive association between PTSD symptoms and suicide mortality even in analyses that were controlled for several mental health diagnoses. This pattern differed from that of previous studies examining PTSD diagnoses and suicide mortality. , , , , Our findings indicate that veterans who receive positive results on the PC-PTSD have an increased risk of suicide mortality. These findings may inform treatment decisions and support the need for continued improvement of suicide risk assessment. We note that our sample consisted of patients who did not have a formal diagnosis of PTSD yet remained at an increased risk of suicide mortality. A PTSD diagnosis requires the presence of symptoms across 4 domains: reexperiencing, avoidance, negative alterations in cognition and mood, and hyperarousal. These symptoms must persist for more than 1 month. However, the presence of subthreshold PTSD symptoms may indicate suicide risk regardless of whether a patient qualifies for a formal diagnosis. In the absence of a PTSD diagnosis, a positive PC-PTSD result may indicate subthreshold symptoms that suggest a patient has an increased risk of suicide mortality. Patients with a positive PC-PTSD result may also have met the full diagnostic criteria for PTSD, which had not been detected until the time of screening. As a consequence, in lieu of waiting for a formal diagnosis, it may be important to consider initiating suicide risk assessment strategies for patients with positive responses on the PC-PTSD. Another reason to use and incorporate the PC-PTSD into suicide risk assessment strategies is that, unlike a medical records diagnosis, the PC-PTSD asks a patient about PTSD symptoms experienced within the past month. The symptom severity of PTSD may fluctuate daily, and this fluctuation has been linked to adverse health outcomes. Although further research is warranted, findings from the present analysis suggest that the PC-PTSD might be a better indicator of immediate suicide mortality risk than a static diagnosis obtained from the medical record. Future research is needed to examine differences in suicide mortality risk among those who receive a formal PTSD diagnosis after administration of the PC-PTSD. Additional risk was associated with a positive response to item 4 (“felt numb or detached from others, activities, or your surroundings”). This symptom has been associated with suicidal ideation in previous research. , , , , A positive response to item 4 may indicate feelings of social isolation or thwarted belongingness, which may be one of the most important factors in suicidal desire, as described by the interpersonal theory of suicide. , This finding highlights the need to improve social support systems and encourage engagement in meaningful activities, which may not only reduce PTSD symptoms but potentially help to mitigate the risk of suicide. In 2018, the VHA implemented a new strategy for risk assessment that included the addition of 1 measure to the PC-PTSD. This measure, item 9 from the Patient Health Questionnaire, asks patients about suicidal ideation. Positive responses to that item have been associated with an increase in suicide risk. , The VHA requires additional risk evaluation for patients with positive responses to item 9. The findings of the present study raise the possibility that positive PC-PTSD results may also be associated with greater suicide mortality risk. As more current mortality data become available, future analyses will assess risk using the expanded instrument to evaluate this issue and inform ongoing VHA health system suicide prevention activities. Limitations This study has several study limitations. We examined PC-PTSD data available in VHA medical records, and these data may not be generalizable to other populations. Although the sample included all screens coded in the administrative data for 2014, we may not have captured every PC-PTSD administered within the VHA health care system in 2014. We noted in the Results section that patients with excluded screens had an increased risk of suicide mortality. Excluding their screens may have inflated the study results, as excluded screens were less likely to have had positive overall results compared with included screens. In addition, we did not assess depressive symptoms, which may be associated with suicide mortality risk, at the time of PC-PTSD administration. The PC-PTSD has been updated based on the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition ; this update, the PC-PTSD-5, has been used in the VHA health care system since 2018. Future research is needed to examine the associations between PC-PTSD-5 results and suicide among patients receiving care in the VHA system. o our knowledge, this is the first study to examine the association between positive PC-PTSD results and suicide mortality. Positive PC-PTSD results, and specifically positive responses on items that describe feeling numb or detached, were associated with an increase in the risk of suicide mortality, which decreased over time. The PC-PTSD may identify distinct risks that are not identified by other assessment procedures used in the VHA or other health systems. |
Assessing the reliability of pediatric emergency medicine billing code assignment for future consideration as a proxy workload measure | 9f33909e-6c0b-47a3-ba4f-edc04f0e1bbb | 10456198 | Pediatrics[mh] | Crowding is a common problem in pediatric emergency departments (PEDs) and can negatively impact patient health outcomes and clinicians’ wellness . Chan et al. attributes PED crowding in part to inefficiencies in the patient flow—namely, the input, throughput, and output factors . The input and output factors, defined as the number of incoming patients and disposition respectively, are generally not under the control of the PED. However, the rate at which patients are treated, known as the throughput, can be improved by optimizing the allocation of resources such as space and staffing assignments . This can be achieved using a proxy measure to quantify PED physician workload, allowing for prediction of resource needs to guide allocation and ensure efficient PED throughput. To date, there has been two proposed measures to estimate PED physician workload; however, neither are validated for workload estimation. The first is the time needed to treat, as measured by the direct interaction time spent between the PED physician and the patient . However, workload is determined by a multitude of different factors in addition to the time needed to treat, including mental demand, physical demand, and psychological stress . Therefore, time needed to treat by itself cannot adequately represent PED physician workload. Furthermore, it is generally not a conventionally collected variable in the PED and is labour intensive to record, making it largely unavailable for academic and administrative purposes. The second measure, sometimes perceived as a surrogate for physician workload is the Pediatric Canadian Triage and Acuity Score (PaedCTAS). This is a triaging tool that evaluates the urgency of the patient’s needs based on their clinical presentation to prioritize access to care in the PED . While the PaedCTAS has been shown to correlate with PED disposition , it was not designed to measure workload, nor has it been evaluated for such purposes. Of note, there is evidence to suggest that using the CTAS (adult equivalent of PaedCTAS) alone is not sufficient for determining physician workload in the general emergency department (ED) setting given the large variability in their workload measure at each triage level . This brings into question the validity of using PaedCTAS, a derivative of the original CTAS triaging tool, to be used as a measure of physician workload. To address the current lack of PED physician workload proxy measure, we propose evaluating billing codes, which are assigned by physicians for compensation either for direct remuneration or shadow estimation of workload and administrative purposes, after each patient encounter based on their impression of the amount of work required to treat the patient. Throughout Canada, many EDs use either a 2 or 3-level billing code system, with greater levels indicating more complexity and work required to manage the patient encounter; some systems also include modifiers which account for other factors such as time of the day, patient age, and procedures performed . With the 3-level system, level 1 is assigned for treatment involving a single organ system or a simple condition, level 2 for conditions which necessitate treatment of at least 2 organ systems with a need for reassessment during the visit, and level 3 for complex conditions requiring prolonged observation and therapy with multiple assessments . In British Columbia (BC) alone, billing code data is used to estimate workload in the fee-for-service setting to allocate approximately $75 million of funding to emergency physicians . Given that billing codes are readily reported and the reliance on billing codes to measure physician workload for remuneration purposes, this variable holds potential to be a proxy measure of PED workload. To assess if physician assigned billing codes can approximate physician workload, we must evaluate the degree of reliability in which PED physicians are assigning these billing codes. Inter-rater agreement of billing codes has been evaluated in other medical specialties and reliability has been found to vary between them . In this study, we aim to assess how reliably PED physicians bill when compared to a billing expert who is also the provincial auditor. In addition, we aim to identify which factors are associated with inter-rater reliability. Study objective and designStudy setting and populationOutcome measuresStudy procedureAnalysis approach We report descriptive statistics to summarize our study sample, using proportions with 95% CI as appropriate. The percentage agreement and Gwet’s AC 2 statistics were used as the measure of reliability in the PED physicians’ billing practices. The AC 2 statistic was chosen for its resiliency against the effects of trait prevalence, where high chance agreement can paradoxically result in low chance-corrected agreement despite relatively higher percentage agreement . The Landis and Koch criterion was used to interpret the AC 2 values which categorizes the chance-corrected agreement statistics as follows: 0–0.20 slight agreement, 0.21–0.40 fair, 0.41–0.60 moderate, 0.61–0.80 substantial, and >0.80 excellent agreement . AC 2 was calculated with linear weighting. We completed univariate logistic regression models to determine the impact of covariates of interest on inter-rater reliability, then adjusted potential confounders. Analyses were performed using R statistical software. Given that there are two raters (PED physicians and the billing auditor) and three categories (billing codes 1, 2, 3), to estimate AC2 within a margin of 0.15 with 95% confidence, a sample size of 90 was required . We added a margin to ensure that we obtain 150 chart visits that meet all our eligibility criteria and requested 300 randomly selected charts meeting specified distribution over time and acuity from the PHSA DARE Office. Upon receipt, we used the Microsoft Excel’s random number generator function and reviewed charts in a randomized sequence to review for eligibility and extract data until the sample size of 150 was met. We conducted a retrospective cross-sectional study at BC Children’s Hospital (BCCH) ED to evaluate the reliability of billing codes assigned by PED physicians compared to the billing code assigned by a billing auditor, who is one of the listed authors of the research group (G.M.) and does not work at the BCCH. The billing auditor selected is an emergency physician and the Chair for the Fee-For-Service Committee within the Section of Emergency Medicine at Doctors of BC, the association representing physicians in BC. Given the billing auditor’s clinical and administrative expertise in emergency medicine, their interpretation of billing code was used as the criterion standard. The primary objective for this study was to evaluate how consistently billing codes are assigned by determining the inter-rater reliability between PED physician assigned billing codes and billing auditor assigned billing codes. Our secondary objective was to identify visit characteristics associated with inter-rater reliability. BCCH ED is a quaternary care referral centre located in Vancouver, BC with approximately 50,000 annual visits . We collected data from a random sample of visits from children aged up to 18 years who visited the BCCH ED between January 1 st , 2018 to December 31 st , 2018 inclusive, provided that the patient did not leave without being seen by a physician and that the physician assigned a billing code to their visit. We used health records provided by the Provincial Health Service Authority Data Analytics, Reporting and Evaluation (PHSA DARE) Office. A timeframe before the COVID-19 pandemic was studied to ensure physician billing practices were unaltered by pandemic precautions such as extra PPE and disease screening. The sample of visits was evenly distributed between months of the year and with representation of all 5 levels of the PaedCTAS scale, with propensity for PaedCTAS 3 and 4 as they generally make up the majority of all PED visits . While our PED is staffed with pediatric emergency medicine physicians, general emergency physicians, and nurse practitioners, our study only included visits which were managed or supervised (when a trainee is involved) by a pediatric or general emergency medicine physician, as other care providers do not assign billing codes. Physicians at BCCH ED are paid on an alternate payment plan and therefore utilize the shadow billing system, whereby billing codes are assigned not for remuneration, but for both individual physician performance monitoring and group contract negotiation. Ethics approval was obtained from the University of British Columbia and BC Women and Children’s Hospital Research Ethics Board and the requirement for informed consent was waived by the two ethical governing bodies. The inter-rater reliability between PED physicians and the billing auditor was evaluated using percentage agreement and Gwet’s AC 2 with 95% confidence intervals (CI) as our primary outcome measure. As the secondary outcome measure, we calculated the percentage agreement and AC 2 values stratified by visit characteristics including triage categories (PaedCATS1-5), patient age (<1y, 1-5y, >5y), whether clinical trainees were involved, time of disposition (day 0800-1800h, evening 1800-2300h, and night 2300-0800h), and disposition (discharged vs. admitted). From the chart review, we extracted the billing code assigned by the PED physician and the clinical variables needed for the billing auditor to assign a billing code. Clinical variable selection was informed by consultations with clinicians and published literature around the subject of physician workload. These variables include those which were found to be strong predictors for workload intensity such as the PaedCTAS score, presentations or comorbidities related to mental health, requirement for ambulance, laboratory and imaging ordered, number of subspecialty consultations, procedures performed, need for sedation, trainee involvement, language barrier, disposition, and length of stay . As well, information which can inform the billing auditor of the clinical context were also collected, such as the patient demographic, chief complaint, the history of presenting illness, physical exam findings, vital signs, and any other progress notes or text relevant to the patient visit. The clinical variables were collected by two trained research students onto the Research Electronic Data Capture platform, a BCCH Research Institute licensed data capture software. The authors had access to patient identifiers, such as the personal healthcare number, during the data extraction which were not collected. To ensure inter-extractor reliability between the students, data extraction training was carried out by a PED physician. Both students separately extracted the data from 15 charts (10% of total sample size) and compared their output for any discrepancies, which were resolved by consensus. This process was repeated until the extracted data between the two students matched for all 15 charts at which point the remaining charts were divided and the data was extracted by each student. In total, 30 charts were extracted in tandem. The data collection was conducted between August to October of 2021. Following data collection, the billing auditor was given the extracted clinical data to assign a billing code. The billing auditor was blinded to PED physicians’ billing codes. requested 300 randomized patient records from the PHSA DARE Office, and reached the sample size requirement of 150 after reviewing 187 charts . The distribution of subgroups across our sample is outlined in . Overall, the percent agreement between PED physician and billing auditor was 68.7%. There was substantial inter-rater reliability (AC2: 0.72 95% CI: 0.64, 0.8). Among the 47 (31.3%) instances where the PED physician and the billing auditor disagreed, the PED physician assigned a lower billing code than the billing auditor 27 times (18%). shows the inter-rater reliability indices for the overall sample size and stratified by visit characteristics. The inter-rater reliability is highest in PaedCTAS 3 (AC 2 : 0.84 95% CI: 0.6, 0.9), age <1y (AC 2 : 0.81 95% CI: 0.7, 0.95), and clinical trainee involvement (AC 2 : 0.79 95% CI: 0.7, 0.9) subgroups. Other subgroups display wide and overlapping CIs and no pattern of changes in the inter-rater reliability index. shows the adjusted and unadjusted regressions exploring the association between individual visit characteristics and inter-rater reliability. After controlling for all other subgroups in the adjusted model, clinical trainee involvement is the only subgroup showing significant association with increased billing code assignment reliability (adjusted OR: 2.2 95% CI: 1.02, 4.9), when compared to visits managed only by the staff PED physician. InterpretationLimitationsOur study found substantial inter-rater reliability in billing code assignment between PED physicians and the billing auditor, which suggests billing codes are accurately assigned. This is an important step in establishing the potential for billing codes to serve as a proxy measure of PED workload. While several subgroups showed association with higher inter-rater reliability, only clinical trainee involvement was found to be associated with significantly higher inter-rater reliability, and this significance persisted when controlling for PaedCTAS, patient age, time of day, and disposition. Several studies evaluating billing practices showed that the amount of experience and time allocated to teaching physicians about billing is associated with increased billing accuracy, such that staff and senior residents tend to have greater levels of comfort and knowledge in assigning billing codes compared to junior residents . These findings are rather intuitive, as more exposure and education regarding a certain topic understandably increases one’s competency in practice. Therefore, given that billing codes in BCCH ED are only assigned by staff physicians, our results finding high inter-rater reliability with the billing auditor is expected. A systematic review analyzing current billing practices to recommend methods of improving pediatric billing accuracy supports this notion, stating that more billing education is a key component to improved accuracy . Other studies that evaluate billing practices, which yielded findings of lower billing accuracy, include residents or recent residency graduates to assess their quality of education, readiness, and the financial impact of inaccurate billing, rather than assessing billing reliability by experienced staff . Our results also show significantly increased odds of higher inter-rater agreement when clinical trainees are involved, which may be explained by a few factors. First, PED clinical trainees’ documentation has been reported to be more complete when compared to staff physicians which may have allowed the billing auditor to have a better context and more accuracy in assigning their billing code, increasing the probability of agreement. Second, clinical trainee education and participation is at times intentionally set aside to prioritize the efficiency of patient flow when the ED capacity is stressed . In these cases, it may be that trainees are more likely be involved in simpler cases which require less interpretation to code. This appears to be reflected in our samples as high acuity cases, which are more likely to be complex, involve fewer trainees than low acuity cases. We acknowledge that the immense complexity of estimating PED workload cannot be entirely addressed using a 3-level billing system. However, until a more comprehensive PED workload measure is developed and validated, it may be the most appropriate and accessible variable for physician workload estimation given the following reasons. First, billing codes are by design meant to estimate the complexity of clinical decision making and treatment, which is demonstrated in their utility as the variable used to allocate millions of dollars to compensate fee-for-service physicians. Second, the 3-level billing code system is widely used in Canada, as it is implemented in BC, Ontario, Prince Edward Island, and the Northwest Territories . Furthermore, compared to the 2-level system used in other provinces such as Quebec, Newfoundland and Labrador, Saskatchewan, and Manitoba , the 3-level system may offer better stratification in estimating the PED workload. Third, a 3-level billing system can be simple to learn and assign in comparison to other existing billing systems, which may be contributing to its high reliability in use by PED physicians. More complex billing systems exist in other specialties which is based off a diverse set of diagnostic or procedural work, such as the International Classification of Diseases or Current Procedural Terminology in the United States, or provincial payment schedules in Canada . These contain thousands of billing codes plus modifiers, which are constantly changing and can often be challenging for physicians to use . Our study results should be interpreted within its limitations. First, the billing auditor’s billing code assignment depends on the quality of the physician’s documentation. Within our sample, the billing auditor flagged seven of the 150 patient records, indicating that there is poor documentation. In two of the seven flagged records, the alternative billing code they would have assigned, had the documentation contained the required details, matched with the physician’s billing code assignment. Therefore, it is possible that improving physician documentation will likely increase the inter-rater reliability, and that our reported level of agreement, based on retrospective documentation, may be conservative. Secondly, further research with additional sample sizes in various visit characteristics is needed to explore the potential association between them and PED physician billing code reliability. Our study does not assess whether the association found between trainee involvement and improved billing code accuracy is intrinsic to the trainee’s charting or if the association can be explained by other variables. Finally, we used shadow billing codes from PED physicians and their compensation is not dependent on billing pattern. Without a direct financial incentive, concerns may arise about the accuracy of shadow billing data . However, a study in Alberta showed that shadow billing does not affect the accuracy at which they are submitted by specialists including pediatricians in urban, acute care hospitals compared to fee-for-service billing . This suggests that the use of shadow billing data in our study is unlikely to affect the validity of our results. In this study, we showed that 3-level billing codes are accurately assigned by PED physicians. This provides a positive first step in the validation of billing codes as a proxy measure of PED workload. With a validated proxy measure, opportunities exist for better optimization of PED resource allocation via workload prediction, which can ultimately improve the throughput. |
A low cost surrogate eye model for corneal foreign body removal | f910b4fa-a9e7-4acb-9608-4ba4e4e88cb5 | 7006147 | Ophthalmology[mh] | Patients with a corneal foreign body (FB) form an important group of people presenting to the Emergency Department (ED) with ocular complaints . This makes it important for doctors working in the ED to be familiar with the procedure of corneal FB removal. ED staffing with junior doctors is a common occurrence worldwide , resulting in this group of doctors seeing many of these patients. Yet, most of them have little to no experience in dealing with corneal FBs. Throughout the past decades, approximately 70% of junior doctors have a lack of confidence in dealing with eye emergencies, both locally and internationally . Additionally, undergraduate training in ophthalmological conditions and emergencies are inadequate and widely variable, further compounding this problem . It is imperative for junior ED doctors to be proficient in the evaluation and management of corneal FBs since errors can potentially cause adverse sight-threatening complications such as corneal perforation [ – ]. However, the removal of corneal FBs is not an ideal procedure to teach on an actual patient, as it can be intimidating for both the patient and doctor. We were unable to find commercially available eye models for educational purposes, so we created a safe and effective model to teach the procedure of corneal FBs removal. Sited in a tertiary academic institution, our ED has an annual census of approximately 110,000 patients, ranging from Patient Acuity Category Status (PACS) of 1 to 3, where a patient with PACS 1 status requires immediate medical attention and a patient with PACS 3 status is ambulant, with mild to moderate acute symptoms . Among the attendances, the number of patients presenting with corneal FBs averages about 250 per year. Our ED is primarily staffed by junior doctors who are at least postgraduate year (PGY) 2 and above. On-site supervision by board-certified emergency physicians are available round-the-clock, and a fully equipped consult room with a slit-lamp is dedicated for use on patients with ophthalmological complaints. There are three medical schools in Singapore, two of which provide undergraduate medical education whilst the other provides postgraduate studies. Of the 3 medical schools, the National University of Singapore (NUS) Yong Loo Lin School of Medicine produces the most number of graduates who join the public hospitals yearly . The ophthalmology curriculum in NUS only includes an hour-long didactic lecture, bedside tutorials, and operating theatre observation. There is a practical demonstration to introduce students to skills such as slit-lamp examination. However, there are no hands-on sessions in the curriculum for training of corneal FB removal . Here, we describe our method of creating a cheap and easy eye model that can be used for training corneal FB removal. The primary objective of this study was to evaluate the effectiveness of this eye model in improving the knowledge and confidence of our junior doctors in the removal of corneal FBs.
We conducted a prospective study between April and November 2016, using our novel, low-cost eye model, among postgraduate learners in the ED of a Singapore hospital. Our hospital is a tertiary academic medical institution with a structured Emergency Medicine residency-training program accredited by the Accreditation Council for Graduate Medical Education International. Ethics approval for waiver of consent was obtained from the National University of Singapore’s (NUS) Institutional Review Board (NUS-IRB reference code: NUS-IRB Review B-16-058). Model construction Training structure Pre- and post- training evaluation Statistical analysis To make our model (see Additional file 1: Video S1), the following materials were used: (1) agar powder, (2) water, (3) table tennis ball and (4) pencil lead shavings (Table ). First, the table tennis ball was cut into half to simulate the hemispherical shape of the eye. Next, pencil lead shavings were scattered into the base of the moulds to simulate corneal FBs. After mixing 3 g of agar powder with 230mls of water, the water was brought to a boil and left until the agar powder completely dissolved. This solution was then poured into the pre-prepared moulds and refrigerated until firm, as seen in Fig. a. If the lead shavings floated upwards after pouring in the solution, they were pushed to the bottom with a pair of tweezers to ensure correct positioning. Excluding the time required to refrigerate, the entire process can be completed within 5 min. Once firm, the agar model was unmoulded. These were made in large batches and stored in a fridge until ready for use. Just before each session, the model was glued onto a box (Fig. b), which was then secured with tape onto the chin rest of the slit lamp as seen in Fig. c. Figure d shows the eye model when the slit lamp beam is cast upon it. Additional file 1: Video of eye model construction..
Junior doctors were divided into groups of 3 and assigned to an ED senior resident for a 2-h hands-on session. The ED senior resident conducting the training sessions would have completed the intermediate exams in Emergency Medicine (either the Membership of the Royal College of Emergency Medicine [United Kingdom]) or the Master of Medicine in Emergency Medicine [Singapore]) and completed at least 2 weeks of clinical rotation in Ophthalmology. The training curriculum comprised of 2 parts. Every participant was required to complete a set of pre-reading materials prior to attending the tutorial. Basic slit-lamp use was then taught together with the technique of FBs removal using a 27-gauge needle. A standard teaching template was provided for each educator’s adherence to ensure every session’s homogeneity. During the teaching session, a set of pre-prepared slides was also used to augment the learning experience.
The junior doctors completed a self-assessed questionnaire (Table ) before and after the session for confidence and knowledge in corneal FBs removal, and in effectiveness of the eye model. The questionnaire was scored on a 7-point Likert scale.
Results were analyzed using Stata 14 (StataCorp LP, College Station, TX). Proportions were expressed in percentages and non-parametric variables were analyzed using the Mann Whitney U test.
Demographics Confidence and knowledge Effectiveness of eye model Using the eye model, the ease of visualization of corneal FB was excellent with a median score of 6 (IQR 5 to 7). The study participants also felt that the model was very effective in teaching the skill of removing corneal FB (median score of 6, IQR 5 to 6) and very realistic in simulating a corneal FB (median score of 5, IQR 4 to 6). Overall, the effectiveness of the eye model in teaching FB removal scored a median of 6 (IQR 5 to 7). Among the participants who had prior experience in removing corneal FBs, they felt that the eye model corresponded relatively well to the real eye in terms of texture (median score 5, IQR 4.5 to 6). Figure illustrates the overall impressions of our participants on our eye model. Participants were asked to omit answering how well the model corresponds to a real eye texture or in simulating FBs if they have not had any prior experience in removing corneal FBs.
A total of 73 junior doctors participated in this study between April and November 2016. Their median age was 26 years old (interquartile range [IQR] 25 to 29 years old), with an overall median PGY of training at 2 years (IQR 2 to 4 years). Table outlines the demographics of all our participants.
Prior to the training session, both the participants’ knowledge in slit-lamp use and corneal FBs removal scored a median of 2 (IQR 1 to 3). After training, both indicators improved to a median score of 4.5 (IQR 4 to 5.5) and 5 (IQR 5 to 6) respectively. Their confidence in slit-lamp use and corneal FBs removal improved from a median of 1 (IQR 1 to 2.5) and 2 (IQR 1 to 2) to a median of 5 (IQR 4 to 5) and 5 (IQR 4 to 6), respectively (all p values < 0.001). Their confidence in assessing the depth of corneal FBs also improved significantly from 1 (IQR 1 to 2) pre-training to 5 (IQR 4 to 5, p < 0.001) post-training. Figure illustrates our participants’ questionnaire responses.
With the use of simple, low-cost and easily available materials, we successfully constructed an eye model that is effective in improving junior doctors’ confidence in removing corneal FBs. It has widespread acceptance amongst the study participants in terms of realism and resemblance to real corneal texture. There have been several described attempts at creating a simulation model to teach this procedure. In 1995, Austin et al. described the use of glass spheres with a film of paraffin, with embedded pieces of metal to simulate corneal FBs. Participants reported improved comfort and skill in removing them . In 2015, Cheng et al. developed a model using unused but expired microbiology agar plates with gravel simulating corneal FBs . In 2016, Gallagher et al. compared different simulation models, using a variety of different materials. In their first model, they used a cardboard box with ink simulating the corneal FBs. In their second model, polyvinyl resin and gelatin were used to create the eyeball, and ground pepper was used to simulate corneal FBs. Their third model made use of a glass sphere and wax to simulate the eyeball, and melted crayon was used as the corneal FB. Their final model was an agar plate simulating the eye, with peppercorn as corneal FBs . More recently in 2017, a task-trainer using ballistics gel, silicone, paint thinner, baby oil, petroleum jelly and cornstarch was created for under USD$75. This task-trainer also included the use of a Styrofoam mannequin head and reported a time of approximately 90 min to complete . There have even been literature describing the use of bovine eyes as a way to teach this procedure . It is thus evident that teaching the removal of corneal FBs is a procedure that can be taught via simulation. Having looked at all the available literature at making a simulation eye model, we feel that our low-cost, low-tech model has an advantage over the others, for the following reasons: [1] our model approximates the consistency of a human cornea well, [2] its hemispherical shape simulates that of the eye ball and allows users to appreciate depth of the corneal FBs, and [3] it is quick, easy, and cheap (under USD$1) to re-create and particularly suited for an environment with tight resources. We have established that the majority of our junior doctors lack experience in dealing with eye emergencies. This problem is not unique to us . Before the commencement of this training curriculum in 2016, teaching of corneal FBs removal to junior doctors was largely opportunistic in our institution. On a regular shift, junior doctors manage patients independently and would consult a senior doctor if they encounter problems. If the senior doctor was occupied with other patients, they may not have enough time to teach the procedure and these patients are then referred to the on-call ophthalmology service for removal of the FBs. Learning opportunity lost aside, this potentially lengthens the throughput time of the patient in the ED, contributing to the perennial issues of overcrowding. Increasingly, we are also seeing patients who understandably, do not want to be “practiced” upon, and frequently ask for an experienced physician to perform the procedure for him or her, making it even more difficult to teach a young doctor this crucial procedure. Using simulation in medical education is now widespread and this phenomenon is in part, contributed by changes in patients’ healthcare expectations, as well as changes in our academic environment, with renewed emphasis on patient safety, making it more difficult to “practice” directly on an actual patient [ – ]. The use of simulation bypasses many of the issues that arise from the traditional “see one, do one, teach one” method: they are readily available at any time, and allow trainees to practice their skills in an environment free of risk . Patient safety is an important element in our daily practice as emergency physicians. This ties in closely with important ethical issues about the appropriateness of “using” actual patients as training resources. Teaching the removal of corneal FBs using our surrogate eye model circumvents such ethical issues and improves patient safety. It is safer for patients and allows doctors to be confident with their skills before performing the procedure on an actual patient. Apart from teaching, educators can also use simulation as a means of evaluating competency in a procedure . Immediate feedback and guidance can also be given during each session without any threat to patient safety . The educational experience is now learner-centered, instead of being patient-centered, as would be appropriate in real-live clinical situations . Simulation based teaching has been shown to be more effective than didactic teaching alone for various subjects and we strongly believe in the effectiveness of our model. Using a simulation model provides useful practice of a procedural skill requiring eye-hand coordination prior to application of these skills in real-life clinical practice. Limitations We acknowledge that the fidelity of simulation training is never completely identical to the real-life patient and that there is no validation on how well agar compares to cornea. For example, it is difficult to simulate a perforated globe using our agar model if the participant makes an error during the removal of the corneal FBs. The real eye is also mobile and the element of blinking in a real patient is difficult to simulate in our model. We have tried to circumvent this problem by showing participants videos during the session and explaining how this would appear under the slit lamp using our simulated model, which we felt was much better appreciated than just reading on their own. Additionally, we have an instructor present to guide and correct each learner, making the teaching individualized and targeted at resolving each learner’s doubts. Given how cheap and readily available agar is, we believe this is still a good initial training tool to provide to junior clinicians. The transparency of our model can be improved, as it is sometimes difficult to assess the depth of the FBs if the particular model used has a cloudier consistency. Additionally, our model is not re-useable, although it can easily be reproduced at a low cost. With these issues in mind, we have now developed a re-usable model using a 3-dimensional computer design to print a human face with eye sockets that can accommodate artificial globes made of gelatin, glycerine and water. This model has thus far been promising in simulating the eye, with an improvement in simulating the transparency of the cornea. However, we feel it is still relevant and important to present our agar model because this new re-usable model may not be as readily available to some as, similar to previously described models in literature, it requires more expertise and specialized equipment to manufacture. There were limitations in our study methodology. Participants were evaluated with a self-administered questionnaire without assessment of objective outcomes such as size of corneal defect after FB removal and these results may not translate to clinical competency in real-life scenarios. Our methodology mainly assessed our learners’ reaction to the training and was unable to completely assess task performance or knowledge objectively. We were also not able to ascertain if using just video training with live teaching and slit lamp training would have resulted in the same improvements without hands-on teaching with our eye model, as this was not part of our study design. To adequately ascertain clinical competency, the learners will have to be assessed while performing the procedure on a live patient with corneal FBs, which would be opportunistic and may not be ethical. As the assessment collected has been de-identified in accordance to our Institutional Review Board’s regulations for consent exemption, we were also unable to trace the individual’s ability to perform this procedure on real patients after the training. Moving forward, we intend to use this study as a proof of concept and will collect data using experienced clinicians (senior emergency physicians or ophthalmologists) to evaluate the fidelity and suitability of our model as a surrogate.
have described an innovative, affordable and easily reproducible method of creating a surrogate eye model. Our model has proven to be sustainable and an acceptable teaching method among junior emergency doctors. Learners expressed greater confidence in managing removal of corneal FBs and use of slit lamp. Given the easy and inexpensive way in which it was constructed, we feel strongly that our model has huge potential applications, especially in training settings where resources are limited.
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Immunoprofiling Reveals Novel Mast Cell Receptors and the Continuous Nature of Human Lung Mast Cell Heterogeneity | 14e94b57-4f21-41a8-b6ca-82ee96247d38 | 8764255 | Anatomy[mh] | Heterogeneity among mast cells has been known for a long time and was first attributed to differential expression of proteoglycans in rodent mast cells, which gave them distinct staining patterns ( ). This led to the division of rodent mast cells into connective tissue mast cells and mucosal mast cells. In humans, mast cell heterogeneity has been based on the expression of mast cell proteases, i.e., cells expressing tryptase only (MC T ) and those expressing both tryptase and chymase (MC TC ) as well as carboxypeptidase A ( , ). These subtypes have been defined using immunohistochemistry, a method that produced binary results, that is, the absence or presence of expression. The MC TC subtype is more predominant in connective tissues such as the skin, while the MC T subset is more prevalent in mucosal surfaces such as the airways and gastrointestinal tract ( ). Mast cells are found in the human lungs in all different compartments, i.e., under the epithelium, in smooth muscle bundles, around pulmonary vessels, in the parenchyma and in close proximity to sensory nerves ( ). Human lung mast cells (HLMCs) have several important functions in health and diseases, such as host defense, induction of acute inflammatory responses, vascular regulation, bronchoconstriction and tissue remodeling ( – ). The heterogeneity of HLMCs was first suggested to be related to differences in their size and function ( ), where a heterogeneity in the response to secretagogues also was reported ( ). It was described that the protease expression within HLMCs differ, with the MC T being the predominant subtype except around pulmonary vessels, where the MC T and MC TC subtypes are found in equal numbers ( ). However, the heterogeneity among HLMCs goes beyond size and protease expression, as demonstrated by the differential expression of certain mast cell-related markers (FcεRI, IL-9R, 5-LO, LTC 4 S, etc.) among the MC T and MC TC populations in different lung compartments ( ). Mast cell heterogeneity has primarily been studied in a binary manner using immunohistochemistry, describing the absence or presence of a given marker. Here, we used a quantitative flow cytometry-based approach to study HLMC heterogeneity, profiling the expression of 332 surface markers and intracellular staining of the proteases tryptase, chymase and CPA3. None of these markers distinctly divided the HLMCs studied into subpopulations. However, several markers showed a high degree of variation within the mast cell population with a nonclustered gradient expression pattern. Six of these markers correlated with each other, revealing the continuous nature of HLMC heterogeneity rather than separation into distinct subpopulations.
Ethical Approval Cell Preparation Flow Cytometry Statistical Analysis The local ethics committee approved the collection of lung tissue from patients undergoing lobectomy, and all patients provided informed consent (Regionala Etikprövningsnämnden Stockholm, 2010/181-31/2).
Single-cell suspensions were obtained from macroscopically healthy human lung tissue as previously described ( ). Briefly, human lung tissue was cut into small pieces and enzymatically digested for 45 min with DNase I and collagenase. Thereafter, the tissue was mechanically disrupted by plunging through a syringe, the cells were washed, and debris was removed by 30% Percoll centrifugation. After preparation, the cells were stained and analyzed by flow cytometry.
The following antibodies were used: anti-CD45-V500 (Clone HI30, BD Biosciences, San Jose, CA, USA), anti-CD14-APC-Cy7 (clone M5E2, BioLegend, San Diego, CA, USA), anti-CD117-APC (clone 104D2, BD Biosciences), anti-FcεRI-FITC (clone CRA1, Miltenyi Biotec, Bergisch Gladbach, Germany), anti-FcεRI-PE (clone CRA1, BioLegend), anti-SUSD2-PE (clone W3D5, BioLegend), anti-CD63- FITC (clone H5C6, BD Biosciences), anti-CD49a-BV786 (clone SR84, BD Biosciences), anti-CD66a/c/e-A488 (clone ASL-32, BioLegend), anti-CD326-BV650 (clone 9C4, BioLegend), anti-CD34-BV421 (clone 581, BD Biosciences), anti-HLA-DR-PE/Cy5 (clone L243, BioLegend), anti-CD344-PE/Vio770 (clone CH3A4A7, Miltenyi Biotec), anti-CD38-BV421 (clone HIT2, BD Biosciences), anti-tryptase (clone G3, Millipore, Burlington, MA, USA) conjugated in-house with an Alexa Fluor 647 monoclonal antibody labeling kit (Thermo Fisher Scientific, Waltham, MA, USA), anti-CPA3 antibodies (clone CA5, a kind gift from Andrew Walls, Southampton, UK) conjugated in-house with an Alexa Fluor™ 488 antibody labeling kit (Thermo Fisher Scientific) and chymase (clone B7, Millipore) conjugated in-house with a PE Conjugation Kit (Abcam, Cambridge, UK). Surface staining was performed by incubation for 30 min at 4 degrees with the antibodies in PBS+ 2% FBS, followed by washing with PBS+ 2% FBS. When using the LEGENDScreen™ human cell screening kit, which contains 332 markers and 10 isotype controls, conjugated to PE (Cat. 70001, BioLegend) that are detailed in , cells were first stained with anti-CD45, anti-CD117, anti-CD14 and anti-FcεRI antibodies for 30 min, followed by washing. Thereafter, the cells were stained with the kit reagents according to the manufacturer’s instructions. The cells from each donor were not sufficient to run an entire screen. Therefore, each kit was run using several donors, and a total of 10 donors were used to run three Legendscreens kits. The presence of anti- FcεRI antibody in the backbone panel did not result in mast cell activation, as measured by surface CD63 expression (data not shown). For intracellular staining, cells were fixed with 4% paraformaldehyde (PFA) and permeabilized using 0.1% saponin in PBS with 0.01 M HEPES (PBS-S buffer). Nonspecific binding was blocked using blocking buffer (PBS-S with 5% dry milk and 2% fetal calf serum (FCS)). The cells were thereafter stained intracellularly over-night with the antibodies in blocking buffer, thereafter washed with PBS-S buffer and PBS+ 2% FBS. The cells were analyzed using a BD FACSCanto (BD, Franklin Lakes, NJ, USA) or BD LSRFortessa, and FlowJo software version 10 (FlowJo LLC, Ashland, OR, USA) was used for flow cytometry data analysis. Fluorescent minus one (FMO) controls (extracellular) or isotype controls (intracellular) were used to establish gates for positive staining ( ).
Statistical analyses were performed with GraphPad Prism software version 7.0b ( , and ) or the Python environment (3.7) with the following packages: statsmodels (0.10.1), seaborn (0.9.0), scipy (1.4.1), pandas (1.1.0), numpy (1.18.1), and matplotlib (3.1.3) ( ). The specific methods used are detailed in the figure legends. * p < 0.05; ** p < 0.01; *** p<0.001; **** p<0.0001.
Expression of Mast Cell Proteases Immunoprofiling of HLMCs Heterogeneous Expression of the High-Affinity IgE Receptor FcεRI Heterogeneous Expression of Cell-Surface Markers With a Continuous Distribution The classic division into distinct human mast cell subpopulations is based on the presence or absence of granule-associated antigens, i.e., the proteases tryptase, chymase and CPA3. We therefore first performed intracellular staining of lung cells and used a flow cytometry-based readout to accurately characterize the mast cell population with respect to the expression of tryptase, chymase and CPA3. Flow cytometry analysis identified CD45 + CD14 low CD117 high HLMCs with the characteristic expression of tryptase ( ). The HLMC population showed a high variation of chymase and CPA3 expression between individual cells and the degree of CPA3 and chymase double positive cells varied considerably from donor to donor ( ). However, no distinct subpopulations were discernable, instead there was a continuous spectrum of expression levels.
Whereas intracellular antigens failed to discriminate distinct subpopulations, we set out to perform an extensive mapping of cell surface antigens on HLMCs to potentially identify distinct subpopulations and novel markers. Flow cytometry analysis characterized the expression of 332 surface markers on HLMCs, using a LEGENDScreen™ human cell screening kit. Since many of the markers are broadly expressed we used CD14 + cells as a reference to selectively enrich for lineage-specific antigens in HLMCs ( ). Well-known monocyte markers such as CD11b, CD11c, and HLA-DR were highly expressed on CD14 + cells, verifying the validity in enriching for lineage-characteristic markers. In analogy, HLMCs expressed high levels of CD117 and FcεRI. Markers with the most significant differences between the HLMCs and CD14 + cells included CD9, CD59, CD274 and CD226 ( ). CD9 is a broadly expressed tetraspanin with a wide variety of functions; in mast cells, it is abundantly expressed and has been implicated in chemotaxis and activation ( ). CD59 can prevent complement-induced cytolytic cell death by preventing assembly of the complement membrane attack complex and has also been implicated in T cell activation ( ). CD274 is also known as programmed death ligand-1 (PD-L1) and can cause blockade of T cell activation ( ). CD226 has received increasing interest in recent years and can play a role in many immunological processes ( ), including enhancement of FcεRI-mediated activation in mast cells ( ). Of the 332 markers analyzed, HLMCs showed significant expression of 102 markers ( ), of which 23, to the best of our knowledge, have not been described on (non-neoplastic) human MCs before ( ).
One of the principal markers for MCs is the expression of the high-affinity IgE-receptor FcεRI. FcεRI expression on HLMCs has been shown to differ among compartments in the lungs, as well as between healthy and diseased tissues. HLMCs from healthy individuals present in the parenchyma are negative for FcεRI, while patients with concurrent asthma show higher expression of the receptor ( , ). In our study, the expression varied considerably between donors and there was no clear separation between the negative and positive FcεRI populations but rather a continuous spectrum of expression and approximately 50% of the donors expressed FcεRI on virtually all MCs ( – ).
The heterogeneity of HLMCs has primarily been studied using immunohistochemistry in a binary manner and they have been divided into the MC T and MC TC subtypes ( , ). How this heterogeneity is reflected by the heterogeneous expression of cell-surface markers has barely been investigated. None of the surface markers investigated in this study, using quantitative flow cytometry, did distinctly and consistently divide the HLMCs into subpopulations (data not shown). However, several markers were expressed with a considerable continuous variation within the HLMC population ( ). Co- stainings of nine of these markers revealed that expression of six of the markers, SUSD2, CD49a, CD326, CD34, CD66 and HLA-DR correlated (r > 0. 4) ( – ). The correlations seen between markers was not due to autofluorescence since there was no correlations in the FMO controls ( – ). In addition, the HLMCs showed no significant staining with the 10 isotype controls ( ). We next investigated if the expression of these markers correlated to the classical mast cell subsets using intracellular staining for CPA3 as a marker for MC TC . Expression of neither SUSD2, CD38 ( ) nor CD344 (data not shown) correlated with CPA3 expression. Since the expression of SUSD2, a marker for stem/progenitor cells ( ), correlated to CD34, that is expressed on circulating mast cell progenitors ( ), we investigated if the expression of these markers could identify MCs in different stages of maturation. When mast cells mature they become larger with an increasing number of granules ( ). We therefore gated SUSD2 low, intermediate and high cells and compared the FSC and SSC values that reflect the size and granularity of the cells. However, SUSD2high cells had higher FSC and SSC then SUSD2low cells ( – ), suggesting that they were larger with a higher granularity and therefore unlikely to be immature MCs. SUSD2 has been linked to proliferation in cancer cells ( ), why we investigated the proliferation status of the cells with the proliferation marker Ki-67. However, in agreement with the fact that mast cells are long-lived cells with low turnover ( ), no staining was observed ( ).
HLMCs have been shown to be heterogeneous; classically, they have been studied using immunohistochemistry in a binary manner, and they have been divided into the MC T and MC TC subtypes based on whether the mast cell proteases chymase and CPA3 are detectable ( , ). By using a quantitative flow cytometry based method we indeed found high variation in chymase and CPA3 expression but no distinct subpopulations were discernable ( ). The apparent discrepancy between our study and previous immunohistochemical studies is probably due to the fact that we have studied the expression in a quantitative manner using flow cytometry, thus finding that there is a spectrum of different expression levels. In contrast, in previous studies the cells have been classified into MC T /MC TC cells in a binary manner depending on the detection limit of the immunohistochemical technique. Although attempts have been made to map cell-surface antigens on HLMCs ( – ), extensive mapping including the heterogeneity of cell-surface antigen expression has not been carried out. In this study, we identified significant expression of 102 markers on the HLMC surface ( ), of which, to the best of our knowledge, 23 are novel mast cell markers ( ). Several of these markers, including SSEA-5, SUSD2, W4A5, CD243, CD111, CD131 and CD164, are described as markers expressed on stem cells. The expression of stem cell markers on mast cells is in accordance with results from the FANTOM5 consortium, in which skin mast cells exhibited similarities with stem cells ( ). In some cases, our results are in disagreement with previously published data; for example, CD4, CD10, CD36 and CD74 were previously shown to not be expressed by HLMCs ( , ). This discrepancy might be explained by differences in the procedures, as in contrast to published data, we did not purify or culture the studied mast cells prior to analysis ( , , , ). Culturing mast cells has been shown to alter their phenotype and expression of cell-surface receptors ( , ). Furthermore, although macroscopically healthy tissue distal from the tumor was used for our analysis, one cannot rule out the possibility that also this part of the tissue is affected by the disease and can have an impact on the results. The expression of FcεRI has been shown to differ in different compartments of the lung, with mast cells present in the parenchyma being negative for FcεRI ( ). We have investigated single cell suspensions from lung tissues without separation of the parenchymal cells. However, we did not observe distinct FcεRI positive and negative mast cell populations but rather a continuous spectrum of expression levels ( ). Additionally, in about 50% of the donors virtually all mast cells stained positive for FcεRI, meaning that we cannot detect any FcεRI negative parenchymal mast cell population in these individuals ( ). This discrepancy is again likely to be due to the different techniques used, immunohistochemistry and flow cytometry, and the different detection limits of the techniques. We also observed a large variation in expression of FcεRI among individuals ( – ), and in line with our results, this has previously been shown also for human skin mast cells ( ). The reasons for this variation could be manifold, as the surface expression of FcεRI can be regulated in many different ways. FcεRI is, for example, upregulated by IL-4 and stabilized on the cell surface by the binding of IgE antibodies ( ). Furthermore, it was described recently that IL-33 downregulates the expression of FcεRI ( , ), indicating that the state of inflammation in the tissue can influence FcεRI expression. Heterogeneous expression of cell-surface markers on mast cells has scarcely been investigated. We investigated the heterogeneity of cell-surface markers in a quantitative manner using flow cytometry and did not find any markers that distinctly and consistently divided the studied mast cells into subpopulations with a bi- or multimodal distribution (data not shown). We did, however, find several markers with considerable continuous variation in expression within the mast cell populations ( ), and co-stainings revealed that six of these markers, SUSD2, CD49a, CD326, CD34, CD66 and HLA-DR, were correlated ( ). To investigate whether these markers are correlated with the classic mast cell subpopulations MC T and MC TC , we costained for SUSD2 and CPA3, but no correlation was detected, ruling out the possibility that these markers are extracellular markers of the classic mast cell subtypes ( ). Recently, CD38 was demonstrated to be differentially expressed in human nasal polyp mast cells where CD38 low were of the MC TC subtype, while CD38 high MCs were a heterogenous pool of both MC T and MC TC cells ( ). In our study, CD38 did not distinctly separate the HLMCs into subpopulations and did not correlate with CPA3; i.e., in HLMCs CD38 could not be used to distinguish the MC T and MC TC subtypes ( ). CD344 did not correlate with the MC T or MC TC profile either (data not shown). Another marker suggested to be expressed on MC TC is the complement 5a receptor CD88 ( ). However, we did not detect significant expression of CD88 on HLMCs ( ). Thus, we were unable to find an extracellular marker that distinguishes the classical mast cell subsets. One of the markers that showed high continuous variation in the HLMCs, CD63, is used as a surrogate marker for mast cell activation ( ), i.e., degranulation. However, CD63 did not correlate to any of the other eight markers investigated, including the 6 markers that show correlation to each other SUSD2, CD49a, CD326, CD34, CD66 or HLA-DR indicating that these markers do not reflect varying degree of activation ( and data not shown). Since two of our six heterogeneity markers that correlate, CD34 and SUSD2, is expressed on stem/progenitor cells ( , , ), we wondered whether these markers could identify cells in different stages of mast cell maturation. However, if that was the case, one would expect cells with high expression of SUSD2/CD34 to be small and contain few granules similar to mast cell progenitors ( ). In contrast, the cells with high expression of SUSD2 had relatively high FSC and SSC values ( – ), suggesting that they were relatively large and granular and therefore unlikely to be immature mast cells. In this context, it is worth noting that mature murine mast cells also express CD34 ( ), and in these cells, CD34 inhibits adhesion and is required for optimal migration ( ). SUSD2 is also expressed in certain cancers, in which it has been linked to proliferation ( ); thus, one could imagine that cells with high SUSD2 expression are proliferating. However, we could not detect any staining for the proliferation marker Ki67 in HLMCs ( ). In summary, we have identified the expression of 102 cell-surface antigens on HLMCs, of which 23 have not been described previously on MCs. Several of these antigens had a high continuous variability in their expression within the HLMC population. The expression of six of these markers correlated to each other and the size and granularity of the cells. Further studies are needed to determine how these cells differ functionally. None of the markers correlated with the intracellular protease expression. Thus, in contrast to the dogma of distinct mast cell subtypes, we demonstrate the continuous nature of HLMC heterogeneitRegionala Etikprövningsnämden, StockholmR, JD, and GN conceived and designed the studies. ER, JD, and AR designed and performed the experiments. ER, DZ, and JD analyzed the data. JS, A-CO, MA-A, MA, and S-ED provided samples. ER, GN, and JD wrote the manuscript draft. All authors reviewed, critically revised, and approved the final manuscript.
This study was supported by grants from the Swedish Research Council (2018-02070 and 2020-01693); the Heart-Lung Foundation; The Swedish Cancer Society; the Ellen, Walter and Lennart Hesselman Foundation; the Tore Nilsson Foundation; the Lars Hierta Memorial Foundation; the Konsul Th C Bergh Foundation; the Tornspiran Foundation; the O. E. and Edla Johanssons Foundation; the Swedish Society for Medical Research; The Centre for Allergy Research Highlights Asthma Markers of Phenotype (ChAMP) consortium funded by the Swedish Foundation for Strategic Research; the AstraZeneca & Science for Life Laboratory Joint Research Collaboration; and the Karolinska Institutet. The funding sources were not involved in the study design, collection and interpretation of data, writing the report or the decision to submit the article for publication.
S-ED reports personal fees from AstraZeneca, Cayman Chemicals, GSK, Novartis, Regeneron, Sanofi, and Teva, for consultancies outside the submitted |
Low-value chronic prescription of acid reducing medication among Dutch general practitioners: impact of a patient education intervention | 94622b5d-758d-44c2-958c-9769a762c798 | 10996147 | Patient Education as Topic[mh] | Dyspepsia is one of the most commonly encountered clinical conditions in general practice, with a pooled prevalence ranging between 15 and 21% of the global population [ – ]. Dyspepsia is generally defined as a symptom complex characterised by a predominant pain or discomfort in the upper abdominal region, such as epigastric discomfort or pain, heartburn or regurgitation . In the Netherlands alone, approximately 800,000 patients reporting symptoms of dyspepsia annually . International assessments of the prevalence of dyspepsia reveal significant variation between countries, with rates ranging from less than 1% to as high as 57% [ , , ]. As dyspepsia mostly is not caused by an identifiable disease or organic abnormalities, it is generally perceived as a harmless condition, in absence of alarm symptoms such as bleeding, anaemia, unintended weight loss, or dysphagia [ – ]. Numerous studies have demonstrated an association between the development of dyspepsia and various lifestyle factors including diet, smoking, alcohol consumption, excessive body mass, and mental state [ – ]. Dutch guidelines for general practitioners (GPs) therefore recommend GPs to provide lifestyle advice prior to treatment with acid reducing medication (ARM), such as antacids or H2-receptor antagonist and proton-pump inhibitors (PPIs) . However, a recent review indicated that around a quarter of the adult population worldwide uses ARM . Additionally, ARM was the most frequently prescribed drug category in Dutch general practice in 2020, with over 2.2 million users [ – ]. Although short-term ARM prescriptions are an effective way to control acid-related disease, the chronic prescription of ARM is only indicated in specific situations. According to the guidelines for Dutch GPs, chronic prescriptions of ARMs should only be considered in patients with Barrett’s oesophagus, Zollinger-Ellison syndrome, or in patients at high risk of gastrointestinal bleeding . However, a recent study showed that around 88% of patients with a chronic ARM prescription in Dutch general practice lacked an appropriate indication, so called low-value prescription . Although PPIs used to be considered effective and safe, there is growing concern regarding their long-term use as it is associated with numerous adverse effects such as vitamin deficiencies, development of multidrug resistance, decreased bone density, and enteric infections [ – ]. Moreover, the use of ARM can cover potential lifestyle risks. It therefore is necessary to reduce the (chronic) prescription of ARMs among Dutch general practitioners. Previous studies have demonstrated the effectiveness of patient decision aids in reducing low-value treatment. Patient decision aids help patients comprehend the potential benefits and risks associated with their treatment options, empowering them to actively engage in healthcare decisions and make choices that align with their values . However, the effectiveness of the introduction of patient decision aids varies. Furthermore, in the context of chronic ARM provision, the existing evidence of their effectiveness is limited. Only one study by Krol et al., showed that the provision of patient information can effectively reduce low-value chronic ARM use through provision of an educational flyer to chronic ARM users . However, the educational materials used were limited to discussing the newly updated GP guidelines on dyspepsia management and did not provide information regarding potential underlying causes, associated risks and benefits of stopping ARM use, or appropriate indications. In this study, we therefore investigated the impact of an patient focused educational intervention containing these elements on the chronic prescription of ARM.
Study design, phases and setting Intervention and recruitment Sample size calculation Randomisation Assessment of the low-value chronic prescription of acid reducing medication Statistical analysis of the difference in prescribing over the two periods We conducted a randomized controlled interventional study and evaluated this using data derived from a subset of practices participating in the Nivel Primary Care Database (Nivel-PCD). The Nivel-PCD contains care data routinely collected from the electronic medical records from 529 GP practices throughout the Netherlands, representing approximately 2 million registered patients . Furthermore, the database contains longitudinal information regarding patient characteristics such as age, sex, GP consultations, diagnoses, and drug prescriptions. Socioeconomic status (SES) scores (on the level of Dutch postal codes) were obtained from the Central Statistical Office (CBS) . Patients were assigned to one of five categories (lowest, below average, average, above average, highest) based on quintiles. Age categories were defined based on the available GP guidelines Diagnoses are recorded using the International Classification of Primary Care version 1 (ICPC-1). Prescriptions are recorded using the Anatomical Therapeutic Chemical classification system (ATC). This study was approved by the relevant governance bodies of the Nivel-PCD (nr. NZR00322.017) and by the Research Ethics Committee of the Radboud University Medical Centre (dossier number 2022–13,579).
The intervention consisted of the distribution of a poster for the waiting room and flyers to be given to patients aiming to inform both patients and GPs with respect to the correct indications for treatment of dyspepsia (Table contains an elaborate description of the intervention materials). After signing up, practices assigned to the intervention group received a package containing 60 flyers and one waiting room poster to use during consultations. The flyer and poster provide a short description of the correct indications for treatment of dyspepsia. Additionally, both the flyers and posters contained a QR-code linking to a decision aid explaining the correct indications and causes of dyspepsia. The intervention materials are added as Supplementary file .
Based on a z-test sample size calculation using the proportion of patients that received an inappropriate chronic ARM prescription observed in an earlier assessment in the Netherlands (88% of chronic ARMs users do not have an indication), an alpha of 0.05, power of 0.80 and an expected reduction of 10%, a minimum number of 28 GP practices (with a mean of 328 patients that are inappropriately using a chronic ARM) were required to achieve significance .
The participating general practitioners were recruited in a blinded manner from the Nivel-PCD. Meaning that the GPs were approached by the Nivel-PCD without receiving information regarding the purpose of the study. After having consented to participation, GPs were randomly assigned to either the intervention or control group. When a GP was assigned to the intervention group, the entire practice was seen as being exposed. GPs assigned to the intervention group received the poster and flyers, to be shared with the patients suffering from dyspepsia. GPs assigned to the control group received nothing. However, it is important to note that the access to the decision aid was not limited to the GPs of the intervention group and their patients, it was freely accessible to anyone through the website Thuisarts.nl .
Our assessment of the amount of ARM users was conducted using a patient-indication lens, as described by Chalmers et al. Implying that all patients that were chronic ARM users were included in our denominator and all patients without indication for chronic use in our numerator. Individuals were considered chronic ARM users when they had received acid reducing medication for at least 180 days in the previous year. We defined a patient’s chronic prescription as being of low-value when for at least 75% of all prescription days there was no clear indication for chronic ARM prescription present . Supplementary file contains an overview of the way we operationalised our assessment of low-value chronic ARM prescription. This part of the analysis was performed using STATA 16 .
To assess the differences in ARM prescriptions we compared the incidence rate of (inappropriate) chronic ARM prescriptions in the same 6 months before and after the intervention (i.e. last 6 months of 2021 and last 6 months of 2022). Our primary outcome therefore would be the odds ratio (OR) of patients receiving a low-value chronic ARM prescription between the pre- and post-intervention periods. For this purpose, we built a multilevel binomial model, with an interaction term between both the indicator of cohort (i.e. 2021 vs. 2022) and an indicator indicating whether a patient was part of a practice belonging to the intervention or control group. We aimed to include random effects for both the patient and practice level when possible. However, we ended up using models only including a practice level because of the limited number of observations on the level of the patient. Generalised variance inflation factors (GVIF) were calculated to test for collinearity among the included variables before multilevel analysis was conducted (Supplementary file ). Patient age, socioeconomic status (SES) and sex were included as case-mix variables in the models, since previous research has shown they could affect the amount of care a patient requires, receives or has access to [ – ]. Patients for which either the age or socioeconomic status was unknown were excluded from the multilevel analysis, but were included in the table showing the general description of both cohorts (as presented in Table ). Following our analysis of the baseline characteristics of the included population, we were forced to exclude patients above the age of 80 from this analysis while no cases of low-value care provision were present, which would result in too little variation on the practice level. We therefore chose to exclude patients aged 80 and above from our analysis, prioritising the recognition of clustering at the practice level over the inclusion of this age group in our model. The pre-intervention period (2021) was taken as reference period. A P -value smaller or equal to 0.05 was considered statistically significant for all analyses, based on two-sided testing. Data analysis and visualisation was performed using R (version 4.4.2) .
A total of 24 practices responded to our call for participation within the recruitment period. These 24 practices were randomly assigned to either the intervention or control group, resulting in 13 practices in the intervention group and 11 practices in the control group. To even out the number of practices in each of the groups, the two practices were randomly selected from the Nivel-PCD to be added to the control group, resulting in a total of 26 participating practices. These additional practices were selected based on the similarities in size and degree of urbanisation compared to the other practices included in our analysis. Tables and provide a general overview of the characteristics, and recorded number of (low-value) episodes in both the intervention and control group. The initial outcomes indicate a slight increase in chronic low-value ARM prescriptions for both the control and intervention groups. In the control group, the proportion of patients with a low-value chronic ARM decreased from 50.3% in 2021 to 49.7% in 2022, and in the intervention group, it increased from 51.3% in 2021 to 53.1% in 2022. Most patients were prescribed PPI’s as subsequent analysis of the types of ARMs used over both periods revealed that the majority of patients used a PPI. In the 2021 and 2022 cohort, 99.7% and 99.3% of the patients received an PPI (ATC-codes starting with A02BC), while 2.1% and 2.4% of patients were prescribed another ARM. Furthermore, 35% of the ARM users included in the 2021 cohort were also present in the 2022 cohort. Conversely, 37% of patients included in our 2022 cohort were also present in the 2021 cohort. Our results also show that the number of prescription increases with age, however the proportion of inappropriate prescribing decreases. This can be explained by the notion that with increasing age, the number of indications for appropriate chronic ARM use also increases. Analysis of the VIF factors before performance of the multilevel analysis revealed that little or no collinearity exists among the variables included in our analysis (Supplementary Table ). Subsequent multilevel regression analysis revealed that albeit the proportions showing to have slightly increased in both the control and intervention group. no significant difference in low-value chronic ARM prescription between the two groups was observed. The odds of receiving a chronic low-value ARM prescription showed to not- significantly differ when comparing the control to the intervention group over the examined periods (Odds ratio: 1.11 [95% CI: 0.84–1.47], p > 0.05). Table contains an overview of the study outcomes after removal of the patients aged 80+, and Table contains the odds ratio resulting from the subsequent statistical analysis.
Our study shows that over the last half year of 2021 and 2022 in both the intervention and control group approximately half of the patients received low-value chronic ARM prescription. This indicates that ARM was still regularly prescribed over the investigated periods. Furthermore, no significant difference in the number of patients receiving a low-value chronic ARM prescription was observed between the control and intervention group (Odds ratio: 1.11 [0.84–1.47], p > 0.05). Additional analysis revealed that in both the 2021 and in the 2022 cohort, the majority of patients used a PPI (ATC-codes starting with A02BC, prescribed to 99.7% and 99.3% of the patients respectively while only 2.1% and 2.4 of patients in either the 2021 and 2022 cohort were prescribed another ARM). This suggests that it is highly unlikely that the lack of an effect following our intervention cannot be ascribed due to a large proportion of patients stepping down from an PPI to antacids. Comparison with other research Analysis absence of effect Strengths & limitations A strength of this study is that it used routinely collected administrative data containing high-quality and clinical information. This use of highly detailed data enabled us to accurately differentiation between appropriate and inappropriate prescriptions of ARM among patients. However, our study is also prone to limitations. Firstly, we were unable to reach the required number of practices to achieve significance according to our power calculation. Despite extensive efforts, we only managed to include 26 of the required 28 practices, making it challenging to draw definitive conclusions regarding the effectiveness of our intervention. Second, there were also some methodological limitations regarding our assessment of low-value chronic ARM prescription among GPs, as discussed in our previous study . There is an inherent uncertainty in identifying whether a prescription is of low-value. Recommendations contain terms that do not map directly to data variables; also, diagnosis and procedure codes may not precisely identify patients for whom care is of low value. For instance, the recommendations regarding chronic ARM use lacked enough detail or required variables which are absent in the data to accurately distinguish appropriate from inappropriate prescribing. An illustrative example is the guideline stating that gastro-protection using a non-selective non-steroidal anti-inflammatory drug (NSAID) is justified if a patient is using a high dosage of a NSAID. However, information regarding the dosage of the prescribed NSAIDs was unavailable in the data used. We were also unable to identify patients suffering from chronic heartburn, as we only had access to diagnosis established within data of the years (and one year prior) included in our analysis. Patients diagnosed with heartburn outside of this period could therefore potentially be missed. More crucially, heartburn often only persists until patients take ARM (albeit via a prescription or obtained over the counter). The use of ARM often resolves the patients’ symptoms, resulting in the removal of the heartburn diagnosis from their medical records, making defining chronic heartburn challenging. Third, unfortunately we are unable to monitor the number of patients that actually accessed or used the monitor following a visit to their GP. We did contact participating practices to obtain an indication of whether or not patients used the decision-aid. Unfortunately, the participating GPs indicated that they did not have insight into whether the patients actually did use the decision aid and reported that patient never mentioned its use in any of the subsequent visits. Finally, the persistent relatively high prevalence of inappropriate chronic ARM prescriptions could be attributed to the perception of ARMs as relatively harmless. ARMs are readily available over the counter at most drugstores in the Netherlands. Thus, it is likely that our assessment still underestimates the true extent, as we could not capture all chronic ARM users in this study, particularly those using non-prescription ARMs.
It seems that the intervention in itself did not alter the inappropriate prescription of ARM among the included GPs. This finding is not unique, however there is quite some variation in the effectiveness of similar interventions addressing low-value ARM prescribing using a patient educational tool exists. In a study of Boster et al., the treating primary care physicians directly discussed the appropriate indications for ARM use with their patients. Using this method, they successfully reduced the patients’ ARM dosage or completely stopped ARM usage in 44% of the identified ARM users within a military hospital over a 6-month period . Apart from this one study, most studies regarding the reduction of ARM use rely on providing patients the tools needed for appropriate self-management of their dyspepsia. These tools included the provision of intensive support by a specialist nurse, the formulation of an action plan and an explanation of the appropriate indications as well as the benefits of decreasing or discontinuing ARM usage. However, the outcomes of these studies vary. For example, both the study by Murie et al. and the study by Coyle et al. managed to stop or reduce PPI use (by 83% and 35%, respectively) by providing patients the tools for self-management of their ARM use, such as formulating an action plan and providing information regarding appropriate ARM use . Conversely, the study by Dibly et al. provided similar support to ARM users, but their study did not show to change ARM use among the included patients . This observation is consistent with a previous study by Batuwitage et al., which demonstrated that providing education to patients about the appropriate indications for ARM use did not lead to a significant change in ARM utilisation . However, it is worth noting that none of these studies specifically focused on chronic ARM users in their intervention evaluation. As previously mentioned, only the study by Krol et al., specifically assess the impact of their intervention on chronic ARM users, and managed to reduce chronic ARM use by 24% in the intervention group compared to 7% in the control group (24% reduction vs. 7%, respectively) . The difference between our study outcome and theirs can probably be that in our study the practices assigned to the control group in our study could also had access to the intervention materials, while these were freely accessible online. This could have led to exposure of the control practices to the intervention, which was not possible in the study by Krol et al., since they only actively approached the intervention practices. This difference could explain why we did not observe a difference in low-value chronic ARM prescribing between the control and intervention groups.
Our intervention did not lead to a significant reduction in low-value chronic ARM prescriptions between the intervention and control group. The present study does show a much lower percentage of low-value chronic ARM users compared to a previous assessment. Our earlier study, which examined chronic ARM use from 2016 to 2019, found that approximately 88% of chronic ARM users in the Netherlands lacked an indication. In the current study, this baseline was 66% . Several possible reasons could explain the lower baseline for the included practices. First, since our previous assessment, a lot of (media) attention such as reports by national newspapers and an item during the eight o’clock news, has been generated on the appropriate use of ARM. Also, the publication of a report by the Dutch National Health Institute discussed the state of (appropriate) care provision for patients with dyspepsia early in 2021. This public attention might have had an effect on the prescription of ARM by GPs . Second, the overarching national campaign started well before our distribution of the intervention materials among the intervention practices. Therefore, we cannot guarantee that before onset of our assessment the included practices (in both control and intervention groups) were not already affected. Third, the participating practices might already have affinity with improving the quality of care provision as they willingly joined the study unaware of the research topic or intervention. These practices might therefore already have a critical attitude towards the (chronic) prescription of ARMs, providing an explanation for the lower baseline observed in our study. Fourth, contact with the different intervention practices a few months after having distributed the materials revealed that the degree of exposure to the intervention varied amongst the intervention practices. Most GPs indicated that they were aware of the existence of the decision-aid. However, we do not know to what extent all GPs in the intervention practices have used the materials when seeing patients with dyspepsia. The fifth and final reason which could explain the absence of an effect following our intervention could be that our intervention was not sufficiently tailored to be effective. Hence, our intervention focussed on explaining the potential causes of dyspepsia and appropriate indications for ARMs use to both GPs and patients. However, as previous research indicated, the provision of low-value care is often the result of an interplay of multiple factors existing on multiple levels (e.g. the patient, healthcare provider and organizational or even medical society context) . Additionally, it shows that the effectiveness of deimplementation strategies and interventions depend on contextual factors, such as workplace culture or economic factors. Factors which we could not control in our intervention. Potentially, our intervention could have shown an effect if we had proactively put more emphasis on the use and implementation of the materials as well as improving knowledge of the existing guidelines. While in the current setup, our intervention heavily relies on the pro-active participation of the participating healthcare providers to improve ARM prescribing; something which has proven hard to monitor.
Our educational intervention did not result in a change in the low-value chronic prescription of ARM, suggesting that (low-value) chronic prescribing ARM remains an important issue in current medical practice. Future research therefore should focus on what is needed for practices to successfully adopt the use of a patient-centred decision aid and reduce low-value chronic prescribing ARM.
Supplementary Material 1. Supplementary Material 2. Supplementary Material 3.
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The Rehabilitation of Partially Edentulous Maxilla With Unilateral Zygomatic Implants: A Retrospective Study up to 23 Years Follow‐Up | 47c40771-8b5c-4b03-9ebb-6bf41e459009 | 11810560 | Surgical Procedures, Operative[mh] | Introduction The zygomatic implant (ZI) treatment is recognized as a graftless solution for edentulous patients with severely atrophic maxillae (Brånemark et al. ). The outcomes have proven successful in the long term, reducing treatment time and providing immediate loading restoration for these challenging patients (Roper et al. ). Originally, the zygoma anchorage solution was developed for patients with maxillary deficiencies, particularly in cases of hemimaxillectomy (Parel et al. ). The reconstruction has demonstrated favorable functional results and patient satisfaction, offering an alternative to the traditional flap and grafting approach (Roper et al. ). However, the application of ZI in the partially edentulous maxilla rehabilitation still lacks evidence. In the early 2000s, a couple of reports detailed the outcome of ZI use with delayed loading in partially edentulous maxillae as an alternative to graft approach (Parel et al. ; Pham et al. ). Subsequently, Davo et al. reported five patients who underwent partial restoration with the combination of ZI and conventional implants (CIs) with immediate loading, demonstrating a 100% 5‐year survival rate (Davó, Malevez, and Pons ). Goker's study involving 32 patients treated with 34 ZIs and 31 CIs for partial edentulous rehabilitation reported no ZI failures over an average follow‐up of 34 months (Goker et al. ). A recent systematic review outlined a protocol for the unilateral ZI treatment involving the placement the ZI splinted with one or more CIs in the edentulous side (Polido et al. ). However, the authors cautioned about the limited scope and number of studies, involving merely 14 patients, for evaluating these treatment outcomes and establishing possible indications (Polido et al. ). As compared to the use of ZI in full‐arch rehabilitation, the biomechanical feasibility of splinting ZI with CI for partial bridges raises concerns due to the scarcity of data (Ujigawa et al. ), and the evidence supporting the use of unilateral ZI rehabilitation, in general, is still significantly weaker than that for established treatments like maxillary sinus floor elevation or short implants. In 2023, the ITI consensus report in ZI critically evaluated unilateral rehabilitation in partially edentulous maxillae, with findings suggesting a need for broader research due to limited studies and short follow‐up periods (Al‐Nawas et al. ). Therefore, the aim of this retrospective study was to evaluate the survival, complications, and patient‐reported outcomes associated with ZI rehabilitation in partially edentulous maxillae in consecutive patients, with a mean follow‐up period of more than 10 years. Material and Methods 2.12.22.32.42.5Study Design and Population This retrospective study included a cohort of patients who underwent rehabilitation in the partially edentulous maxilla with the ZI between November 1999 and February 2020 in the Department of Implantology and Maxillofacial Surgery of Medimar International Hospital in Alicante, Spain. The follow‐up period ended in February 2024 with a minimal 36 months. The manuscript was prepared according to the Strengthening the Reporting of Observational Studies (STROBE) in Epidemiology (Vandenbroucke et al. ). The participants signed a general informed consent form as part of the hospital's standard protocol prior to the treatment, which adhered to the principles outlined in the Declaration of Helsinki on clinical research. The committee board of Health Centers, Services and Establishments, Hospital Vithas Medimar (Number 80), approved the use of human data for the study. The inclusion criteria for ZI placement were as follows: Patients with partially edentulous maxilla with insufficient bone height and width in an edentulous area involving more than three teeth; Patients with a history of unsuccessful bone grafting or failure of CI treatment; maxillary posterior/anterior bone height ranging between 1 and 3 mm (Division d of Misch's Classification, Misch ); Insufficient bone width in the posterior/anterior maxillary area to place regular diameter implants without additional massive bone grafting or insufficient bone height for CI even with a tilted approach; Requirement for immediate prosthesis loading or refusal of bone grafts. The exclusion criteria for ZI placement were as follows (Davó et al. ): General contraindications for implant surgery; History of radiation therapy in the head and neck in 1 year (> 70 Gy); Current heavy smoking (> 20 cigarettes/day); Restricted mouth opening (< 3 cm); Untreated maxillary acute or chronic sinusitis. Preoperative Evaluation A panoramic and computed tomography (CT) or cone beam CT (CBCT) scan was carried out for diagnosing the degree of maxillary atrophy and planning treatment with dedicated software (Nobel Clinician; Nobel Biocare AB, Göteborg, Sweden). Atrophy was categorized according to Misch's Classification (Misch ). Occlusal relationships, mouth opening, intermaxillary distance, and status of the mandibular dentition were registered. Patients diagnosed with periodontitis who should retain the affected teeth were treated with antibiotics preoperatively. Patients diagnosed with maxillary sinusitis were treated before proceeding with the implant surgery. Patients with a smoking status were educated on the possible negative impact of smoking on the success of treatment. From 2006 onward, the ZI trajectory was planned according to a prosthetically driven implant positioning using the anatomy‐guided approach (AGA) (Kämmerer et al. ). Implant distribution adhered to the general principle of achieving optimal occlusion load, avoiding cantilever, and maintaining the integrity of the prosthetic reconstruction. In the CI planning, if the bone of the maxillary tuberosity was sufficient, the regular‐sized implant was placed using an undersized drilling protocol to improve primary stability, while being splinted with ZI. If the tuberosity could not accommodate an implant, a pterygoid implant was angled at 45°–60° during insertion into the dense pterygoid bone, where the anchorage in the cortical bone engaged the pterygoid plate, also enhancing stability (Rodríguez et al. ). The position and numbers of ZI and CI depended on the patient's atrophy: Patient presented three tooth gaps; at least two implants were planned (one ZI in the posterior zone and at least one CI in the anterior zone) (Figure ). Patient presented four– six tooth gaps: at least three implants were planned (either one ZI combined with two CIs or two ZIs combined with one CI). When possible, one ZI was placed in the first molar site and two CIs in the premolar sites. In patients with insufficient residual bone in the premolar region, a tuberosity or pterygoid implant was placed (Figures and , Rodríguez et al. ; Sun et al. ). Patient presented more than six teeth gaps: at least four implants were placed (one ZI and three or four CIs) (Figure ). Surgical Protocol All surgeries were performed by the same surgeon (R.D.). From 1999 to 2016, 17 surgeries were conducted under general anesthesia; from 2016 onward, nine subsequent operations were managed with local anesthesia (Ultracin; Aventis Pharma, Paris, France), which was infiltrated in the maxillary vestibulum, around the area of the zygomatic bone, 1 cm palatally to the bone crest, without a need for IV sedation. The zygomatic area was exposed via an incision in the posterior maxilla, followed by a vertical releasing incision anterior to the surgical site. To improve the visibility of the drilling direction and the starting point at the crest, a small lateral bone window was made with spherical diamond burs (Komet Dental, Lengo, Germany). If needed, the maxillary sinus membrane was carefully elevated. ZIs were directed toward the zygomatic bone, anchoring them in the maxillary residual process and the zygomatic bone. From 1999 to 2006, the classic intra‐sinus technique described by Branemark was used for placing ZI (Figure ). The original technique kept the implant platform palatal to the crestal ridge, followed the zygomatic crest into the sinus, and engaged in the zygoma (Brånemark et al. ). The sinus membrane was carefully dissected through a small lateral bone window to avoid membrane perforations. After 2006, the AGA technique was used for ZI insertion: the ZI trajectory was chosen according to the relationship between the ZI body and the maxillary sinus lateral wall, positioning the implant into intra‐sinus, extra‐sinus, or lateral wall of the maxilla (Figures , Davo et al. ). The alveolar process and the zygomatic bone were prepared with three drills used sequentially: a spherical bur and a cylindrical bur (ø 2.9 mm, Nobel Biocare AB or Straumann, Basel, Switzerland; or ø 3.5 mm, Nobel Biocare AB). Drilling was performed under constant saline irrigation to prevent overheating. ZIs were engaged at the zygomatic bone level avoiding impingement in the orbital cavity or the infra‐temporal fossa. ZIs (Nobel Biocare AB, Göteborg, Sweden or Straumann, Basel, Switzerland) were inserted with a contra‐angle handpiece with the torque preset above 35 Ncm, with the final adjustments for proper placement made using a manual wrench. After implant placement, multi‐unit abutments (MUA; Nobel Biocare AB or Straumann) were placed, and the wound was closed with interrupted resorbable sutures (Vicryl Ethicon, Ohio, USA). Postoperatively, patients were given oral amoxicillin/clavulanic acid (875/125 mg) (GlaxoSmithKline, London, UK) or clindamycin (300 mg) (Pfizer, New York, USA), twice a day for 1 week; ibuprofen (600 mg) (Pfizer) 4 times a day during meals for 1 week (patients were instructed not to take ibuprofen in the absence of pain); xylometazoline hydrochloride (nasal decongestant) (Novartis, Basel, Switzerland) 1 mg, five drops twice a day for 2 weeks; and 0.2% chlorhexidine rinses (Isdin, Barcelona, Spain) twice a day for 2 weeks. All complications and adverse events were recorded. Immediate Loading, Prosthesis Design, and Follow‐Up For the patient who met the immediate loading criteria (insertion torque over 35 Ncm) and required immediate restorations, an impression was taken, and an acrylic bridge with temporary abutments was delivered within 48 h (Davó, Malevez, and Pons ). Immediate bridges had light contact in centric occlusion and no contact in lateral or protrusive movements. Patients undergoing immediate loading were scheduled for follow‐up appointments for occlusion checks and oral hygiene monitoring at 2 weeks, 1, and 3 months before the final impression was made. Three months post‐implant placement surgery, impressions were taken and a screw‐retained implant‐supported fixed bridge was delivered (Figures and ). For treatment monitoring, panoramic radiographs were taken annually at each follow‐up visit, while CT or CBCT scans were performed for patients presenting symptoms suggestive of complications, such as orofacial pain, swelling, local inflammation, infection, or sinus disorders. The prosthesis was removed either during the annual follow‐up or in response to any complications reported by the patients. Study Endpoint and Statistical Analysis The primary outcome measure of this study was the survival rates of ZI. Implant failure was defined as the loss of implant integration or unsolvable maxillary chronic pain/sinusitis or complications of the implant‐prosthetic complex resulting in the removal of the implant. The secondary outcomes were complications, such as sinusitis, local inflammation, soft tissue recession, and prosthetic mechanical problems. The perception of the Oral Health‐Related Quality of Life (OHRQoL) was assessed by means of the self‐administered oral health impact profile (OHIP‐14) after loading of the definitive prosthesis (Slade ). It consisted of 14 questions, to which a score from 0 to 4 points could be assigned (totaling 0–56), with lower scores indicating a better quality of life. The normality of the data was evaluated using the Shapiro–Wilk test, and homoscedasticity was verified with Levene's test. Both assumptions were satisfied, allowing us to proceed with a standard t ‐test. The t ‐test was used to calculate the t ‐statistic and p ‐value to compare the mean OHIP‐14 scores between two groups of patients: those with a follow‐up of more than 10 years and those with a shorter follow‐up. A p ‐value of < 0.05 was considered statistically significant. Analyses were conducted using the R and SPSS statistical software packages. Results Although initially the study included 24 patients, 3 were excluded (2 did not attend follow‐ups and 1 died due to unrelated cause), resulting in 21 remaining (12 male and 9 female) with the mean age of 54.5 ± 9.3 years. They received a total of 27 ZIs, which had a mean length of 43.8 ± 4.7 (range 35–52.5 mm) and comprised 21 Nobel Zygoma TiUnite (Nobel Biocare AB, Göteborg, Sweden) and six Straumann Zygomatic implants (Straumann, Basel, Switzerland). Five patients received two ZI‐supported bridges (one on each side), while 17 were treated with only one ZI‐supported bridge. Forty‐eight CIs, with a mean length of 12.9 ± 1.7 mm (range 10–20 mm), were placed and splinted to 27 ZIs. Prior to ZI surgery, three patients had a history of unsuccessful outcomes from previous treatments, which included one maxillary lateral sinus floor lift and guided bone regeneration (GBR) failure, and two experienced implant failures. In relation to Misch's Classification, all patients presented division d in the ZI implanted site (Misch ). Patient and implant data is shown in Table . The mean follow‐up period of ZI was 10.3 ± 5.7 years (range 3.2–23.4 years). Three patients (14%) had a mean follow‐up from 3 to 5 years, 9 (43%) from 6 to 10 years, and 9 (43%) over 10 years. 3.13.23.3Implant Survival and Prosthesis Successful The survival rate was 100% for ZI and 97.9% for CIs with only one CI lost 1 year after placement. A majority of patients (17; 80.9%) were eligible and desired immediate loading. They received 21 of ZIs and 40 of CIs, which were loaded with 21 bridge prostheses (80.7%). Those patients (5; 23.8%) who did not request immediate restoration had delayed loading. The implant distributions and rehabilitation units are provided in Table . Twenty‐six implant‐supported fixed bridges were delivered ( n = 26), with only one prosthesis failure. One patient initially underwent unilateral rehabilitation on the partially edentulous left side, achieving 8 years of functional loading. Subsequently, the remaining teeth were extracted, and the patient received an additional ZI and a CI on the right side. These implants were then splinted with the left‐side implants to facilitate a full‐arch reconstruction. Nineteen patients received 24 (92.3%) zirconia bridges as final restoration, and two patients had metal and acrylic bridges (7.7%). To avoid extensive cantilevers, 11 patients had seven CIs placed in the pterygoid region (with 1 patient receiving a 30° angular MUA, 3 receiving a 17° angular MUA, and 2 a straight MUA) and five in the tuberosity (with all five receiving a straight MUA). Additionally, 5 patients (23%) with five bridges had a cantilever of one crown (1 first molar and 4 s molars) due to insufficient bone in the posterior maxilla. This approach was also taken in the case that had an implant failure. Surgical, Biologic, and Mechanical Complications No significant surgical complications were reported during the procedures. One ZI experienced a buccal soft tissue recession of 3–4 mm after 3.2 years of placement, for which implantoplasty was performed. Two patients experienced local orofacial inflammation at a mean of 4.5 years after placement, manifested as pain and facial swelling, but they recovered with anti‐inflammatory medication. Sinusitis was not observed, neither through the type of the acute symptoms nor radiographic diagnostics. Furthermore, one abutment screw fractured 5.2 years post loading. Patients Reported Outcomes All patients (100%) completed the OHIP‐14 questionnaire. The mean score was 1.19 ± 1.99. Patients with a follow‐up of more than 10 years ( n = 9) showed lower mean scores, reflecting a more favorable perception of their quality of life as compared to those ( n = 12) with a shorter length of follow‐up (0.79 ± 0.67 vs. 1.50 ± 2.58), although differences were not significant ( p = 0.21). Discussion The present study assessed the clinical outcomes of the use of unilateral ZI with CI for rehabilitation in partially edentulous atrophy maxilla, demonstrating a high survival and successful rate. To our knowledge, this is the first study that focused on this treatment modality and the accompanying patient‐reported outcomes, furnishing an average observation period exceeding 10‐year. The findings suggest that this treatment is predictable and its complications are manageable, offering a viable alternative for patients with previous failed implantation or grafting treatment. Moreover, it could enable the provision of immediate restoration for patients meeting the necessary criteria and requirements. Nowadays, the majority of the evidence of unilateral ZI treatment stems from patients using ZI‐supported obturators to treat post‐hemimaxillectomy defects (Molinero‐Mourelle et al. ). Clinical outcomes for these patients have varied widely, with overall survival rates ranging from 77% to 100% (Hackett, El‐Wazani, and Butterworth ). This variation might be attributed to tumor recurrence, thickness of the soft tissues, or loss of osseointegration (Chrcanovic, Albrektsson, and Wennerberg ). The primary cause of failure in this treatment may be the lack of ridge anchorage and recurring inflammation around peri‐implant tissue. In the present study, which focused on continuous maxillae, all patients had atrophic residual ridges, but without loss of continuity, and soft tissues around the ZI head were able to remain stable. This could explain the better survival observed in partially edentulous patients compared to those with maxillary defects. Previous unsuccessful experiences with sinus floor elevation or implant treatment were among the criteria for enrolling patients in the study. Sinus floor elevation in cases of insufficient posterior bone height is still considered the gold standard for implant therapy. However, the patients with the extremely severe bone atrophy usually need with a combined horizontal and/or vertical GBR (Corbella, Taschieri, and Del Fabbro ). Reliability of these procedures depends on several risk factors that may affect the surgical procedure and outcomes, such as the presence of sinus septa, membrane thickness, vascularity, types of defect, and dehiscence (Testori et al. ). ZI is considered a valid option for patients with a severely atrophic edentulous maxilla who have experienced treatment failures or complications. Moreover, while most evidence continues to support staged rehabilitation involving lateral sinus lift procedures, only a limited number of studies have demonstrated the feasibility of achieving immediate loading in cases with limited bone quantity. In the present study, 80.9% of patients opted for the immediate loading protocol, which was possible due to the primary stability of the implant. Therefore, ZI solutions for the partially edentulous maxilla are not limited to cases of implant/grafting failure but also can benefit patients requiring immediate function. The qualification of surgeons performing zygomatic implant procedures was addressed in the ITI ZI Consensus, which emphasizes that clinicians must possess the necessary skills and experience to effectively manage potential difficulties and complications (Al‐Nawas et al. ). Although major complications, such as orbital penetration, appear to be rare, the surgical technique for placing ZI still presents significant challenges due to the zygoma's irregular shape and the length of the implants (Kämmerer et al. ). This complexity makes ZI placement one of the most demanding tasks for dentists and oral surgeons. Particularly in partially edentulous cases, the limited operational space for preparing the implant site poses a substantial difficulty. Surgeons must carefully protect the opposing teeth, which may obstruct the drilling trajectory and complicate implant placement. In the author's experience, the use of a contra‐angle handpiece is highly reliable for unilateral ZI procedures in partial edentulism. The surgical navigation system appears to be an excellent tool to enhance the safety and precision of ZI placement (Wu et al. ; Fan et al. ), which improves the learning with increased practice (Wang, Zhuang, et al. ). Although to date no study has specifically focused on utilizing this technique for the unilateral ZI approach, the reports of ZI use in hemimaxillectomy cases implied acceptable accuracy with the navigation approach (Wang, Fan et al. ). This study portrays the evolution of surgical techniques over a 23‐year period. Prior to 2006, three ZIs in three patients were placed using the Brånemark intra‐sinus approach, palatally entering the residual ridge and reaching the zygoma bone through the maxillary sinus (Brånemark et al. ). Subsequently, all other placements adhered to the anatomy‐based AGA, such as intra‐sinus, extra‐sinus, and in‐the‐sinus‐wall techniques (Davo et al. ). Recent systematic reviews have shown that both the Brånemark and AGA techniques demonstrated high implant survival across 25 studies (Kämmerer et al. ). However, the Brånemark intra‐sinus approach was associated with a higher risk of sinusitis and peri‐implant inflammation than AGA. In the present study, sinusitis was not observed, likely due to the specific parameters, including the limited number of patients and the use of unilateral procedures, which may have contributed to this outcome. Additionally, the intra‐sinus technique was avoided in cases where the patient's sinus concavity was not pronounced (Davó and Fan ). Larger studies with a more extensive patient cohort are needed to fully assess the potential risk of sinusitis in cases of unilateral ZI rehabilitation. The concept of splinting in ZI rehabilitation was introduced in both the classic and quad approaches to achieve cross‐arch stabilization for immediate rehabilitation and definitive prosthesis in completely edentulous patients (Vrielinck et al. ; Davó et al. ). In a recent study by Davo, involving 56 patients with the quad approach, a 97.7% survival rate was reported after 8 years (Davó et al. ). All patients received an immediate loading protocol, suggesting that immediate restoration could offer benefits in stabilizing ZIs through cross‐arch stabilization. Finite element analysis showed that splinting only two ZIs unilaterally can lead to overload under masticatory forces (Ujigawa et al. ). Therefore, the splinting of unilateral ZI with at least one CI remains a fundamental treatment principle. In the present study, a minimal placement approach was applied in six patients who presented with a gap of three teeth, utilizing a single ZI and a single CI. As a result, the placement of two implants was considered sufficient for rehabilitation. Moreover, if an additional CI had been feasible, the use of a ZI would not have been necessary. However, in Goker's study, which involved 34 patients treated with unilateral zygoma (Goker et al. ), three patients (two with two ZIs and one with one ZI) underwent exclusively ZI rehabilitations, indicating a need for further investigation into the long‐term feasibility of such treatment. The use of regular‐length CIs in conjunction with ZIs in the severe edentulous maxilla has been recommended since the early studies by Prof. Branemark and Prof. Malevez (Brånemark et al. ; Malevez et al. ). Both studies demonstrated that short implants had a lower survival rate compared to regular‐length implants when splinted with ZIs in the edentulous maxilla. This protocol has also been followed by the author in unilateral ZIs rehabilitation. More recently, Vrielinck and colleagues (Vrielinck, Blok, and Politis ) reported cumulative survival rates of CIs in ZI treatments at 10, 15, and 20 years, with survival rates of 90.5%, 81.6%, and 67.7%, respectively. Significant risk factors for CI failure included bruxism, overdenture design, and the use of implants shorter than 10 mm. Based on this evidence, the use of short implants in combination with ZIs is not recommended. Patients treated with ZI rehabilitation experienced immediate prosthetic reconstruction in both function and anatomy, achieved through less invasive surgery (Al‐Nawas et al. ). The present study employed OHIP‐14 questionnaires to assess overall satisfaction, yielding a normalization level (1.19 ± 1.99) that is comparable to the score following lateral maxillary sinus augmentation (2.4 ± 3.7, Schiegnitz et al. ), which stands at 1.19. The limitations of the present study included its retrospective design and the small sample size (Talari and Goyal ). Additionally, CT/CBCT imaging was only systematically documented for patients with major complications, making it impossible to accurately assess the trajectories of all ZIs. Further clinical studies (especially randomized control trials) should assess the feasibility of unilateral ZI treatment compared with extra short implant rehabilitation or maxillary sinus floor augmentation in the partially edentulous maxilla. Conclusion The ZI treatment represented a predictable option for patients with partially atrophic edentulous maxilla who have experienced previous graft or implant failures, or who require immediate loading. Splinting ZI with CI in the restoration was considered important in unilateral ZI treatment. Complications were infrequent and could be managed effectively, while the patient‐reported oral health‐related quality of life indicated normalization. Shengchi Fan: conceptualization, data curation, investigation, writing – review and editing, writing – original draft, methodology, formal analysis, project administration. Ruben Davo: conceptualization, methodology, data curation, investigation, visualization, writing – original draft, writing – review and editing. Bilal Al‐Nawas: writing – review and editing, supervision, project administration. Eduard Valmaseda Castellón: writing – review and editing, supervision, formal analysis. The ethical approval from the review board of Hospital Medimar Internacional to use human data for the study was obtained. Dr. Ruben Davo serves as an advisor and global speaker for Nobel Biocare; Dr. Eduard Valmaseda‐Castellón is a recipient of grants, including non‐financial support, and/or personal fees from MozoGrau, Inibsa Dental, Dentaid SL, BioHorizons Iberica, Laboratorios Silanes, Geistlich Pharma AG, and Mundipharma. Other authors declare no conflicts of interest. |
One swallow does not a summer make: Twenty years of challenges and achievements of family medicine in Mozambique | 186ec14c-0712-4b6d-b34e-c149172feb0f | 8905403 | Family Medicine[mh] | Only eight family physicians (FPs) have been trained in Mozambique. Ten, if we count two Mozambicans who graduated outside the country. Given the size of our population (31.3 million in 2020), this number is extremely small and we certainly still have a long way to go before we can say that our specialty impacts the health status of our population. This number could have been higher if it was not for so many difficulties experienced in the short life of family medicine (FM) in Mozambique. The first training programme in FM began in 2000 at the Hospital Geral Polana Caniço in Maputo, with the Spanish Cooperation taking care of the operations and academic management. In 2009, the programme was closed, having trained only four FPs. Only in 2015 did the programme resume its activities, with one resident starting his training that year and two more in 2016. There are currently two programmes in the country: one in Maputo and another in Nampula, which has not received any residents yet, despite having the accreditations and capacity to train new FPs. Despite this uncertain and timid beginning, the current national scenario is very positive for the growth of FM and primary healthcare (PHC). To keep taking the right steps, we need to analyse the current political scenario and the health context in which we live. This case study is the result of a series of virtual world-café meetings amongst the authors using the World Health Organization’s Operational Framework for Primary Health Care as a theoretical model. It aims to describe the strengths, opportunities, weaknesses and threats of the current scenario that can influence the development of FM and PHC in Mozambique.
Mozambique is a country with many social, economic and health needs. The country ranks 22nd on the Fragile States Index list and is experiencing an incomplete epidemiological transition scenario, where the burden of morbidity resulting from infectious diseases competes with an increasing prevalence of non-communicable chronic conditions. With just over 30 million inhabitants and a GDP per capita of US$467, we still have high maternal (289 per 100 000 live births) and infant mortality rates (74.2 per 1000 live births) and a prevalence of HIV of 11.5%, greater than most chronic non-communicable diseases. To deal with this high burden of morbidities, we have an extremely small ratio of doctors per population – just one doctor for every 10 000 inhabitants. As a result of the comprehensive training, the work of the family doctor, when integrated with other professionals in the PHC team can be decisive in meeting the health needs of the population in Mozambique. Certainly, the skills developed over the four years of training makes them more capable of meeting the demands of the population. On the other hand, this same training makes local authorities see these FPs as skilled professionals who can occupy positions in the Ministry of Health (MoH) or provide managerial support in public services. Even when such duties are intermittent, the hours available for teaching and taking care of patients is reduced. In addition, professional recognition is not accompanied by financial compensation for any medical specialty in Mozambique. Despite the quality training that we seek to offer to our residents, much of what is learned cannot be applied in clinical practice. Community health clinics lack the most basic resources and supplies for comprehensive patient care. This hinders the population’s confidence in primary care clinics, making patients and families seek care in more resourced hospital services. Considerations for the near future There is a growing movement of young FPs in Mozambique, as a result of the reactivation of the Maputo Residency Programme and the creation of the Mozambican College of FP in 2010. Despite the optimism and engagement, this group is still too small to generate political changes and modify practices in universities, health services and public administration. Fortunately, the current MoH’s agenda has been trying to fix the lack of trained human resources in our country, inviting medical societies to jointly design a plan for expanding residency training for all medical specialties with FM occupying a prominent seat at this table of negotiations. This initiative will create new training sites in remote areas far from the capital, following successful experiences from other countries. In this way, instead of attracting young doctors from small cities to be trained in Maputo, the necessary resources will be deployed to 10 healthcare facilities in five provinces of the country, improving the quality of these services. The aim is to make future generations of FPs settle in the places where they were originally trained, increasing the number of specialists in areas that have no FP today and improving their distribution throughout the country. This MoH initiative represents the beginning of a paradigm shift in medical education in our country, which still values tertiary hospitals and specific medical specialties as more important than the work of generalists in the community. Despite this favourable political scenario, there are major challenges to achieve this goal. Initially, the aim was to train 750 new FPs by 2025, but this number proved to be impractical because of structural and financial shortcomings. Currently, 10 health facilities in five provinces are listed as potential sites to become new residency programmes in FM. If that happens, in 2022 we will have 60 vacancies for residents spread throughout our country: 9 in Maputo province, 12 in Sofala, 12 in Zambézia, 12 in Tete and 18 in Nampula, in addition to the five vacancies in Polana Caniço. New preceptors must be hired to teach and practice in these centers. The number of FPs about to graduate will not be enough to meet this demand. In 2022, five new FPs will graduate, but there will be no one graduating in 2023. Only in 2024 and 2025 we will have eight and seven new specialists graduating, respectively. In the short term, it is almost certain that Mozambique will need to rely on the Cuban International Cooperation to remediate this shortage of trained preceptors. In order to strengthen this initiative, it will be crucial to face another major challenge in the short term: building and validating a competency-based curriculum for FM in Mozambique. This will help us to create objective parameters to guide both the training and practice of FP and make the expansion of residency training in FM uniform amongst all six training sites. We also need to make our specialty more present and active in medical schools so that this transformation is sustainable in the long-term. Students have very little contact with FM whilst studying at medical school. Their contact with patients in the community happens under the supervision of nurses and technicians (technical professionals responsible for most of the primary care delivered in Mozambique) and takes place in health facilities where they get contact with health programmes focused on maternal and child care, HIV and AIDS, tuberculosis and vaccination. Students rarely have the opportunity to follow medical appointments during these visits and when this happens, it is not a FP running the consultation. The Eduardo Mondlane University Health Center is the training site for residents in FM in Maputo. It serves students, university employees, employees’ families and residents of the neighborhood. Unfortunately, medical students only have contact with what we call community health. This discipline presents a simplistic view of what PHC and FM really are. Medical students and other fellow professors at the university end up discrediting the specialty, seeing it as lacking glamour and excitement. This misconception about the work of FPs needs to be changed and it relies on the efforts we have been making, seeking greater integration with other medical specialties and other healthcare professionals. In addition, we need to improve teaching and learning experiences in FM at the residency programme and medical school. If we succeed in this endeavour the next generations will fully experience FM and will start to see primary care as a desirable place where medicine can be taught and learnt by any healthcare student. This new generation of FPs will have a lot of work ahead, advocating for the specialty with politicians and decision-makers. The momentum created by the resurgence of the residency programmes in 2015, the promotion of this new generation of FP and the current political scenario creates a favourable environment for the sustainable expansion of FM. Collaboration with colleagues from abroad, international internships, continuing medical education, faculty development activities and capacity building for research in PHC are key elements for FM to flourish in Mozambique. Finally, optimisation studies would be helpful to better plan the investment of the scarce financial resources we have. The information described here is summarised in presenting the strengths, opportunities, weaknesses and threats of the current scenario of FM and PHC in Mozambique, categorised according to the World Health Organization’s 14 levers for the development of PHC.
As the saying goes: ‘One swallow does not a summer make’. Perhaps eight swallows can, with small victories each day, make PHC in Mozambique less based on verticalised healthcare programmes and more based on comprehensive, efficient and person-centred care.
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Evaluation of dental emergency during the COVID-19 pandemic in the Social Rehabilitation Centre of San Francisco Kobén (Campeche, Mexico) | 6ab18394-2356-4c1c-9376-7618821e6b8e | 11474841 | Dental[mh] | Figures for the medical and dental care of the population of the Francisco Kobén Social Rehabilitation Centre (CERESO) showed that the inmates face a proportionally lower rate of dental morbidity and mortality when compared to the data provided by the World Health Organisation (WHO) and the official data of Campeche State . This is probably due to greater access to healthcare, given that there are little or no limitations on conventional dental, medical and mental healthcare provisions thanks to the presence of a clinic inside the centre supported by the State and Federation, while members of the general public are obliged to attend a clinic. Inmates are entitled to comprehensive healthcare, which constitutes one of the responsibilities of the State, from the moment they enter prison . Inmates of the CERESO - with or without mental disorders -, receive high quality preventive and corrective medical and dental care linked to social rehabilitation during imprisonment. The Francisco Kobén Social Rehabilitation Centre offers a medical and dental care programme, supported by the department of special programmes of the Secretary of State for Health, which offers access to the service for all inmates. As soon as they enter the centre, they undergo an initial assessment, after which the patient is informed about his/her medical-dental health and is shown intraoral images used as content for education on dental health (preventive activities). The images also enable us to create additional evidence to complement diagnosis and prepare an adequate treatment plan. The State and the Federation are committed to offering an effective response to inmates’ health problems, given that many of these persons rarely make use of traditional public health resources once they are released. It is our obligation as health professionals to educate inmates and their families about prevention, diagnosis and treatment, to do away with social myths and panic related to newly emerging diseases such as SARS-CoV-2 (COVID-19) and provide adequate dental care through effective screening in prisons. The dental health services progressively adapted to the COVID-19 pandemic. The SARS-CoV-2 virus severely disrupted dental procedures around the world. The risks of infection to dental care personnel and cross infection between patients and any person in the dental care setting were high during this period of the pandemic, due to the close proximity of the dentist and patient and the generation of aerosols created during commonly used procedures and treatments , . Almost all dental procedures involve a high risk of infection for dentists and patients due to the propagation of aerosols. As a result, public health agencies issued guidelines to improve the control of infection, the use of personal protective equipment and limited care provision to the emergency services . This article arose from the need for scientific evidence that would support dental health personnel in prisons, given that at the most critical moments of the pandemic there were inmates who filed legal remedies for dental care considered by the health personnel themselves to be basic and not urgent, placing the health of the personnel and their families at risk for not respecting the medical-dental criteria for a real dental emergency. Therefore, distinctions were made between a false and real dental emergency during the SARS-CoV-2 outbreak for an improved and effective screening of inmates at the San Francisco Kobén CERESO.
We designed an observational descriptive, cross-sectional and prospective study for a sample of 100 inmates of the San Francisco Kobén CERESO. The data was gathered at the prison dental clinic, the male and female participants signed an informed consent letter , for voluntary inclusion in the study during the SARS-CoV-2 outbreak from February to October 2020. A previously validated version of the “Assessment of a True Dental Emergency” questionnaire was used. A pilot test was also used to assess the validity of the measurements, which were found to be highly feasible for application to a Mexican population. The Cohen kappa coefficient was used to assess inter-examiner and extra-examiner reliability, giving a reliability of k = 0.98. All the inmates who went to the clinic to ask for the dental service were included, whole all those who requested other medical services were excluded. After undergoing triage, all the inmates who went to the San Francisco Kobén CERESO, were given the questionnaire and instructions on how to complete it. Each question was read out loud to persons who could not read or write, and the investigator or a nursing auxiliary filled in the questionnaire. All the information gathered was then dumped in a database with the SPSS v.21 software for analysis.
The study included 100 inmates of the San Francisco Kobén CERESO in Campeche, 96% of whom were male and 4% were female, with an average age of 39 years, at a standard deviation of 11.16, with a minimum value of 20 years and a maximum of 77. Depending on the offence committed, 20% correspond to federal jurisdiction and 80% to common jurisdiction. At the time of the survey, 51% of the inmates had been sentenced and 49% were awaiting a final ruling. 26% of inmates were single, 27% cohabited with a partner, 36% were married, 9% divorced and 2% were widowed. 10% reported that they were illiterate, 26% had completed primary school, 42% had completed secondary education, 16% had a secondary school diploma and 6% were graduates. 91% declared that they consumed drugs, 58% consumed marihuana and 33% cocaine. As regards oral hygiene, 42% of inmates reported that they brushed their teeth three times a day, 52% twice a day, 5% once and just 1% stated that they brushed their teeth four times a day. When examined, 4% of inmates showed good, 69% moderately good and 27% bad oral hygiene. The oral examination showed the following: 26% presented dental caries; 55% supragingival tartar; 8% coronal fracture; 6%, dental abscess; 1% impacted third molar; 2% occlusal wear; 1% cold sores and 1% edentulism. The figures for slowly progressing chronic-degenerative diseases showed that 14% of the inmates suffered from the following; 7% presented diabetes, 6%, high cholesterol and 1%, human immunodeficiency virus. All the patients who suffered from these diseases were receiving treatment. 23% of the patients who went to the dental clinic at the CERESO were prescribed drug treatment in accordance with their condition. Assessment of a real emergency showed the following: 59% of patients who requested treatment presented pain while 41% did not. The figures for inmates with pain showed 8% with mild pain, 20% moderate, 8%, severe, 17% very severe and 6% the worst possible pain. The average period from when pain started to appear was 8.32 days, and figures showed that 50% of inmates reported between one and five days of pain prior to the appointment. 18% presented abscesses, of which 6% were pulp infections and 12% were periodontal. 15% of the interviewees presented inflamed gums. Only 4% presented facial inflammation, 1% reported facial inflammation of eight days, the remaining 3% just one day; 16% of inmates presented fever, which in this case was considered to be a temperature of over 37 C. The period when symptoms were present ranged from 1 to 30 days. 28% had difficulties in swallowing solids or fluids. 20% of the participants in the study presented difficulties in opening the mouth (lockjaw); 16% reported previous maxillary or mandibular trauma caused by: being struck with an open hand (4%), being struck with a fist (8%), a blow with a blunt instrument (1%), being kicked (2%) and falling to the floor (1%). Assessment of emergencies at the dental clinic during the SARS-COV-2 pandemic, 84% were found to be a false emergency, and only 16% were real, according to the results of the survey ( ). The gender variable distribution showed that 4% of the women and 80% of the man reported a false emergency. 16% of male cases were real emergencies ( ). The inmates who were studied and reported a real emergency at the clinic showed the following: 6% belonged 20-31 year old age group; 5% were 32-41 years old; 3% were 42-51 and 2% formed part of the 52-77 year old group ( ).
Studies of COVID-19 in odontology, especially in prisons are very rare, if we consider its impact on global health and the economy, given that the disease is a new one (Coronaviridae Study Group of the International Committee on Taxonomy of Viruses) . What studies there are lack homogeneity, randomness and repetition, which makes it difficult to come to decisions about the focuses and actions that are most appropriate for controlling disease transmission and limiting the potential consequences, which means that the articles that have been recently published have gaps and questions that can confuse dentists, especially those who work in general practice (with no speciality). This may be due to the fact that the articles that are published are specific to each dental speciality and are individual in nature . Before carrying out any kind of emergency dental care, the professional involved needs to be aware of the evolution of the pandemic in their area in order to provide effective care. Training of support staff on the importance of precautions and self-care is also recommended. The training should bring together concepts and improve preventive measures that help to improve dental care while mitigating the spread of this new virus . Professional public health measures in each country are dynamic; some countries can go as far as to block all dental care while other countries may not. Given the large amount of literature on the possible transmission of the virus in asymptomatic patients , , all patients should be treated as potentially infected and all dental practices should include adequate disinfection and control protocols. Many contexts of dental healthcare, such as mobile dental services for schools, remote communities, residences for the elderly, prisons, refuges for the homeless and refugee camps, and dental services in low-resource settings where lack of supplies is a constant challenge, make protective measures almost impossible. The risks of infectious aerosols are the basic factors behind all the changes made to current dental healthcare practice. This may mean the end of dentistry as we currently know it. At this stage of the pandemic, dentistry needs a concept of ongoing dental services that go as far as possible to avoid procedures that might lead to the production of infectious aerosols. Current evidence indicates three possible main routes of transmission of the virus in dental care settings: direct transmission from inhaling the virus via coughing, sneezing or droplets; via ocular, nasal and oral mucous membranes and via contaminated surfaces . All these routes of transmission are made easier and possibly amplified by aerosols created in a wide range of dental procedures , . The Occupational Safety and Health Administration (OSHA) considers that working environments where aerosols can be produced create a high or very high risk of infection from COVID-19 . The first experiences in dental care in China during the COVID-19 pandemic were instructive and revealing: they took swift and daring measures to contain the pandemic, including limited emergency services at a tertiary care centre offered under highly stringent precautions . The UK and other countries also established emergency care centres specialising in dental treatment , . However, the level of service infrastructure and provision was insufficient for general dental practice or training programmes in oral health in the USA or other parts of the world. The situations in developing countries are different from those in the first world. Today we know that the dental services of many countries with economic difficulties faced the challenge of a massive outbreak with general dental clinics, which normally (pre-pandemic) carried out all types of treatments ranging from sealants to tooth extraction. Now there is sufficient knowledge of which treatments should be carried out during a pandemic and which should not. The inmates at our prison received optimal medical, dental and psychiatric care during their incarceration, with or without signs of any orofacial disorder, prior to their social rehabilitation, thanks to the existence of a medical, dental and mental healthcare programme, backed up by the department of special programmes of the Secretary of State for Health. Such programmes enable all inmates to have access to services from the moment they enter prison, to receive a comprehensive medical, psychological and dental assessment, and be informed about their general and dental health status. Clinical evidence is used to offer information about orofacial problems, and clinical evidence is generated from intraoral images to draw up and complete the diagnosis, prepare and adequate treatment plan that backs up a dental health education and welfare programme. The fact that inmates are incarcerated enables dental care to be regularly and constantly maintained, including primary care services for oral health, via programmed appointments that enable health professionals to offer organised medical and dental care to every inmate. The first short-term benefit is the acquisition of better oral health habits, while also enabling the provision of basic and necessary dental procedures geared towards recovering oral health. In the mid-term, once the inmate’s dental health has been improved, the next stage is to motivate and supervise inmates to enable them to control their own oral health. Finally, in the long-term inmates can enjoy the benefits of good oral health and care for their teeth, which may in turn make their period of incarceration more bearable and enable them to more effectively reintegrate into society, with the corresponding impact on their emotional, economic and social status. However, the pandemic caused by the SARS-CoV-2 virus made it necessary to modify medical and dental care and, in accordance with the recommendations of the WHO and the Secretary of State for Health, hospitals were converted into centres for COVID-19, and the CERESO was no exception to this rule. The work of prison health professionals is to guarantee a level of health for inmates that matches that of members of the community, and the willingness of these professionals is not enough to bring about this end is not enough, a commitment on the part of administrative and political authorities is also required . To sum up, a validated version of the Assessment of a True Dental Emergency was applied in order to establish priorities for real dental emergencies. Bu doing so it was possible to delay and reorganise orofacial services without violating inmates’ human rights and carry out a real screening process for urgent cases in accordance with the health protocols established by the WHO and the essential oral health services of the Secretary of State for Health, to contribute as much as possible to containing the spread of the SARS-CoV-2 virus in the San Francisco Kobén CERESO.
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Laparoscopic vs. Robotic transabdominal adrenalectomy- propensity matched analysis and learning curve | 5b0eae4c-ba3b-4cbf-b941-f2b4a57c98c7 | 11950101 | Surgery[mh] | While LA is the standard of care for small adrenal tumors [ – ], more and more specialized centers have switched to RA in recent years . The widespread availability of robotic devices has encouraged centers to take advantage of the benefits of robotic surgery, such as the ability to perform precise movements in a limited working space, the reduction of tremor, orientation errors due to camera fixation, three-dimensional optics with a higher resolution and stereopsis, and of course the ergonomic benefits for the surgeon . However, RA has yet failed to show clear improvements with regard to estimated blood loss, conversion rate or complication rates compared to LA, while costs suggested to be higher than laparoscopic surgery . To date, there are only a few studies with small patient cohorts, thus, overall evaluation of possible advantages of RA remain unclear. A recent meta-analysis compared 747 RA to 415 LA and did not report a significant difference in conversion rate, estimated blood loss, morbidity and mortality with a slight shorter hospital stay of 0.5 days (2.88 d after LA comparted to 2.38 days after RA) and a longer operation time. The authors concluded that RA is a safe and effective procedure, but more studies are needed to discriminate the differences. We changed our own standard to RA in May 2023 and wanted to analyze costs (including disposables), outcomes as well as learning curves of RA compared to LA after one year.
Study design Cost parameters Propensity matching Statistical analysis The local ethics committee in Wuerzburg approved the study (No 88/11). Patients undergoing minimal invasive LA or RA (daVinci XI system) at the Department of Surgery, University Hospital Wuerzburg, between January 2018 and December 2023 were recruited from our prospective database (ENSAT register). Bilateral LA or RA were excluded. With 60 minimal invasive adrenalectomies performed per year the university hospital is regarded as specialized, high volume center . All patients were divided into two groups according to the type of surgery (LA versus RA). Baseline patient’s characteristics included age, body mass index, (BMI), gender, the Charlson comorbidity Index, tumor side localization, hormonal status, tumor size and prior abdominal surgery. The operations were performed by two robotic-experienced surgeons (> 50 robotic procedures) and experienced surgeons in LA (> 100 procedures). In addition, we analyzed the rate of conversion, operating time as well as complications according to the Clavien-Dindo classification. Perioperative parameters included, intraoperative RR fluctuations (> 160mmHg, < 90 mmHg), length of stay, early complications (within 30 days), postoperative C- reactive protein levels on the first and third and postoperative day. Furthermore, the difference in hemoglobin levels on the preoperative day and on the first day after the operation to validate intraoperative blood loss and mortality (within 90 days) were recorded.
In order to analyze the expenditure for each procedure, cost parameters were collected by the controlling department. These included the cost of consumables (surgical equipment such as robotic instruments, amortization of robotic and laparoscopic equipment, ultrasound dissectors, drapes, sutures and sterile supplies) and hospital-related costs (e.g. the charge per minute of surgery, the cost of a day in intensive care or a day on the regular ward).
To exclude possible confounders of the results, we used a propensity score matching analysis for lesion size and tumor localization, hormonal secretion status, BMI, and previous abdominal surgery.
We used IBM SPSS 28.0 (IBM SPSS, USA) for statistical analysis. Chi²-test (indicated by * in the tables) as well as single factor variance were used to detect differences between LA and RA. Significance was assumed for p < 0.05. Descriptive analyses included mean (MV), minimum (min), and maximum (max) values given as range, percentage, and standard deviation.
Baseline characteristics Perioperative results of RA and LA Propensity matched analysis of laparoscopic and robotic adrenalectomy Learning curve of RA Cost analysis of RA compared to LA A detailed analysis of the medical disposables used for either LA or RA showed, that disposable materials in RA are slightly more expensive. In LA the overall cost of medical disposables and amortization of instruments is 608.86 € compared to 713.76 € in robotic surgery. But due to the shorter length of stay over all costs are in total in favor of robotic surgery where average costs went down to 3466.36 € compared to 4536.35 € in LA.
During the study period (2018 and 2023), 313 patients underwent minimal invasive transabdominal adrenalectomy. 23 bilateral adrenalectomies were excluded. Of the remaining 290 patients, 263 underwent LA and 27 RA. In the first years, all patients underwent LA, while RA was introduced in May 2023, with most of the patients receiving robotic-assisted surgery for the rest of the observational period. Baseline characteristics of laparoscopic operated patients ( n = 263) and robotic patients ( n = 27) are comparable and shown in Table . Although there were significantly more women (23/27 (85.2%) in RA compared to 156/263 (59.3%) in LA) and tumor size was larger in the laparoscopic group (3.5 cm compared to 2.5 cm), other relevant factors such as age, BMI, Charlson comorbidity index, hormone secretion and previous abdominal surgery did not differ.
The perioperative outcomes of patients who underwent LA or RA were analyzed and are shown in Table . Perioperative morbidity and operation time did not differ in RA, though operation time was slightly longer in LA (72.39 ± 25.65 min in the laparoscopic vs. 81.50 ± 52.60 min in the robotic group; p = 0.12). In addition, perioperative markers such as C- reactive protein or hemoglobin levels (as a marker of perioperative blood loss) did not show significant different gradients, nor relevant fluctuations in RR were observed. However, length of stay (3.50d ± 1.81d) and stay on intensive care (0.41d ± 0.58d) was significantly shorter in RA compared to LA (4.61d ± 2.75d length of stay and 1.0d ± 1.79d stay on intensive care; p = 0.04) (Fig. ). An overall low comprehensive complication index according to the Clavien-Dindo classification with 4.65 ± 13.0 points in the laparoscopic group compared 5.84 ± 12.9 points in the robotic group revealed that minimal invasive adrenalectomy is a surgical safe procedure with an overall low morbidity. None of the patients died within 90 days after surgery. Three patients in the laparoscopic (1.9%) and one patient (3.7%) in the robotic group were reoperated due to postoperative hematoma or bleeding. Furthermore, two patients were converted to open surgery in the LA and none in RA.
To exclude possible confounders on outcomeior abdominal surgery (Table ). This propensity-matched analysis revealed that there were no significant differences between LA and RA. In contrast, the trend of a shorter operation time in LA was even less pronounced if prior operations, tumor size, localization and BMI were taken into account with an operation time of 83.90 ± 33.18 min in LA compared to 81.50 ± 52.60 min in RA. Furthermore, the significant shorter length of stay was even more relevant in the propensity matched group, where laparoscopic patients stayed 2 days longer than the robotic patients.
The learning curve of RA of the two attending, robotic-experienced surgeons is shown in Fig. . The average operation time went down from 91 ± 22.3 min to 59.4 ± 9.8 min after the 6th operation.
Minimal invasive, LA is the surgical standard of care for adrenal tumors. While to date LA is well established to date, evidence on RA remain limited and important questions unanswered. Here we provide a substantial verification on the robotic approach in a large patient cohort of patients including propensity score matching. Based on that, our study demonstrates that both RA and LA are safe and feasible surgical procedures in a referral center but length of stay is shorter in RA. Although in our cohort RA did not show a distinctive advantage in our cohort, there might be benefits that should be considered. Firstly, in the long term the operation time might be shorter in RA. The duration of a RA after the learning curve was significantly shorter compared to LA. This is in line with other groups, who have also reported a shorter operative time . This fact, in contrast to many other robotic procedures, might be easily explained by the ability to articulate the instruments and the use of a fourth arm for additional counter traction especially on the right side with limited retroperitoneal space. The relatively short learning curve of RA must also be taken into account. In LA, a significantly shorter operative time has been reported after 30 to 40 procedures, while other papers have shown a similarly short learning curve in RA compared to our study [ – ]. However, our results may be biased because the surgeons were experienced in both LA (more than 100 adrenalectomies per surgeon) and robotic surgery (more than 50 robotic procedures per surgeon). Experienced surgeons may adapt quickly to RA, but inexperienced surgeons may have difficulty overcoming the learning curve. At last costs must be considered. Robotic systems might have limitation, since acquisition and maintaining costs are high, however, careful evaluation of its financial implications is needed. While other groups postulated that RA has lower complication rates compared to LA [ – ], they concluded that despite the higher costs of RA is cost-effective and economically sustainable in high-volume centers especially when performed for challenging cases . Similarly, our cost analysis showed that robotic surgery is only slightly more expensive than laparoscopic surgery when using an ultrasonic dissection device for LA. Furthermore, when total hospital costs are considered, RA is cost effective due to the shorter length of stay. In conclusion, our study supports the fact that RA is as safe and feasible as LA. It may also have advantages over laparoscopic adrenalectomy in terms of shorter length of stay, shorter learning curve and similar costs.
The present study has limitations. First, the study was conducted in a referral centre for adrenal pathologies. Second, the study was retrospective. However, the strengths of the study are the large sample size for a rare disease and the large collection of clinical data.
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Impact of molecular diagnostic tests on diagnostic and treatment delays in tuberculosis: a systematic review and meta-analysis | 32f255f2-a1c8-45f0-9f22-8a96e4bafe21 | 9748908 | Pathology[mh] | In the last two decades, there has been a global push to end the tuberculosis (TB) epidemic by setting aggressive targets with the End TB Strategy . Nonetheless, in 2020, there were an estimated 9.9 million TB cases and 1.3 million deaths, of which an estimated 40% went undiagnosed . These missed diagnoses, made worse by the ongoing COVID-19 pandemic, perpetuate transmission and present significant challenges in ending TB . Implementing diagnostic tools that improve detection and reduce diagnostic and treatment delays is critical in overcoming these gaps in TB care . GeneXpert MTB/RIF® and MTB/RIF Ultra® (Xpert) and line probe assays (LPA) are commercial nucleic acid amplification tests (NAATs) that have good diagnostic accuracy with the capacity to diagnose drug sensitive (DS-TB) and drug resistant TB (DR-TB) within 1–2 days of sample processing . Anticipating improvements in accurate and timely TB diagnosis, these NAATs were recommended by the World Health Organization (WHO) . Since then, unprecedented efforts have been made by National Tuberculosis Programs (NTPs) across the globe to scale up these tests and included them as part of the routine TB diagnostic algorithms . These NAATs have proven to have high accuracy, and research has increasingly focused on studying their actual clinical impact . While there are systematic reviews on the diagnostic accuracy of Xpert and LPAs , and others that separately describe diagnostic and treatment delays experienced by TB patients , no study has summarized the impact of NAATs on reducing time delays in diagnosis and treatment of TB. Therefore, the main objective of our systematic review was to summarize the available quantitative evidence on the impact of NAATs on diagnostic and treatment delays compared to that of the standard of care for DS-TB and DR-TB. As the secondary objective, we investigated the potential sources of heterogeneity on the effect estimates, including the period the tests were used (pre-2015, post 2015), empiric treatment rate, HIV prevalence, healthcare level, and type of study design (randomized controlled trial, observational study design). We also describe methodological areas of concern in assessing time delays, an aspect that has not been adequately addressed in previous systematic reviews of diagnostic delays in TB. Study selection criteria and operational definitionsStudy search strategy, study selection, and data extractionQuality assessment of time delay estimatesData synthesis and meta-analysis We calculated overall medians and IQRs of diagnostic and treatment initiation delay for each diagnostic test (Xpert vs. smear, LPA vs. any culture DST methods) from the medians and means reported by the individual studies. Additionally, using the extracted raw data, we applied the Mann–Whitney U test on overall medians to determine the statistical significance of the median time estimates between the index and comparator tests. We assumed no confounding in the primary studies. We then conducted a meta-analysis using the quantile estimation (QE) method developed by McGrath et al. to assess the absolute reduction in diagnostic and treatment initiation delay using NAATs . The method involves estimating the variance of the difference of medians of each study and pooling them using the standard inverse variance method. Time to event data are non-normally distributed variables that are primarily reported in medians and IQRs. As units of delay measurements (days) were uniform across all studies, the effect size was chosen to be the raw difference of medians in time delay for both diagnostic and treatment initiation delays. We used a random effects model because the studies differed importantly in characteristics that may lead to variations in the effect size . Between-study heterogeneity was estimated by the method of restricted maximum likelihood. Since this method requires complete data from median (or mean), IQR (or SD), and sample size, studies that did not report all the data points were excluded for the analysis. Given the multifactorial nature of the studies, we also evaluated the heterogeneity based on the I-squared statistic, where a value greater than 75% is considered to be considerably heterogeneous . We conducted subgroup analyses to identify possible sources of heterogeneity and to assess key factors (pre-2015 vs. post-2015, RCT vs. observational, etc.) that can variably influence the magnitude of our effect size estimate. We specifically chose 2015 as our cut-off time point not only because this was the cut-off for the original systematic review but also enough time had passed since the recommendation to see the effects of the implementation of NAATs in research studies. Further, we assessed for “small study effects” and publication bias with funnel plots followed by Egger’s test to determine their symmetry. We managed and analysed the data using Microsoft Excel 16 (Microsoft Corporation, USA) and R version 4.1.1 (R Foundation for Statistical Learning, Austria). Prior to the review, we developed a conceptual framework for classification of essential time delay components and definitions (Fig. ). This framework standardized time delays and provided structural guidance in assessing time delays reported in the studies included in this review. We defined diagnostic delay as the time between initial patient contact with a clinic or sputum collection to reporting of results. Treatment delay was defined as the time between results and initiation of anti-TB treatment. And the combination of diagnostic delay and treatment delay was referred to as treatment initiation delay . Our review focused on the impact of the World Health Organization (WHO)-recommended rapid diagnostics (WRD), specifically Xpert® MTB/RIF and MTB/RIF Ultra assay (Xpert) and GenoType MTBDR plus and Inno-LiPA RifTB (both referred to as LPA here on), because of their rapid uptake at the global level . Several other tests have been recommended since 2020, but we did not include them in our systematic review because data is still limited . We included only peer-reviewed studies that assessed time delays in the process of diagnosis and treatment of DS-TB and DR-TB with the index test as NAAT and a respective comparator test (e.g., smear for Xpert and culture DST for LPAs). We did not restrict our studies based on geography, settings, language, or type of study design. We excluded studies if they: (1) did not include primary data; (2) did not report all data necessary for meta-analysis; (3) were reviews or modelling studies; (4) only reported ‘run-time’ or turnaround time of the test (e.g., “2 h to run” Xpert test); and (5) focused on childhood or extra-pulmonary TB. For conference abstracts, we contacted the authors to see if there was a manuscript in preparation to obtain relevant data. Similarly, we requested original data from the authors when a study did not report time delay estimates as per our study requirements. The present systematic review is an update to the systematic review published in the lead author’s (HS) doctoral thesis in 2016 . The original and updated search were undertaken on January 31, 2015, and October 12, 2020, respectively. We identified eligible studies from MEDLINE, EMBASE, Web of Science, and the Global Health databases that included terms associated with time, like “delay” and “time to treatment” (see Additional file for the complete search strategy). We also consulted references of included articles and previous systematic reviews focusing on the diagnostic accuracy of NAATs, and experts in the fields of TB diagnostics to identify additional studies not included in the database search. After removing duplicates, two reviewers (SGC, ZZQ, or HS—original review; JSL, JHL, or TG—updated review) independently screened titles and abstracts, followed by full-text review for inclusion (HS, SGS—original review; JSL, JHL—updated review). Any discrepancies were resolved by consensus or, in case of the updated review, a third reviewer (HS, TG). Google Forms (Google LLC, Mountain View, CA, USA) was used for the initial review, but in the updated review, this data was incorporated into Covidence (Veritas Health Innovation, Melbourne, Australia) to manage the review and extract data . The data extraction tools were pilot tested, using five studies in the full text review pool, prior to conducting full data extraction. A set of reviewers (HS—original review; JL, JHL—updated review) extracted the data before it was examined by separate reviewers (SGS—original review, TG—updated review) to resolve any discrepancies in the extracted data. We extracted data on study design, geographic setting, operational context, time delays for both the index and comparator tests, and delay definitions. Units of time were converted into the number of days. An example data extraction tool is available in Additional file . Unlike quality assessment tools for diagnostic accuracy studies, there is currently no established method or checklist that can be used to assess the quality of studies investigating time delays or time to event study outcomes . Therefore, we developed a matrix of key methodologic and contextual information necessary to determine the usefulness and comparability of the time delay reported. These included (1) provision of a clear definition of measuring time delay and reporting the time delay estimates (“delay definition”); (2) use of appropriate statistical methods to report and assess changes in time delays (“statistical methods”); (3) evaluating time estimates alongside patient-important outcomes (“patient important outcomes”), which included culture conversion, TB treatment outcomes, infection control and/or contact tracing. The provision of a clear delay definition was a binary variable with “Yes” and “No” options, where “Yes” indicated that the time delay term was defined clearly indicating its start and end time points with the delay estimate. The other two quality indicators were ranked on a high–medium–low scale. For the statistical method assessment, high quality studies evaluated the distribution of time delay and whether it used proper statistical methods [randomized controlled trial (RCT) or propensity score method for observational studies] that adjust estimates for proper comparison with a measure of variance to assess time delays between the index and the comparator test. Medium-quality studies evaluated the distribution of time delay with uncertainty estimates but did not use appropriate statistical methods for comparative assessment of time delays. And low-quality studies neither evaluated the distribution nor compared the time delay. For patient-important outcomes, high-quality studies analysed the relative risk or odds of improvement in culture conversion with the amount of time saved in TB treatment initiation. Medium quality studies reported time estimate alongside patient-important outcomes but without direct analysis, and low-quality studies did not consider patient-important outcomes at all. Search resultsDescription of included studiesQuality assessment of time delay estimatesImpact of NAATs on delay For DS-TB analysis, 12 studies were included in the primary analysis for diagnostic delay, and 18 studies were included for treatment initiation delay. The overall median diagnostic delay for smear and Xpert were 3 days and 1.04 days, respectively. The overall median treatment initiation delay for smear and Xpert were 6 days and 4.5 days, respectively. A random effects meta-analysis of the difference of medians showed that the use of Xpert did not show a statistically significant reduction in diagnostic delay [1.79 days (95% CI − 0.27 to 3.85)] compared to smear but showed a statistically significant reduction in treatment initiation delay by 2.55 days (95% CI 0.54–4.56) (Figs. and ). For DR-TB analysis, 13 studies were included in diagnostic delays and 12 studies were included in treatment initiation delays. The overall median diagnostic delay for culture DST and LPA were 54 days and 11 days, respectively. The overall median treatment initiation delay for culture DST and LPA were 78 days and 28 days, respectively. A random effects meta-analysis of the difference of medians showed that, in comparison with culture DST, the use of LPA significantly reduced diagnostic delay by 40.09 days (95% CI 26.82–53.37) and treatment initiation delay by 45.32 days (95% CI 30.27–60.37) (Figs. and ). I 2 value of 99.79% and 97.22% for diagnostic and treatment initiation delay indicated considerable heterogeneity. Comparing the studies from the two different phases of the review (pre-/post-2015), we found no statistical significance in the reduction of diagnostic delays but observed statistical significance in the reduction of treatment initiation delay with a median difference of 2.54 days (95% CI 0.45–4.62) for post-2015 studies and 5.04 days (95% CI 0.09–9.99) for pre-2015 studies. Similarly, subgroup analysis based on study design showed a statistically significant reduction in treatment initiation delay in the RCT group [2.85 days (95% CI 1.16–4.55)] but not in the observational group [1.67 days (95% CI − 1.70 to 5.05)]. When classifying studies by the healthcare systems level, Xpert did not provide meaningful reduction in treatment initiation delay regardless of the location of its placement: 1.27 days (95% CI − 1.45 to 4.00) for primary health care centres and 5.27 days (95% CI − 1.06 to 11.60) for tertiary hospitals. When grouped by POCT status, Xpert test implemented as a POCT service showed statistically significant reductions in treatment initiation delay compared to non-POCT programs. All sub-group analyses with greater than 2 studies showed I 2 values greater than 89%, suggesting considerable heterogeneity (Tables and ). After removing duplicates, we identified 14,776 (original review—7995; updated review—6781) titles and abstracts eligible for title and abstract screening. Of these, 323 were selected for full text review during screening. A total of 45 studies (26 DS-TB and 20 DR-TB) with relevant time delay estimates were ultimately included in this review (Fig. ). Of the 45 studies included in this review, 21 (81%) DS-TB and 15 (75%) DR-TB studies were conducted in Low-and Middle-Income Countries (LMICs) (Tables and ). One study had estimates for both DS-TB and DR-TB . Overall, half of the studies (17 DS-TB, 7 DR-TB) were conducted in the African region with over two thirds of those in South Africa (n = 15). HIV prevalence was reported by 31 (19 DS-TB, 12 DR-TB) studies, of which about half (16 DS-TB, 4 DR-TB) reported a HIV prevalence of over than 50%. Amongst the DS-TB studies, 7 studies (27%) implemented Xpert as a point-of-care testing (POCT) program, and 15 studies (58%) implemented Xpert on-site, within walking distance of a primary care program or a laboratory. The studies had considerable methodological heterogeneity in the definitions of time delays. When classifying reported time delays according to our operational definitions and by study design, no study reported all sub-components of time delay. All studies evaluating treatment delay used TB treatment initiation time but start and end points for diagnostic delay varied across studies (Tables and ). Overall, 13 of the 45 studies did not provide a clear definition of the time delay estimates reported (Table ). Amongst studies included in the DS-TB analysis, 6 (23%) studies employed a randomized control trial (RCT), and 2 studies (8%) were quasi-experimental using pre- and post-implementation study designs. One study used a single-arm interventional pilot study (4%), and the remaining 15 studies were observational (58%). All the studies in the DR-TB analysis were observational. In the use of proper statistical methods for measurement and reporting of delay estimates, 18 studies ranked high, 23 ranked medium, and 2 ranked low. In the evaluation of time estimates alongside patient important outcomes, 7 ranked high, 18 ranked medium, and 18 ranked low. In all funnel plots (Additional file ), there were several studies falling outside of the 95% CI, impacting the visualized asymmetry. This may be due to considerable heterogeneity (I 2 > 99%) of the studies. However, Egger’s tests—used to assess whether there are systematic differences between high- and low-precision studies—demonstrated no clear evidence of “small study effects.” (p = 0.085–0.462). Principal findingsStrengths and limitationsWhile there are several patient-important impact measures for new diagnostic tests , time delay estimates provide direct measure of the timeliness of TB care. To our knowledge, our systematic review of 45 studies is the first to comparatively synthesize and quantify reductions in delays in diagnosis and treatment of DS and DR-TB when the WHO recommended NAATs are used instead of smear (DS-TB) or culture DST (DR-TB). Our random effectives meta-analysis of the differences of median times showed that the use of NAATs improved treatment initiation delay for patients investigated for both DS and DR-TB; however, this benefit was not seen for diagnostic delay for DS-TB (Xpert vs. smear). We also found that the degree of benefit in reducing delays in using NAATs for TB care was highly variable and dependent on how the tests were implemented (e.g., laboratory-based vs. POCT), differences in study design to evaluate impact of NAATs on TB care delays, and large variations in how delays were defined and quantified. In principle, Xpert and smear are “same-day” tests; therefore, expected reduction in diagnostic delays may be limited for Xpert. As such, in our meta-analysis, we did not find significant reduction in diagnostic delays when using Xpert compared to smear [1.79 days (95% CI − 0.27 to 3.85)]. For treatment delays, our analysis of 18 studies showed that Xpert reduced treatment initiation delays for DS-TB by 2.55 days (95% CI 0.54–4.56) compared to smear, but the degree of this effect was highly variable depending on how and where Xpert was deployed within the health care system. Particularly, in our sub-group analysis, we found that the use of Xpert as non-POCT (at any levels of health system) did not show meaningful improvement in DS-TB treatment initiation delay. Moreover, the ‘hub-and-spokes’ model—where patient samples for Xpert from several community health centres (spokes) are referred to a centralized laboratory (hub) in the system—for Xpert testing evaluated in earlier studies has shown limited impact on improving and optimizing the timeliness of TB care due to operational barriers causing further delays , de-prioritization of Xpert use as an initial test in the national algorithms , and continued high empiric treatment rates in certain settings. In contrast to DS-TB, use of LPA for DR-TB care had resulted in large reduction in delays for DR-TB care. Our meta-analysis results found that use of LPA drastically reduced overall DR-TB care delays by 45.32 days (95% CI 30.27–60.37). This was mainly due to prolonged delays associated with conventional DR-TB diagnostics (culture DST) that takes weeks to diagnose and treat DR-TB patients. However, reduction of these delays were not solely due the implementation of the technology alone. In an earlier phases of LPA implementation in South Africa, use of LPA for DR-TB care were much restricted and centralized at higher levels of the health and laboratory system, and caused treatment initiation delays of more than 50 days . DR-TB care delays gradually improved to 28 days (IQR: 16–40) through the 3-year DR-TB care decentralization program, which included streamlining LPA testing in the clinical practice (years 2009–2011). Moreover, studies from settings with more established healthcare infrastructure (e.g., China and South Korea) also found that operational challenges diminished the potential benefit of rapid molecular testing in improving DR-TB care delays . For the meta-analysis, we used the Quantile Estimation (QE) method because it had excellent performance in simulation studies that were motivated by our systematic review . One advantage compared to more traditional approaches based on meta-analysing the difference of means is that the QE method uses an effect size that is typically reported by the primary studies (i.e., the difference of medians) rather than one that must be estimated from the summary data of the primary studies (i.e., the difference of means). However, our meta-analysis results should be interpreted with caution because considerable statistical power was lost when restricting to studies that presented all the necessary data for estimating the variance of the difference of medians. Also, the high level of clinical (e.g. participants, outcomes) and methodological heterogeneity (e.g. study design, defining and reporting of time delays) in the studies included in our review translated into high I 2 values in all of our meta-analyses results, making generalized interpretation of our summary estimates difficult. We also advise caution in the interpretation of our subgroup analyses because these confounders often complicate the interpretation and lead to wrong conclusions . Delays in TB care occur due to a wide range of patient and health systems risk factors. Studies included in our review did not comparatively assess and adjust for risk factors associated with time delays for both the index (Xpert or LPA) and the comparator (smear or culture DST). This may be because time delay estimates were not the primary outcomes in most of the studies, and thus lacking proper analytical assessment of these outcome measures. Therefore, we were limited to sub-group analyses on key study-level attributes (e.g., HIV prevalence, empiric treatment rate, Xpert placement strategy, and study design), which were highly heterogenous and in many cases, inconclusive in showing that Xpert improved delays in TB care. Moreover, our findings are subject to potential confounding issues—at both health systems (e.g., differences in healthcare system infrastructure, TB care practices, implementation strategies of the index tests) and patient level factors (e.g., symptom levels, age, care-seeking behaviours)—which may bias our effect estimates (number of days reduced in diagnostic and treatment initiation delays) towards or away from the null. Given these reasons, generalizability of our findings may be limited. Likewise, our review underscores a need for more research investigating health systems and patient factors that can impact delays in TB care during and after the implementation of diagnostic tests and strategies that aim to improve the timeliness and quality of TB care. Lastly, despite carrying out comprehensive searches and considering non-English studies, we may have missed some studies in our review. Therefore, we cannot rule out potential publication bias. In our study, we also investigated consistencies in defining and reporting of time delays across studies with a framework developed as part of our study (Fig. ). In our quality assessment of the studies reporting time delay estimates (Table ), we found considerable heterogeneity in defining time delays and close to 30% of studies (13) reported delay estimates without providing clear definitions. Many of the studies included in our review used the same terms to define different components of the delay. For instance, “turnaround time”, “time to detection”, and “laboratory processing time” were used to describe the time from specimen receipt by the lab to test result at the lab, while others employed these same terms to define diagnostic delay, time from specimen collection to notifying the clinic of the test result. In addition, several studies included in our review did not include or inappropriately reported uncertainty ranges (e.g., no IQRs or reported means with IQRs). As time data may be highly skewed, standardizing the practice of reporting delay estimates as medians with their variances or other measures of spread (e.g., IQR or range) can help facilitate synthesis of these studies. Many of these issues have been previously reported by other systematic reviews on TB care delays and our findings reemphasizes the importance in standardizing how TB care delays are defined, measured, and reported . The global rollout of NAATs has dramatically changed the landscape of TB diagnosis in high TB burden settings with improvements in the TB diagnostic infrastructure and the quality of TB prevention and care programs. Our systematic review findings suggest that implementation of NAATs have resulted in a noticeable reduction in delays for TB treatment compared to the conventional methods. However, these improvements did not fully realize the potential benefits of NAATs because of health system limitations . Additionally, we identified methodological concerns in reporting of time delay estimates and emphasize the need to standardize and promote their consistent reporting. Additional file 1. Systematic review search strategy. The detailed search strategy for each database searched for this review. Additional file 2. Funnel plots. Outputs from the analysis of risk of bias. Additional file 3. Data extraction tool. The template for the data extraction tool. |
Evaluation of the Accuracy of Cameriere, Modified Cameriere and Willems and Blenkin-Evans Methods for Turkish Children | 01451b74-55c1-462e-bb85-9da55f1eb760 | 11806340 | Dentistry[mh] | Children and adolescents may encounter situations such as forced marriage, illegal adoption, human trafficking, involvement in crime, or losing their families due to major natural disasters or wars. These circumstances can lead to becoming undocumented migrants or losing documents that prove their identity. In such cases, the absence of chronological age (CA) information highlights the importance of determining physiological age. , Furthermore, under Turkish law, individuals' criminal responsibility can vary significantly depending on their age group, with ages 12 and 15 being particularly crucial distinctions. In dentistry, determining physiological age is crucial for diagnosis and treatment planning, especially for paediatric and orthodontic specialists. Physiological age is based on the developmental level of various systems and tissues. Due to factors such as race, hormones, genetics, and nutrition, there can be discrepancies between an individual's CA and physiological age. Various methods have been developed to estimate physiological age from radiographs, such as evaluating skeletal or dental development. , Each dental age estimation method is generally developed based on individuals from different ethnic backgrounds. The Demirjian Method (DM) was established using the French-Canadian child population, while the Willems Method (WM) is a modification of DM tailored to the Belgian-Caucasian population. The Blenkin-Evans Method (BEM) was adapted from DM to suit Australian children. These methods are applied using the mineralisation chart created by Demirjian et al. On the other hand, the Cameriere Method (CM) is a measurement-based approach initially developed for Italian children and subsequently modified by Kış et al. for Turkish children (Modified Cameriere Method (MCM)). , , , , , , It is known that different genetic and environmental factors can lead individuals with the same CA to have other body characteristics and developmental levels. Therefore, before using an age estimation method for legal purposes in any population, it is essential to validate the accuracy and applicability of the method for that specific population. This study aims to compare the agreement between dental age estimated using the Cameriere method (CM), Modified Cameriere method (MCM), Willems method (WM), and Blenkin-Evans method (BEM) on panoramic radiographs of children from Karaman province and its surroundings in the Central Anatolia region of Turkiye with their CA. The study seeks to identify which method provides the closest estimate of the actual age of children in our region, aiming to assess the suitability of these methods for age estimation and serve as a reference for future studies. Ethical approvalStudy population and sampling criteriaCalculation of chronological age (CA)Application of Cameriere's method (CM)Application of the modified Cameriere method (MCM)Application of Willems method (WM)Application of Blenkin and Evans method (BEM)Statistical analysisThis cross-sectional retrospective study was conducted according to the Declaration of Helsinki and approved by the Karamanoğlu Mehmetbey University Faculty of Medicine Local Scientific Medical Research Ethics Committee with protocol number 09-2023/13. Before radiography, written informed consent was obtained from all patients' parents. No personal information other than the patient's date of birth, gender, and the date of the radiograph was used in the study. This study included 953 individuals aged between 6 and 14.99 years to evaluate all 4 methods who presented for routine examination and follow-up at the Karamanoğlu Mehmetbey University Faculty of Dentistry between August 2021 and September 2023. All digital panoramic images examined in the study were obtained using a PCH-2500 (Vatech, Gyeonggi-do, Korea) digital panoramic x-ray machine with 60 to 75 kVp, 5 to 10 mA, and 15 s radiation time parameters. A total of 953 panoramic radiographs were saved in 'TIF' (Tagged Image File) format from the faculty's radiograph archive to the study computer (3.10 GHz Intel 10th Generation i5 with 8 GB RAM, Windows 10 Professional operating system, and a 21.5-inch flat panel color screen (Lenovo ThinkVision S22e-20), 1920 × 1080 pixels) and categorised into folders based on age groups and gender. The inclusion criteria were as follows: • No anomalies or bilateral pathologies in the maxillofacial region or history of trauma, • No history of orthodontic treatment, • No bilateral permanent tooth missing, decay, restoration, root canal treatment, or apical pathology, • Systemically healthy individuals, • Non-migrants (Those whose TR Identity Number does not start with 99 or 98 or who do not have a temporary asylum card) Of these, 337 panoramic radiographs were excluded from the study due to not meeting the inclusion criteria (n = 26 for bilateral tooth missing/decay/root canal treatment, n = 14 with a history of trauma, n = 7 with a history of orthodontic treatment, n = 10 with pathology, n = 280 radiographs were not Grade 1 quality based on the UK National Radiological Protection Board, n = 0 immigrants). The diagnostic quality of the radiographs was evaluated by an oral and maxillofacial radiologist with 8 years of experience (M.G.) based on the UK National Radiological Protection Board criteria. Only Grade 1 radiographs were included in the study. As a result, the 616 individuals included in the study were divided into 18 groups as follows: girls aged 6-6.99 years (6F) and boys (6M), girls aged 7 to 7.99 years (7F) and boys (7M), girls aged 8 to 8.99 years (8F) and boys (8M), girls aged 9 to 9.99 years (9F) and boys (9M), girls aged 10 to 10.99 years (10F) and boys (10M), girls aged 11 to 11.99 years (11F) and boys (11M), girls aged 12 to 12.99 years (12F) and boys (12M), girls aged 13 to 13.99 years (13F) and boys (13M), girls aged 14 to 14.99 years (14F) and boys (14M). For this study, the power value calculation was based on a study by Şahin et al. using the G*Power 3.1 software package (v.3.1.9.7, Universitat Kiel, Kiel, Germany). With a 5% margin of error, a 95% confidence interval, and an effect size of 0.25, the sample size needed was determined to be a minimum of 486 across 18 groups for a power value of 95%. CA was calculated by subtracting the date of birth from the date the radiograph was taken. For ease of calculation, it was expressed in decimals (e.g., if the individual's age was 10 years and 3 months at the time of the radiograph, they were included in the 10-year age group as 10.25 years). The apical width of the left mandibular 7 teeth (Ai, i = 1, . . ., 7) and the length of the teeth (Li, i = 1, . . ., 7) were measured using the Image J program (Image J, NIH, Maryland, USA) with × 150 magnification on panoramic images as shown in . These measurements were then substituted into the relevant formula (Age = 8.971 + 0.375g + 1.631 × 5 + 0.674N0 – 1.034s – 0.176sN0) to determine the individual's Estimated Age (EA) (xi = Ai/Li (i = 1, . . ., 7), X5 = A5/L5, s: the sum of the Ai/Li of the open apices, g: 1 for boys and 0 for girls, N0: the number of teeth with complete root development). For teeth with two roots, the apical widths of both roots were summed to calculate the tooth's Ai. The apical width and tooth length data used for the Cameriere method were substituted into the relevant formula modified by Kış and colleagues for Turkish children (Age = 9.876 + 0.361g + 0.663N0 – 0.927s – 0.057N0s) to determine the individual's EA. The mineralisation stages of the left mandibular 7 teeth on the panoramic radiographs were assessed using the mineralisation chart created by Demirjian et al., and the developmental stages of the teeth were determined. The scores from the gender-specific tables created by Willems et al. were summed to determine the individual's EA. The measurement data used for the WM were also utilised for the BEM. The developmental stages of the teeth labeled as A, B, C, D, E, F, G, and H in the WM were converted to 1, 2, 3, 4, 5, 6, 7, and 8, respectively, for the BEM. Simplified Maturity Scores (SMS) were obtained by summing these numbers, and the individual's EA was determined using gender-specific tables prepared by Blenkin and Evans. For all methods, if a tooth in the left mandibular region was missing, decayed, had dilaceration or apical pathology, or had fillings or root canal treatment, the measurements of the corresponding tooth on the right mandibular side were used instead. After the initial measurements for each method, 62 randomly selected individuals from the same study group were repeated under the same conditions by the same researcher, a paediatric dentist with 8 years of experience (T.N.Ş.), four weeks later to assess intra-observer reliability. The RANDBETWEEN formula was used for randomisation in Microsoft Excel (Microsoft Corp.). Inter-observer reliability was statistically evaluated by comparing the measurements taken by a second observer (M.G.) under the same conditions using the same radiographs with those taken by the first observer (T.N.Ş.). All statistical analyses for the study were conducted using SPSS 27.0 (IBM Inc). Descriptive statistics were presented as the mean absolute error, standard deviation, median, minimum (min), and maximum (max). The Kruskal-Wallis test was used to compare the estimated age values across age groups. Pairwise comparisons between age groups were performed based on the critical difference post-hoc analyses of the Kruskal-Wallis test. Spearman's Rho correlation analysis was applied to determine the correlation values between the estimated age values obtained from different methods. To assess intra-observer and inter-observer reliability, the Intra-Interclass Correlation Coefficient (ICC) analysis was conducted for the apical width and length measurements in the CM and MCM, for the developmental stage data of the teeth used in the WM and BEM, Kendall's Tau-b, Somer's d, and Gamma analyses were performed. A type-I error value of p<0.05 was considered statistically significant in all analyses. A total of 616 children were included in the study, with 52.2% being girls (n = 321) and 47.8% boys (n = 295). The distribution of the number of patients across the age groups was relatively balanced, as shown in . The age groups with the highest number of individuals were observed to be 14 years (14.6%) and 8 years (13.1%) among girls, and 8 years (13.2%) and 13 years (13.2%) among boys. The EAs obtained using CM, MCM, WM, and BEM were compared for each gender and age group. Significant differences were found for the ages estimated by each method for both genders ( p < .001). Consequently, the test statistics of the applied analyses were compared. Accordingly, the test statistics for girls were as follows: CM (293.49), MCM (292.48), WM (290.82), and BEM (289.67) . When the test statistics in males were analysed, it was observed that the highest result belonged to CM (267.36). After that, the EA obtained with MCM (267.20), WM (262.02) and BEM (260.19) were very close to each other . According to the correlation analysis, a very high correlation was found between the actual age values of the individuals and the age values obtained by different methods in both boys and girls. In girls, the highest correlation value was calculated between CA and EA obtained by CM ( r = 0.957). In boys, CM was the EA most highly correlated with CA ( r = 0.951). Very similar correlation values were obtained with MCM ( r = 0.950). For both genders, the highest correlated values among the methods were found between CM and MCM ( r = 0.996). The differences between the CA values of the individuals and the age values estimated by different methods were taken. The difference values were compared according to age groups. In girls, the difference between CA and CM-derived EA values was significantly different ( p < .001). Up to 10 years of age, the difference values were negative, whereas positive age values were observed in the following age groups. The highest mean difference was observed in patients aged 14 years. The lowest mean age difference was observed in patients aged 8 years. Since MCM age values were estimated to be greater than CA values, a positive mean age difference was obtained only for the 14-year-old patient group. The mean values were higher than the mean values of the difference with CM. The mean difference between MCM-EA, WM-EA and CA values differed significantly between age groups ( p < .001). However, since the mean values obtained for the age groups were close to each other, only the 13th and 14th age groups and the 7th and 10th age groups had significantly different age group pairs in pairwise comparisons. The lowest difference was observed in the 13th age group, and the highest difference value was observed in the 11th age group. Age differences estimated by BEM significantly differed between age groups ( p < .001). Entirely a large number of pairwise comparison results were obtained. A positive age difference was found only for the 14-year-old age group. The lowest age difference was for the 9-year-old age group. When analysed as all girls regardless of age group, the mean CM difference was 0.22, the mean MCM difference was -0.49, the mean WM difference was -0.45, and the mean BEM difference was -0.20 . In boys, the mean difference between CA and age values obtained with four different estimation methods showed a significant difference between age groups ( p < .001). The highest difference between age groups was realised with CM. The mean values of the 10, 11 and 14 age groups generally differed from the other age groups. The mean differences with MCM were generally close to each other ( p < .001). However, it was observed that the value of the 14th age group was different in general. A significant difference was observed between age groups for WM ( p < .001). However, since the mean values of 6, 10, 11 and 14 age groups were very close to zero and even the difference value of 10 age group was 0, it was understood that the prediction was quite good. The mean values of age difference for BEM showed a significant difference between age groups ( p < .001). The values of the 6 and 14 age groups were quite close to CA. The 6th age group value generally differed from the other age groups . When analysed as all boys without considering age groups, the mean CM difference was calculated as 0.17, MCM difference as -0.52, WM difference as -0.43 and BEM difference as -0.12 . For CM, MCM, WM, and BEM, the percentages of accuracy for girls-boys were 77.88% to 78.3%, 69.15% to 68.81%, 61.68% to 67.11, and 73.83% to 75.93 in the absolute difference values within 1 year, respectively . The relationship between age groups and EA-CA of the four methods is presented in . In the boxplot, horizontal lines are located at the median of data inside boxes; box height gives the interquartile range, and whiskers show the range. Accordingly, EA closest to the CA was obtained using the WM in groups 6F, 11F, 6M, 10M, and 11M; the CM in groups 7F, 8F, 9F, 10F, 12F, 7M, 8M, and 9M; the MCM in group 14F; and the BEM in groups 13F, 12M, 13M, and 14M. The average measurement time of Observer 1 was calculated as 2 minutes 09 seconds for one patient with CM, 2 min with MC, 1 minute 12 seconds with WM, and 51 seconds with BEM. Accordingly, as seen in , BEM was the most practical method, requiring the least time. While CM, WM, and BEM were found to be generally valid for this sample group, the MCM method was thought to need more studies. To evaluate intra- and inter-observer agreement, the apical opening width and tooth length measurements used in the methods were performed three times on 62 individuals. For all measurements and methods, both intra- and inter-observer agreement was excellent, above 0.90 (range: 0.941-1.000). The purpose of age determination is to determine an individual's physiological age in the absence of information about the CA of the individual or forensic situations in a way that does not cause legal losses. For this purpose, teeth or bones are utilised. A wide variety of factors, such as gender, race, environmental and geographical factors, systemic diseases, syndromes, congenital disorders, endocrine disorders, and nutritional disorders, are known to play a role in bone development. The sequence and timing of teeth eruption are known to be less sensitive to environmental influences or endocrine disorders than skeletal growth and maturation. Therefore, dental age determination methods were evaluated in this study. Although radiographic methods are considered simple, rapid, reliable, and the most suitable methods for age determination in terms of cost/benefit, they still have some ethical concerns due to the potential side effects of X-rays. , Therefore, this study aimed to eliminate this situation by using panoramic radiographs taken for diagnosis or treatment from patients who had previously applied to our institution in the faculty archive without additional radiation. The study preferred healthy individuals between the ages of 6 and 14.99, and the exclusion criteria used for individuals with radiographs were the same as in similar studies due to the limitations of the methods evaluated in the study. , Also, the maximum age that can be measured according to the methods is as follows for boys and girls, respectively: CM:14.06 to 13.69, MCM:14.87 to 14.51, WM:16.03 to 15.99, BEM 14.6 to 14.33. It is known that different genetic and environmental factors can cause people with the same CA to have other body characteristics and developmental levels. In countries like Turkiye, where people of diverse ethnic backgrounds coexist, it is crucial to extensively test and validate age estimation methods across a wide range of populations and regions to ensure their reliability. WM and BEM methods are dental age determination methods prepared by researchers with data from their populations. , The table Demirjian et al. prepared in both methods determines the developmental stages of teeth. CM is a measurement-based method ready for the researcher's population who developed this method and allows the calculation of the EA of the person by placing the data in a particular formula. Kış et al. similarly modified the formula of Cameriere et al. for Turkish children. While examining the suitability of these methods for the study group, the same tooth development stages data were used for WM and BEM methods, and the same apical aperture and length data were utilised for CM and MCM. According to the literature review, no other study was found that evaluated the suitability of the MCM formula for the Turkish population other than Kış et al. and no other study that evaluated the BEM for Turkish children other than Çarıkçıoğlu & Değirmenci. In addition, to the best of our knowledge, this study is also the first to compare these two methods with WM and CM for Turkish children. Therefore, it is believed that the MCM requires further validation through more tests before it can be reliably applied to Turkish children. While DM is known to overestimate the CA of Turkish children, WM, a method developed from DM, gives results that are closer to CA in most studies and more accurate than DM in terms of age determination. , , Therefore, WM was preferred instead of DM in this study. While it was the closest method to CA for males (0.08), it was underestimated for females (-0.45). Gulsahi et al., in their pioneering study of CM for the Turkish population, reported that CM is relatively fast, inexpensive, practical, and user-friendly, which can be used in a forensic situation. We believe this may be due to genetic similarities between Italians and Turks. Among the studies comparing WM and CM for the Turkish population, a study conducted with 400 children aged 6 to 14.99 years in the Central Blacksea region population (0.76 and 0.49 years with WM and -0.10 and -0.17 years with CM in girls and boys, respectively) and another study conducted with 636 children aged 6 to 14.99 years living in the Thrace region reported that CM gave results closer to CA than WM. In a study conducted with 1024 children aged 6 to 15.99 years living in the Northwest Anatolia region (0.1 and 0.35 years with WM, -0.53 and -0.48 years with CM for girls and boys, respectively) and in a study conducted with 330 children from Central Anatolia aged 5 to 15.90 years living in the same region as our study (-0.062 and -0.05 years with WM, -0.55 and -0.6 years with CM for girls and boys, respectively) WM was found to be more appropriate than CM. In this study, the EA-CA difference was found to be -0.45 and -0.43 with WM and 0.22 and 0.17 with CM for girls and boys, respectively, and it was observed that CM gave results closer to the actual age for both genders. This is because the samples in the other studies had different regions of residence. In contrast, in the study where the same region was evaluated, the number of samples and age groups were different. In forensic medicine, the acceptable accuracy of EA-CA for persons up to adolescence has been reported to be ± 1.00 years. , In other words, in addition to the mean difference, the percentage of absolute difference values of that difference within a year is also significant for evaluating the suitability of a method for that population. Ozveren et al. (77.3% for girls and 84.6% for boys) and Hato et al. (79.9% for girls and 80.6% for boys) reported that CM provided a higher percentage accuracy in absolute difference values within 1 year compared to WM. This is similar to the current study (77.8% for boys and 78.3% for girls). On the other hand, Çarıkçıoğlu & Değirmenci (81.5% for girls and 76.6% for boys) reported that WM had a higher percentage of accuracy than CM. This may be because, in addition to using samples from different climates, different methods may give more appropriate or highly inaccurate results in age groups. The limitations of this study include the inclusion of children from a single region and the lack of time measurement during the application of the methods, which resulted in subjective assessments. Therefore, if a method is to be evaluated or modified for a specific population, it should be assessed with the largest possible sample size. In a country like Türkiye, where various ethnic backgrounds coexist, individuals from multiple regions should be included in the study to ensure broader representativeness. If groups are evaluated separately, the closest results to CA for both sexes were obtained with CM in the Central Anatolia region, around the Karaman province. However, when the mean age difference for all age groups is evaluated by gender, the closest results to all individuals' CA were obtained using the BEM. This is thought to be because, for the CM, the maximum age limit is 14.06 years for boys and 13.69 for girls. MCM modification for the Turkish population was the method with the highest mean difference from CA for our study group. For this reason, if it is desired to modify a valid age determination method for a population, it is thought that children from different provinces should be included in the sample group, especially in countries like Turkiye, where many different ethnic origins live together. Idea/Concept: Şahin, Güleç; Desıgn: Şahin; Control/Supervısıon: Şahin, Güleç; Data Collectıon and/or Processıng: Şahin, Güleç; Analysıs and/or Interpretatıon: Şahin, Güleç; Lıterature Revıew: Şahin, Güleç; Wrıtıng the Artıcle: Şahin, Güleç; Materıals: Şahin, Güleç. None disclosed. |
Perspectives of Child Life Specialists After Many Years of Working With a Humanoid Robot in a Pediatric Hospital: Narrative Design | 015608e3-d85b-40ac-b604-f5e7fcd5eec5 | 7714644 | Pediatrics[mh] | Overview Humanoid Robots Humanoid robots are increasingly recognized for their potential to support health care in pediatric hospital settings . One of the first studies published on the use of a robot during a medical procedure began in 2011 and was focused on MEDi. This study showed that MEDi, when programmed to distract children and teach them breathing as a coping strategy, reduced children’s pain and anxiety during vaccination . This same robot also made children and their parents smile, helped children to cooperate during the procedure and to remember the robot more than the needle, and seemed to empower effective coping . When MEDi was used to support children undergoing blood tests, parents reported that their children experienced reduced pain . MEDi was then used with a variety of other medical procedures, such as intravenous starts, tube removal, and dressing change, with similar effects on pain and fear . Other studies produced comparable results in various areas throughout the hospital using this robot . Other studies have also suggested that children in a health care environment are enthusiastic about engaging with a humanoid robot , particularly children with autism . This accumulating evidence of children’s enjoyment of humanoid robots and the ability of such robots to calm children’s pain, fear, and anxiety aligns well with the supportive role that CLSs have in providing medical procedural support to children to help reduce their distress. Indeed, the integration of a humanoid robot into CLSs’ daily practice is increasingly endorsed . At the same time, a body of literature about the impact of humanoid robots on children’s hospital experiences is drawn from studies of the perspectives of parents and children. To our knowledge, no research has been conducted to capture the experiences of CLSs charged with incorporating humanoid robots into their day-to-day pediatric support practices. In this study, we conducted an in-depth examination of the experiences of CLSs using a humanoid robot in their daily work and our purpose is two-fold: (1) we aim to contribute to the understanding of the human-robot interface as part of supporting children in their hospital experiences, and (2) we will provide practice-relevant illustrations of the use of a humanoid robot in CLS practice. We present this study beginning with a description of the background of the CLS practice environment that provides the study context. We then provide a rationale for our narrative approach to conducting our study using semistructured interviews with CLSs, followed by a detailed illustration of CLSs’ experiences with using humanoid robots. We conclude with a discussion of our conceptual contribution and our practice-relevant recommendations.
Child life specialists (CLSs), also known as play specialists and child life therapists in various countries, work toward creating a friendly hospital environment to facilitate children’s healing . The role of CLSs is multifaceted, whereby professionals with university education, including internship experience, help infants, children, youth, and families cope with illness, injury, and treatment . The broad scope of the CLS role entails supporting people with a range of challenges, needs, and backgrounds every day. Despite the many tools in the form of play, behavior management strategies, and education , providing support to sick children and their families, when they may be facing the most difficult time in their lives, is a monumental responsibility. In an effort to provide CLSs with another tool to use in supporting children, we introduced a humanoid robot (NAO robot produced by Softbank Robotics) named MEDi (programmed with cognitive behavioral strategies) to a children’s hospital.
Study Context Study Design Sample and Recruitment Data Collection Ethical Considerations Data Analysis A team of 4 analysts—the first 2 authors (TB and JP) and 2 RAs—analyzed all data. All 4 analysts have experience and education in interview data collection and analysis. The RAs had participated in another research project pertaining to the use of MEDi; thus, all 4 analysts were familiar with the subject of this study. We used a conventional thematic analysis approach to focus on generating themes from the content of stories shared by participants during interviews . At the same time, this analysis is semideductive as themes were also shaped by the interview questions we posed to the participants. The third author (BL) has an extensive qualitative background and worked to enhance the credibility of the findings, consistent with the principle of triangulation of investigators. BL was not involved with the research until the analysis phase and used this distance to offset the closeness that the first 2 authors (TB and JP) had in the study. Analysis began with authors independently listening to the recordings and reading and re-reading the transcripts. Following Corbin and Strauss , we independently identified concepts evident in each interview and made analytic notes (open coding). We then met to discuss these notes for each interview and then across interviews and were able to see each other’s perspectives and insights into and interpretations of each interview. We discussed detailed and frequently occurring responses that allowed us to draw out links and commonalities between codes, which we then organized into agreed-upon themes (axial coding). We deepened our analysis with the help of BL. BL is a seasoned qualitative researcher who was not involved in the implementation of this study and who comes from a different disciplinary background. As such, BL helped us triangulate investigator and disciplinary perspectives brought to bear on our analysis, which involved combining some themes into broader themes and resequencing some themes as components of the broader themes. We then conducted a member check by contacting all the participants to discuss the summary of our findings. TB and JP met with participants in a focus group format and invited them to reflect on their experiences, add more information, and share disconfirming thoughts. They confirmed that the findings represented their perspectives. Notes were maintained on these processes as an audit trail to further contribute to the trustworthiness of the findings.
In 2014, 4 MEDi robots (NAO robots produced by Softbank Robotics) were introduced to Alberta Children’s Hospital, a 141-bed and 90-clinic tertiary care center seeing visits from 90,000 children per year, located in Western Canada. This introduction was based on research evidence of the impact of the robot on patients at this hospital. It became the first children’s hospital in North America, and perhaps in the world, to use robots for inpatient units and ambulatory clinics, tests and procedures, educational and orientation sessions (or happy visits ), short or long admissions, and with any child where it seemed feasible. At any one time, any one or more of its 4 robots were used throughout the hospital for various purposes. The robot is 22.5 inches tall and weighs approximately 12 lb . It is programmed with a variety of behaviors, such as telling stories based on themes of encouragement, playing interactive games to teach coping skills such as breathing, dancing to attract children’s attention to elicit smiles and laughter, and friendship behaviors such as fist bumps to build rapport. CLSs can also type text into the tablet that operates the robot to have it speak with animations in response to a child’s questions/comments. Pearson and Beran provide a full description of MEDi behaviors. Since the introduction of MEDi in 2014, the team of CLSs regularly using MEDi shared many stories, experiences, and emotions with one another. It became evident that CLSs held compelling and detailed insights into how MEDi was affecting children, their parents, other health care professionals, and themselves. These accumulating and diverse anecdotal accounts added impetus to our plans to conduct this study.
To gain detailed insights into CLSs’ experiences with using MEDi to interact with children and their families, we used a narrative design. Narrative designs are built on the belief that people use stories—defined as consequential linkings of ideas or events —to give others access to the richness of their experiences. We collected CLS stories about using MEDi by conducting semistructured individual interviews. The semistructured design of our interviews was used to keep interview conversations focused on experiences using MEDi while affording flexibility to follow the energy of participants by asking additional questions and prompting participants to more fully describe the experiences they considered interesting and important. Participants’ stories gave us access to narrative elements of their experiences using MEDi, such as settings, problems, actions, and resolution . We were then able to bring individual participant stories side by side to draw out links and broader themes that tie stories together.
We used purposive sampling to select the CLSs who were familiar with MEDi. At the time of recruitment (June to October, 2019), not all members of the child life team were regularly using MEDi in their work owing to responsibilities other than supporting medical procedures. In addition, as CLSs sometimes change roles, only those with the most extensive and recent experience with MEDi within the year before data collection were included in the study. This type of sampling enabled us to select participants according to their ability to inform our purpose of understanding their varied and detailed experiences . The child life team was informed of the study by the team lead (fourth author, SB) during a staff meeting. An email was sent by the researchers to the 7 CLSs most experienced in working with MEDi, inviting them to participate in the study. The email invitation to participate contained information about the purpose and requirements of participation. All 7 CLSs agreed to be interviewed. All were women and had been CLSs for a period ranging between 1.3 and 29 years, with an average of 17 years of experience.
Interviews were conducted in a private meeting room at the hospital where all the CLSs worked. Interviews were scheduled according to the CLS’s availability. Signed informed consent was obtained at the beginning of each interview. Participants were told that although their names would not be included in research reports, the small, in-depth nature of this study might make them identifiable, especially to readers who know the participants. The first CLS participant interviewed was the second author (JP), given that she is also a CLS with 5 years of experience working with MEDi and was responsible for leading the integration of the robot into the child life team at this hospital. JP was interviewed by the first author (TB). This first interview and yielding data served as a pilot of the interview process during which the authors confirmed that the interview questions were meaningful and could be responded to within a standard 90-min research interview time frame. After this first interview, TB and JP together conducted the subsequent 6 interviews. These 6 interview participants worked with JP and were aware of TB’s role in introducing MEDi to their hospital workplace. Furthermore, TB and JP worked to maintain awareness of the separation between their own understanding and the descriptions given by participants by holding a stance of curiosity and openness. As such, we were attuned to various responses both between ourselves and the participants and across participants . All interviews were audiotaped and transcribed verbatim by a research assistant (RA). The interview duration ranged from 1 to 1.5 hours. The interview guide questions were open ended and invited story sharing and descriptions of actions, feelings, and thoughts about working with MEDi. Experiences were explored, and direct questions were sometimes asked for clarification of details . Questions included asking about positive and negative experiences associated with using MEDi, how they use MEDi, others’ reactions to MEDi, the impact of MEDi on the hospital environment, and thoughts about the future use of MEDi.
JP’s collegial relationship with participants and TB’s introduction of MEDi to participants’ workplace required special consideration for all phases of the research. These relationships were disclosed to the university ethics board and to the manager at the hospital who approved this study. Furthermore, per our description of data collection, participants were reminded of the respective roles of TB and JP in relation to the topic being studied and told that their responses would not affect their daily responsibilities. We also assumed that as JP had been working with MEDi and her CLS colleagues for 5 years, negative experiences with MEDi would have been discussed between CLSs as these experiences occurred. However, we sought to create openness for participants to share negative experiences with using MEDi during interviews by explicitly requesting reflections about negative experiences. Moreover, to reduce concerns about encouraging positive responses and/or discouraging negative responses from participants during interviews, TB and JP made efforts to create a relaxed atmosphere, expressing their hope that the CLS participants would enjoy sharing their thoughts, challenges, and the range of feelings they have had in their work with MEDi. Finally, the length of the interview was adjusted according to participants’ energy and scheduling needs, and the participants were told that they could decline to answer any questions that made them feel uncomfortable.
Themes Incorporating a Robot: Navigating From Fear to Friendship MEDi as a Source of Connection and Support for Patients MEDi as an Adaptable Resource for CLSs Working With the Limits of MEDi CLSs described the limits of MEDi. Being unable to meet high expectations people held for MEDi and managing mechanical and operational difficulties were mentioned. CLSs described health care staff and patients as surprised at how MEDi seemed to surpass their expectations of what would typically be seen in a hospital. This could lead to high, difficult-to-meet expectations for what MEDi can do. MEDi could be viewed as a quick fix for any child who was anxious during a medical procedure. CLS1 explained that if a child was struggling during a procedure, he would be called to come and fix it magically . Very nature of MEDi could contribute to conflicting expectations. As one of the CLSs noted: It’s hard to put the words around it too because it’s not something that belongs in any other category–toy, person, tool. CLS7 Indeed, MEDi could bring expectations of being able to perform any human function, such as responding to spontaneous conversation. When MEDi was unable to respond this way, people expressed disappointment. Children with repeated visits sometimes asked for more songs and games than MEDi was programmed to perform. For example, MEDi was able to play segments of some popular movie songs but was not continually updated as new movies were released. Thus, MEDi created high expectations and then was unable to meet additional expectations. As with all machines, operating MEDi sometimes entailed technical difficulties. Because a tablet was used to operate MEDi through the network of a nearby router, the connection to the router was sometimes lost because of interfering signals from other machines. This problem was manageable once CLSs learned how to reconnect to the router. However, CLSs expressed that although MEDi is kid-friendly , it is not always user-friendly . MEDi is designed and programmed to appear and act human-like, and most CLSs described MEDi in relational terms relative to patients and even relative to themselves. However, MEDi is a machine consisting of computers, wires, speakers, motors, and a variety of other parts. Machines break down or fail. CLSs expressed being frustrated, yet learning how to implement MEDi as a therapeutic tool, regardless; that is, they described how the breakdown can be presented to the child as a human quality in the same way that humans are not always on our game . They would also say, for example, that MEDi was not feeling well today and share this reflection with the child about not always feeling well, as a way of normalizing this experience.
In this section, we most often refer to the robot as MEDi, as it is a trademarked name used by hospital staff and patients. The robot is also referred to as he rather than it to acknowledge that this former pronoun is more predominantly used by CLSs. In addition, in the interest of presenting our study concisely, we removed disfluencies in participant descriptions. Through our thematic analysis, we generated 4 major themes. The first theme incorporating a robot: navigating from fear to friendship reflects the processes involved in learning to use a humanoid robot in a pediatric setting for therapeutic support. The second major theme is MEDi as a source of connection and support to children, and this theme comprises 3 subthemes: (1) MEDi as a welcoming and comforting presence, (2) MEDi as a friend, and (3) MEDi as encouraging and motivating. The third major theme is MEDi as an adaptable resource for CLSs, and this theme comprises 3 subthemes: (1) using MEDi to distract and teach, (2) using MEDi to facilitate challenging situations, and (3) using MEDi to facilitate collaboration within and beyond the CLS team. The fourth and final theme is working with the limits of MEDi.
Of the 7 CLS participants interviewed, 4 had been on staff at the time of the robot’s introduction to the hospital. They described their initial feelings of uncertainty with CLS1 stating, “I didn’t really get what it was all about.” Feelings of initial fear and frustration as well as excitement and curiosity were shared. For example, CLS2 described how she had been scared as shit . Fear and uncertainty pertained to how to operate the robot—feeling nervous about breaking it and the frustration that occurred if the robot did not respond. These participants also reported a lack of understanding of how MEDi could be used with children. They felt embarrassed when MEDi did not work in front of a patient or his/her family. One participant had been determined not to use it, saying her initial response was, “…no not for me” (CLS4). However, CLSs were emphatic in their descriptions of a turning point or a process of trial and error as they gained confidence in operating the robot and watched its impact. One of the CLSs said: It took some time and a lot of conversations to figure out where and how it [MEDi®] would or where it would fit best. CLS1 This CLS also explained that the children themselves showed ways in which the robot could be used: …they sort of led the way in finding a way it [MEDi®] can be helpful and therapeutic to them. CLS1 CLSs learned how to rely on their own skills of managing situations where the robot did not work properly. For example, CLS4 described becoming able to “…switch gears if I have to, whereas I think at first I was really intimidated by oh my gosh, if he breaks down…” For her part, CLS4 described gaining familiarity saying: So now I get it. I get how we can use him. It took me a while to get there, but I get that he can be used well in our profession. CLS4 CLS4, the participant who had initially indicated that the robot was “not for me,” summarized her current thoughts about the robot stating, “I think we’re friends.” Gaining comfort and confidence took investment from CLSs both to learn how to use the technology and open-mindedness in being willing to try something new. This frame of mind is ongoing and yields more possibilities. One of the CLSs noted: …the more we give MEDi® to do, the more we can use him in different ways. It’s a process of growing and learning and figuring it out because there’s no manual on this. There’s no manual on what you do. You have the ability, skills, knowledge, and expertise but you have to figure out how to apply that teaching every time...it’s always a different day. So that’s the process of discovery with MEDi® too. CLS6
MEDi was described as a source of connection and support for children. We found that the depth of the support and connection afforded by MEDi varied, and we created subthemes to distinguish depths of support and connection according to (1) MEDi as a welcoming or comforting presence, (2) MEDi as a friend, and (3) MEDi as encouraging and motivating. MEDi as a Welcoming and Comforting Presence MEDi as a Friend MEDi as Encouraging and Motivating was portrayed by CLSs as a welcoming presence. CLS3 referred to MEDi as “…a friendly face” that contributes to “creating a positive experience in the hospital.” CLSs spoke of how MEDi could set the stage for future positive experiences: I think there’s huge recognition now more than ever how important positive experiences at the hospital are for a child’s future visits here. CLS7 I think he [MEDi®] opens doors to treatment in the future with or without him and kind of helps to...build those relationships and rapport. CLS5 CLS1 shared a story of having ridden the elevator with MEDi in a sitting position on a cart. A child who had never seen MEDi before entering the elevator and immediately placed her hand on the cart and, on exiting the elevator, walked alongside for a while. CLS1 explained that MEDi seemed to be on this child’s level. CLS7 stated that she “…genuinely believed kids look at me as though he [MEDi ® ] is on the same level as them.” During patient visits, the CLSs described MEDi as a comforting physical presence: We know that a comfort position at the hospital is when a child sits between the parent’s lap, and MEDi® sits between the child’s lap and they hold on to him. CLS7 CLSs noted that MEDi’s very presence seems to calm children: ...sometimes I’ve had it where he [MEDi®] doesn't say or do anything. He’s just there in the room and it seems to work. It seems to help which amazes me that he can just be sitting there and being hanging out there and he just seemed so friendly and comforting that he just has to be there you know. CLS3 CLS7 noted that MEDi afforded a comforting source of continuity as she described a patient who was a frequent flyer who had had many visits to the hospital over an extended period. This frequent flyer patient would request to see MEDi during her visits. CLS1 also noted that MEDi could become part of children’s visits; however, CLS1 pointed out that the children did not necessarily actively interact with MEDi. Nevertheless, the presence of MEDi mattered: ...I’ve had situations where kids are completely happy with a MEDi® who’s turned right off. They’re just...oh this is so cool, and you know they just push MEDi® around on the cart and he's not even talking or anything. CLS4
The nature of the support and connection that MEDi could provide to children was presented by CLSs as friendship. Some CLSs said that because MEDi greets children by name, a bond of friendship is formed: I think she [4-year-old girl] formed this little bond with him [MEDi®]. CLS7 Another CLS recounted a patient reaction to the personalized connection MEDi offers: …and he [MEDi®] knew her [the patient’s] name and he said, “congratulations” and just her eyes lit up …, “Oh my goodness! How does he know me? That’s so cool...My brother is gonna be so jealous.” CLS5 The friendship style nature of connection is reinforced by children referring to MEDi as he , rather than it . Furthermore, friendship style connections persist even though children are well aware that MEDi is programmable and inanimate. One CLS described: It’s funny how or interesting how kids will connect to him. In medical day treatment a kid was watching me program him. I showed her how we program him. We were picking things together. Yet, she still talked to him and was annoyed when he didn’t respond right away. And so, I think it’s interesting how they can connect even though they know it’s programmed from this tablet or that we’re using this tablet to help run him that they’re still engaged with him as like a person. CLS5 Another CLS used the word love to characterize her sense of children’s fondness for MEDi: The little four-year-old that I already talked about, I think that was it where I really saw the value of MEDi® and the collaboration that it offered with physio...she [the child] fell in love with MEDi®. CLS2
MEDi was described as encouraging and motivating children to do activities they would not otherwise do. One of the CLSs presented MEDi as instilling courage: And he does give kids courage. I think there’s a lot of kids we’ve seen that gain courage from seeing MEDi® do something and recognizing that they can do it too. CLS7 A particularly tender moment was shared by one child, according to a CLS: She [female patient] looked at her mom and she said, “MEDi® thinks I’m brave.” CLS7 Children were described as seeming to implicitly trust MEDi and believe that words of encouragement that come from MEDi are sincere. MEDi helped children manage during procedures that they had formerly found to be extremely difficult. CLS1 shared a story of a young boy with autism who would not sit on the dentist’s chair. After meeting MEDi, this boy agreed to sit on a chair with MEDi in his lap. The boy cooperated with all the dental procedures, and afterward, the mother blurted to CLS1, “That’s not my son,” as she could not believe that her son had allowed the dental assistant to touch and examine his mouth. Another CLS expanded with a story of the surprise over the influence of MEDi expressed by health care staff; the staff had planned to sedate a boy for a procedure that entailed removing approximately 40 bumps that covered his body. The CLS described: He [the boy] had played with MEDi® throughout the whole procedure...when I was leaving everyone who was involved was so impressed and the plan was to try it this time and then maybe go back to doing this procedure in the future under sedation. But then they called me a little while later and booked me for another date to come back with MEDi®. CLS1 CLS2 shared a related story of a girl who would not respond to the physiotherapists who were encouraging her to sit up, build some core strength, and get out of bed. However, the child responded when the CLS went into this child’s room with MEDi: Well that kid almost leaped out of her bed and she has tubes hanging out of every orifice. So, it was a challenge to slow her down, but then physio and I collaborated. CLS2 Another CLS used the word power in explaining the surprising impact MEDi has: …Because that [medical procedure] was such a hard one and it was right in the beginning when we really didn't know the power of MEDi® let’s say. The parent’s reaction that, “Oh my God, we’re actually going to get this [medical procedure] done. We’re going to get them through day surgery,” was phenomenal…that event actually opened up our eyes to what, how MEDi® can affect patients. CLS6 One of the CLSs extended the idea of how MEDi affected patients as she described the impact of MEDi on her own children. She noted that her own children met MEDi when her son was in hospital: He thinks it’s amazing that I get to work with a robot. My daughter wants to be a CLS when she grows up. CLS7
MEDi is an adaptable resource used by CLSs to achieve the therapeutic goals they set on the basis of their understanding of the child’s needs, the ability of MEDi, the parents’ behaviors, the needs of the health care staff, and their own expertise in child development. In other words, CLSs took many factors into account and used MEDi to help achieve their goals. Some CLSs always take MEDi with them to see children, whereas others stated that they first ask the parents if MEDi seems a good fit to their child’s interests. As CLSs worked to determine if and how children would find robot behaviors desirable, they explained how MEDi can be used in many situations and in different ways that allow them to be creative. We display CLSs’ use of MEDi according to the subthemes of (1) using MEDi to distract and to teach, (2) using MEDi to facilitate challenging situations, and (3) using MEDi to enhance collaboration. Using MEDi to Distract and to Teach Using MEDi to Facilitate Challenging Situations Using MEDi to Enhance Collaboration Within and Beyond the CLS Team using MEDi to distract the child’s attention away from the medical procedure; helping the child to cope with anxiety, fear, and discomfort; and allowing the health care professional to complete the medical task more easily. One CLS spoke of the distinct distracting potential of MEDi: ...the child won’t look down won’t notice the poke, will be too busy watching MEDi®. He’s a great distraction ‘cuz unlike the iPad, he’s real and in front of you. He’s 3D. You can see him. CLS3 When and how CLSs introduced MEDi in distraction for procedural support situations depended largely on the child’s age. When asked to use the robot with children aged 3 years and less, CLSs noted exercising additional caution knowing that in this stage of child development, children are often unable to separate fantasy from reality and may be afraid of the robot. For older children and adolescents, CLSs described drawing on the robot’s technical attributes. One CLS spoke of how she used MEDi to engage a long-term teen patient: He’s older so it’s less about the songs and dances at the age which he was really intrigued and captivated by kind of how it works - robots in general. CLS5 Another CLS explained how she would tailor the intervention involving MEDi to the age of the patient: At times, I’d introduce it to an older child and more from the scientific side approach...giving them the control over the tablet...using that teaching mode as their form of distraction...which is just as therapeutic... CLS2 Although CLSs reported weighing countless variables and striving for the highest standards of professionalism, several noted that not every effort to incorporate MEDi as a distraction was successful. One CLS clearly described a difficult situation: I think mom had met MEDi® and wanted us to use MEDi® but then MEDi® kind of did end up being that extra noise and confusion in the room...which sometimes happens where if he’s not distracting and he’s dancing...we needed more calm which is not all on MEDi® cuz I’m the one running MEDi®...In that situation it didn’t work, it just added more chaos and instead of that distraction piece or calm that we needed. CLS5 According to CLS6, sometimes you realize, “MEDi ® is being one extra voice added on to the layer of voices in there …that it’s just not working out as a distraction or support.” In these cases, CLSs explained how they would shift to alternative forms of distraction or other coping strategies, debrief with families following the intervention, and adjust future plans for support accordingly. As an alternative to distracting children from procedures, CLSs used MEDi to teach children specific coping strategies to use during procedures. In one particular application, MEDi instructs, demonstrates, and plays a game to encourage children to slowly inhale and exhale their breaths. Several CLSs mentioned this application. By learning these breathing strategies from MEDi, children can develop courage and confidence in dealing with procedures. CLS7 concisely described how this can work for children: “If MEDi ® can do it, I can do it too.” CLSs also used MEDi to teach through play-based interventions. CLSs dressed MEDi as a patient and interacted with the robot in a child-friendly manner to explain why certain medical procedures were needed and what would be involved. CLS6 explained, “We do the vitals on MEDi ® first and then on the patient.” With MEDi cast in the role of patient, children would care for the robot in the way that the health care professional would care for them. MEDi-as-patient then responded with positive feedback about how gentle and caring the child had been. In this way, children can learn that the health care staff have positive motives even though sometimes they have to perform painful medical procedures.
CLSs described using MEDi in challenging medical situations such as those that can accompany supporting children with autism spectrum disorder (ASD). CLSs described how children with ASD could benefit in particular ways: I definitely think that...kids on the autism spectrum love MEDi® because they can connect with him. CLS3 Children with ASD may find it easier to negotiate their behaviors in interaction with a robot rather than in interaction with other people who tend to express a great deal of verbal and social information that may overwhelm the sensory processing systems of children with ASD. One CLS summed up this idea by saying: I’ve noticed that children with autism tend to engage with the robot more than with me. CLS1 CLS7 expanded on the idea of using MEDi in challenging situations as she described a patient who had an audiology examination. This patient’s grandmother brought her to her examination because there was stress in the family and that day was also the patient’s birthday. CLS7 described wanting to make sure the experience would be “super fun for her and we have the robot.” She brought MEDi to the examination area, and MEDi was carrying a balloon and a present for the patient. CLS summarized, “I think it just worked.” This CLS summarized how a good fit between what a child needs and what MEDi could provide can yield incredible results: But I do think that first, when we pair him [MEDi®] up with the right kid, he can make them do just about anything. CLS4 Although CLSs adapted their use of MEDi according to the needs of patients and used MEDi to distract, teach, and manage challenging situations, they also remained open to new possibilities. One CLS described: I think of MEDi® as being another tool...when I’m trying to decide the best way to meet a family’s needs or the needs of a situation...I wonder if we could blow bubbles. I wonder if they’re allowed to go outside...I wonder if there’s a place here for MEDi®. CLS4 Keeping MEDi’s potential in mind was conveyed by CLS3 who described MEDi as, “malleable and adaptable to whatever the situation needs him to be” and notes being often surprised. CLS3 initially thought MEDi was useful for certain children for specific reasons; however, then she began noticing that children would interact with MEDi in other ways that seemed to leave them comforted and relaxed. This CLS further explained, “So, I kept being surprised at different things throughout…,” noting that, “Kids can make him [MEDi ® ] what they need him to be and that’s what I was most amazed by him throughout getting to know him is that he’s not this cookie-cutter.” Indeed, the ways in which MEDi could be adapted made him a preferred resource distinct from others. One CLS said: I would use MEDi® before other approaches now because I know I have MEDi®...and I’m not going to do [teach] deep breathing on my own. CLS3
MEDi was discussed not only in terms of his therapeutic benefits for children but also for how he affected adults. MEDi was referred to as part of the CLS team. CLS7 said, “…it’s a collaborative effort and I think he's part of that team.” CLS7 drew a distinction between MEDi and other technology-based resources referring to how she and her colleagues, like the children, refer to MEDi as he rather than it : I look at the iPad as an object that can provide distraction. Whereas I do tend to use MEDi® as more I guess, a part of our team...I can call him a he. I wouldn’t call my iPad a he. CLS7 Indeed, in sharing stories of how MEDi affected patients, CLS3 even said that she loves MEDi. MEDi being viewed as part of the team was evident when CLS4 spoke of the need to care and advocate for MEDi: You have to have somebody who can look after him...he can’t just be left in a cupboard then people just grab him when they need him. He needs somebody to advocate for him and look out for him. CLS4 MEDi also helped facilitate collaboration with colleagues beyond the CLS team, as illustrated by CLS2, who described working with colleagues from other departments of the hospital in efforts to optimize the benefits of using MEDi: The cystic fibrosis team that I worked with, especially...she [healthcare staff] loved it when MEDi® came by…she got excited and where she got to the point of requesting MEDi® for certain kids when they do their morning assessments if we knew a certain child was going for blood work. She would tell the child about it saying, “You know, [the CLS is] around to help you with blood work today and she can go get MEDi®.” So, she would tell me and then we would make that happen in the morning. So, she was a great referral for that. CLS2 Several CLSs recount how new working relationships were formed because of MEDi: ...in some of these clinic areas we weren’t very involved and our first involvement came to be because of MEDi®…the robot was the point of interest, caught people’s attention and then we started talking about ways in which we could integrate the robot into these areas. CLS1 Many of the CLSs enthusiastically reported a unique collaboration with a particular physician and feelings of pride in this accomplishment: I think the fact that [he] books his clinics around our schedule speaks to that. CLS7 CLS4 noted potential of MEDi to provide a valuable distraction as she shared her impressions of working alongside this physician in this clinic with MEDi for the first time: ...I was just so blown away by what a great fit that was for all of these boys that were having some pretty uncomfortable surgeries while they were awake...For them to walk into an operating room and see a robot and [say] “what is that?” and then for us to be able to keep them distracted during all of that. ...to me, that’s super powerful… CLS4 CLS7 spoke broadly about the collaborative influence of MEDi on the CLS reputation in the hospital as she indicated MEDi as having, “increased our popularity in general at the hospital” and that she feels like a celebrity because MEDi attracts so much attention and comedy. Indeed, CLS6 pointed out the energy that MEDi could catalyze among adults that extends to dancing with MEDi: ...we concentrate a lot on what MEDi® does for patients but it’s amazing how much MEDi® does for staff or parents...maybe we take it for granted a little bit, but you know having all of the staff watch you and they’re actually laughing or they’re doing the Gangnam Style [dance] with MEDi®. I think that speaks volumes of the effect that MEDi® could have on people I guess in general. CLS6
Principal Findings Strengths and Limitations Conclusions This is the first study to examine the professional life of CLSs working alongside a robot. At first glance, the themes may appear as surprising to the reader as the experiences were to the CLSs. Working with a humanoid robot for social interactions with a vulnerable population is not an everyday experience for most people. Our aim is to offer a glimpse into what new experiences may be created with the introduction of a humanoid robot in a children’s hospital. This study is a change in direction from our previous quantitative research examinations of the impact of the MEDi robot on children undergoing various medical procedures. Having obtained some empirical evidence of a reduction in patient distress , it seemed timely to next look at the nature of this experience through the eyes of the professionals who are orchestrating the operation of the robot. As CLSs continue to forge new ground, we will continue to come alongside with a research lens to present to the world what this strange new humanoid robot phenomenon is all about.
Over several decades, traditional doctor-patient paternal approaches to health care have been moving toward a model of patient-engaged care . Health care that takes into consideration the holistic needs of a patient, orients support toward those needs, and empowers children to feel brave—even in the most challenging medical situations—now represents the highest-quality health care experience possible . Coming alongside this approach to health care is our introduction—almost 10 years ago—of a humanoid robot in a pediatric hospital. By examining stories about this implementation from the perspectives of the CLSs who regularly use the robot, we illustrate pediatric patient support that is provided at the level of the child and that we believe constitutes an exemplar of patient-engaged care. Our evidence of patient-engaged care is in the form of 4 major themes of how CLSs experienced the integration of a robot into their daily practice of supporting children and families at a Western Canadian children’s hospital, and these themes represent a holistic, empowering approach to pediatric care. The first theme, navigating from fear to friendship, is reminiscent of an explorer in the process of discovering new territory. As the daily use of a humanoid robot in a hospital setting to provide support to children was innovative and not previously done at the time it was introduced at Alberta Children’s Hospital, there were no resources or previous learning to serve as a guide. Rather, CLSs took bold risks in trying to use the robot in various ways and types of medical situations, with patients having different backgrounds—never knowing what reactions would occur from patients, parents, and health care staff. It was a risky adventure in an emotionally charged environment. As with any exploration into the unknown, the CLSs reported uncomfortable and even frightening feelings. These reactions are typical when learning something new, especially when presenting something new to an audience of children, parents, and health care staff who are facing a challenging medical event. We submit that patient-engaged care is enhanced when care providers join patients in taking risks. In this case, the CLSs who are encouraging children to be brave and face scary and painful medical challenges are also willing to take risk in trying a robot when the outcome is unknown. Although we explored how CLSs’ perceptions of working with MEDi shifted over time, we did not ask specific questions about how they thought children’s reactions to repeat visits with MEDi may have changed. There were some suggestions (eg, children developing a bond over time and becoming more comfortable and the robot setting the stage for future positive experiences); however, these experiences need to be more thoroughly addressed in future research. The second major theme about MEDi as a source of connection and support to children reflected how the robot could inspire comfort, friendship, encouragement, and motivation. These positive sentiments were suggested in a study on the same robot that interacted with children with diabetes . Drawing on MEDi’s friendly appearance, CLSs brought MEDi into a relationship between themselves and the child. They used MEDi almost as an extension of themselves by having the robot speak encouraging phrases accompanied by expressive behaviors. A line of inquiry that emerges from this theme is whether CLSs select phrases and behaviors to play on the robot that they themselves would use. Alternatively, CLSs may create a unique identity in the robot and play the actions that the robot itself would seem to personify. What is clear is that CLSs worked in a patient-engaged manner to determine what children need to feel stronger and supported, and CLSs learned how to use the robot in ways that met those needs. Perhaps the reason that the robot could be used as such an effective support is its ability to provide physical comfort. Even its mere presence can create a focal point for children to be distracted from negative feelings. Indeed, Alemi et al provided some preliminary evidence that a robot may reduce children’s anger. MEDi’s size and human shape may invoke patient-engaged feelings of warmth and comfort. CLSs’ relational presentation of this human-like object can tantalize children’s imagination and spark their sense of connection with MEDi. CLSs’ sense of delight in how children interacted with the robot seemed to motivate patient-focused professional energy on the parts of the CLSs. Although they used MEDi to provide support, such as they would with tools, for example, iPads, bubbles, and so on, they reported distinctly positive feelings associated with MEDi as they spoke of MEDi as a he and talked about MEDi as a member of the team. It is almost as if the adults saw the robot as more than a robot just as the children did. CLSs described MEDi as an adaptable resource for use to distract, teach, facilitate challenging situations, and promote collaboration with health care staff. By situating the robot as expressing kindness, care, and affection toward children, children’s rapport and bonding with the robot created the platform from which a variety of strategies could be used. The result, as one CLS stated, was to make them do just about anything . They used MEDi in playful and instructive ways to empower children to cope with the distress of being in a hospital. They had the robot talk and act toward the child in any way they thought the child needed to the extent of personifying MEDi in different roles such as play partner, companion, or mentor. Although objects other than robots such as toys, dolls, and stuffed animals can also be used to provide support , the difference is that the robot (as controlled by the CLS) responds to what the child says and does. This special interactional experience affords creativity for CLSs. CLSs described extraordinary patient-engaged moments such as the robot celebrating a patient’s birthday with a gift and balloons on a day that anyone would feel overwhelmed by family and medical challenges. Not only did children experience these tender moments, the impact extended to health care professionals and parents as well. For example, how often is anyone seen dancing in a hospital? Our final theme reflects the complexity of how CLSs are able to work with the limits of the robot. The surprise of meeting a robot created expectations and the desire for more human-robot interactions. Known as expectation discrepancy , people can experience disappointment when they realize a resource’s limited capabilities. CLSs may want to be aware of this potential outcome and preplan and creatively seek a variety of uses for MEDi so that MEDi’s impact can continue to be positive. For example, MEDi can be programmed with a variety of empirically supported psychosocial support strategies and relaxation techniques. As technology advances, more user-friendly programming capabilities can also enable CLSs to create any text and behaviors at the moment they wish to play them. In learning how to operate the robot, CLSs do not typically have training in using such a technologically sophisticated device. Thus, as more people become familiar with MEDi’s implementation in daily practice, this learning can be passed along to new users. As researchers with some familiarity of the experience using a humanoid robot for pediatric support, we too struggle to understand and put into words the contradiction of what type of entity the robot is. Perhaps what is more surprising though is that despite our ignorance, CLSs have, nevertheless, managed to resourcefully use this unusual tool as a means of supporting one of the most vulnerable populations in our society.
As this study was conducted at only one hospital, the results may not be transferable to other hospital settings where the same or other robots are used. A strength in terms of transferability, however, is that all of the CLSs who had the most experience using the robot agreed to participate in this study. These results may, therefore, be relevant to other CLSs working with a robot. Furthermore, CLSs offer distinct insights that are rooted in the multidimensional experience inherent to the demanding role of the CLSs. Our results are further limited given that interviews took place at one point in time and relied on recall, which is likely biased by memory. In addition, the familiarity between the researchers and the participants may have influenced responses in the interviews, despite our attempts to limit this effect. Thus, we see these results as suggesting patterns and themes to be explored further in future research. We recognize that despite deliberately deciding on approaching the data collection and analysis with open minds, our own social and cultural understanding of the CLS environment cannot always or easily be set aside. However, it is also true that the history and investment of the first 2 authors (TB and JP) in MEDi offers us a distinct position from which to understand and interpret participant meanings both in the moment during interviews and later as analysts.
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Longitudinal | cac528d6-8e32-4b89-b95f-440d4816462e | 11598993 | Biochemistry[mh] | CoV-2) infection, continues to be a challenge to the healthcare system in several countries . Since COVID-19 emergence, in addition to vaccination, several efforts have been made to improve the diagnosis and prognosis of severe and critical cases and to control the spread of the infection . However, due to high viral transmission rates, new SARS-CoV-2 genetic variants of concern are still emerging, resulting in the maintenance of viral spread, affecting vaccine performance and altering the pattern of COVID-19 clinical manifestation . It is now well established that the pathogenesis of SARS-CoV-2 is multisystemic . Although the majority of cases are mild to moderate and the mortality rate ranges from 2 to 5% of symptomatic cases, it is estimated that at least 65 million people have developed post-acute COVID-19 symptoms. This number is probably higher due to undocumented or misdiagnosed instances . The persistence of symptoms for more than 3 to 4 weeks after acute SARS-CoV-2 infection has been classified as “long COVID” or “post-acute COVID-19 syndrome” . The clinical manifestations of long COVID include muscular, cardiovascular, neuronal, and endocrine disorders, reflecting the impact of SARS-CoV-2 infection on multiple organs at the acute phase . Importantly, these symptoms were not restricted to subjects that developed severe COVID-19, and they are also observed after mild and moderate cases, and even in asymptomatic infections . These data indicate that virtually all cases of SARS-CoV-2 infection represent a risk of developing some long COVID sequelae. However, the mechanisms associated with the clinical evolution to post-acute symptoms are poorly understood. The pathophysiology of long COVID may be related to immune and metabolic responses induced by acute infection . Metabolomics studies have associated the disturbances triggered by acute SARS-CoV-2 infection with alterations in plasma metabolites, such as lipids, glucose, one carbon, and mainly amino acids. These systemic alterations reflect the extension of inflammation, lesion, and dysfunction of target tissues, ranging from moderate to fatal cases of COVID-19, and they could be used as biomarkers for the prognosis of disease outcomes in the acute phase [ , , , ]. In addition, the development of insulin resistance, diabetes, and other metabolic disturbances observed in the post-acute phase indicates that metabolic dysfunction may persist even after viral clearance [ , , , ]. The characterization of the saliva metabolome can also help describe the metabolic pathways involved in the clinical evolution of COVID-19 . The epithelial cells of the oral mucosa and salivary gland express the cellular receptor angiotensin-converting enzyme 2 (ACE2), and they are potential sites for the initial replication of SARS-CoV-2 . Thus, saliva metabolites may reflect direct changes in the target sites of infection, derived from activated immune cells in the mucosa and oral microbiota responses . Furthermore, changes in saliva metabolism also follow metabolite fluctuation in blood, reflecting systemic alterations in pathophysiological conditions . The literature is still scarce when it comes to longitudinal evaluation of the metabolic responses related to the post-acute phase of COVID-19, mainly in subjects with mild and moderate disease. In the present study, we conducted a 1 H Nuclear Magnetic Resonance (NMR) metabolomics study in symptomatic subjects presenting mild and moderate respiratory symptoms to investigate longitudinal changes in saliva metabolome between the acute and the post-acute phases of SARS-CoV-2 infection. Moreover, we investigated how these metabolic alterations observed in COVID-19 subjects were associated with the degree of replication, vaccination status, and the presence of comorbidities. Our approach contributes to the description of post-acute alterations in host metabolism that may be associated with long COVID.
2.1. Study Design and Participants 2.2. Ethics Approval 2.3. Saliva Sampling and Processing 2.4. NMR-Based Metabolomics 2.5. Statistical Analysis Recruitment took place between April and July 2021 at the Center for COVID-19 diagnosis, presently Núcleo de Enfrentamento e Estudos de Doenças Infecciosas Emergentes e Reemergentes (NEEDIER) at the Federal University of Rio de Janeiro, Rio de Janeiro, Brazil. At the time of admission, 119 symptomatic adults (≥18 years of age) were included in this study 2–5 days after the beginning of symptoms after the screening test for viral antigen. SARS-CoV-2 diagnosis was confirmed by nasopharyngeal swab Real-Time PCR (RT-qPCR) . Viral RNA was extracted using the Maxwell 16 viral total nucleic purification kit system (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Viral RNA was detected using the SARS-CoV-2 (2019-nCoV) CDC qPCR probe assay (Integrated DNA Technologies, Coralville, IA, USA), targeting the SARS-CoV-2 N1 and N2 genes and the human RNase P gene. All reactions were paired and performed in a 7500 Thermal Cycler (Applied Biosystems, Foster City, CA, USA). A SARS-CoV-2 RT-qPCR result was considered positive if both targets (N1 and N2) were amplified with a cycle threshold (Ct) value ≤ 37. Subjects were grouped into SARS-CoV-2 positive (n = 63; COVID-19 group) and SARS-CoV-2 negative, presenting non-COVID respiratory symptoms (n = 56; non-COVID group). Subjects returned 45 days after the first visit, corresponding to n = 32 in the COVID group and n = 20 in the non-COVID group ( A). The first visit corresponded to the acute phase of COVID-19 (T1) and the second visit corresponded to the post-acute phase of COVID-19 (T2). In both T1 and T2, saliva samples were collected in the morning (from 8 a.m. to 11 p.m., local time) through auto sampling, and clinical and demographic data and previous diagnosis of COVID-19 were obtained via structured forms.
The present study was approved by the local ethics review committee from Clementino Fraga Filho University Hospital (CAAE: 30161620.0.0000.5257) and by the national ethical review board (CAAE: 30127020.0.0000.0068). Informed consent was obtained from all subjects involved in this study.
For saliva sampling, participants were asked to briefly wash their mouth with water for prior hygiene of the oral cavity, and sampling was performed by spontaneous salivation in a sterile universal collector containing 1.25% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) for viral inactivation. Only participants who did not consume food up to one hour before the collection were included in this study. Samples (approximately 1 mL of each participant) were immediately transferred to Eppendorf tubes and centrifuged at 4 °C at 14,000× g for 45 min to remove cellular debris. All samples were stored at −80 °C until the analysis.
2.4.1. Sample Preparation and NMR Acquisition 2.4.2. NMR Spectra Pre-Processing and Assignment Saliva samples were thawed and diluted 1.2-fold in a sodium phosphate buffer solution and deuterium oxide to a final concentration of 50 mM phosphate buffer, 16% deuterium oxide, and 0.2 mM DSS, pH 7.4. A total of 600 μL of diluted samples were transferred to the 5 mm NMR tube. NMR spectra were acquired on a Bruker Advance III spectrometer at 500 MHz with a temperature of 298 K coupled to an automatic sample changer and cooled to 280 K. One-dimensional 1 H spectra were acquired with suppression of the water signals using the excitation sculpting pulse sequence . Broad resonances from the protein background were suppressed using the Carr–Purcell–Meiboom–Gill pulse sequence (CPMG) as a transverse relaxation filter with 32 loop counters and a delay of 0.001s. Therefore, the effective T2 delay of the transverse relaxation filter was 68.68 ms; 32,768 complex data points were acquired per transient, for a total of 1024 transients. The spectral width was set to 19.99 ppm, resulting in an acquisition time of 3.27 s per free induction decay (FID). The relaxation delay was set to 1.74 s.
Spectra were pre-processed using MetaboLab software v. 2022.0726.1733 . Prior to the Fourier transform, the FIDs were apodized using an exponential window function with 0.3 Hz line broadening and were then zero filled to 131,072 real data points. After the Fourier transform, each spectrum was manually phase corrected, followed by a spline–baseline correction. Spectra were referenced to the DSS signal. Signal-free regions (0.29 to −5.2 and 14.79 to 9.73), water signals (5.16 to 4.59 ppm), ethanol signals (1.08 to 1.29 ppm), and triton signals added for viral inactivation (8.61 to 8.26, 7.25 to 7.09, 6.86 to 6.60, 3.72 to 3.54, 2.94 to 2.80, 2.21 to 2.13, 1.71 to 1.52, 1.08 to 1.01, 0.8 to 0.3 ppm) were excluded. Additionally, noise filtering was performed, and anything below 5 times the noise threshold—measured between 9.5 and 10 ppm region—was discarded. Spectra were aligned using the Icoshift algorithm , which is integrated into Metabolab software. Spectral data were then binned with a 0.005 ppm interval (32 data points), as this ppm interval while correcting for small peak shifts and promoting spectral smoothing keeps all spectral information. The resulting table presented 1132 variables/bins. For the comparison of each individual metabolite between groups, metabolites’ intensity was used after performing spectral binning. The spectral bins that were chosen for comparison were the ones with the highest intensity, accounting for the whole peak, and also had no overlapping signals. All spectra were normalized using probabilistic quotient normalization, and Pareto scaling was applied prior to multivariate statistics. The TOCSY 1 H- 1 H was uploaded on the COLMAR for the assignments. The peak report of all assigned compounds can be seen at https://spin.ccic.osu.edu/index.php/colmarm , session ID 1399-nBZFhqa0aQ, (accessed on 31 March 2022). The Human Metabolome DataBase (HMDB) and Chenomx NMR Suite 8.2 ® program (Chenomx Inc., Edmonton, AB, Canada) were also used for the assignment of metabolites. presents the 1 H NMR assignment information for the metabolites.
Data distribution was analyzed using the Shapiro–Wilk test, and continuous variables with non-parametric distributions were represented as medians and compared using the Mann–Whitney test. Categorical variables were compared using the chi-square test with absolute (n) and relative (%) frequencies. The processed NMR spectra data were analyzed using the MetaboAnalyst platform , applying multivariate statistical principal component analysis (PCA) and a heatmap. The heatmap was generated with normalized data using the Euclidean distance measure, the clustering method Ward, and the variance IQR method. All metabolites found in this study were subjected to Metabolite Set Enrichment Pathway Analysis (MSEA), available on the Metaboanalyst 5.0 platform, and were compared with the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. The affected pathways were deemed significant when p < 0.05. For univariate statistics, Mann–Whitney and Kruskal–Wallis tests were used to compare non-transformed NMR data. Graph Pad Prism ® software version 8.0.1. was used for all analyses. p values < 0.05 were considered to reject the null hypothesis.
3.1. Subjects’ Demographic Characteristics and Clinical Parameters 3.2. Saliva NMR-Based Metabolomics Shows Specific Metabolite Fluctuation from the Acute to the Post-Acute Phase of COVID-19 3.3. Longitudinal Changes in Metabolites in the COVID-19 Group Were Associated with BMI and Vaccination Status, but Not with Biological Sex 3.4. Longitudinal Changes in Metabolites from the Acute to Post-Acute Phase Were Associated with SARS-CoV-2 Replication We then investigated whether the metabolic alterations observed in T2 in the COVID-19 group could be associated with viral load detected in the respiratory tract during the acute phase. For this purpose, qPCR Ct (cycle threshold) values were used to classify subjects that presented higher viral load (high; Ct < 18.9 cycles) or lower viral load (low; Ct > 18.9 cycles) ( A,B). In the subjects classified as low ( A), only feeble longitudinal changes were detected in T2 compared to T1, as opposed to what was observed in the high group, with several metabolites changed in T2 ( B). It is important to mention that increased levels of BCAA in T2 were only observed in the low group ( A). Lastly, subjects in the COVID-19 group were stratified according to the persistence of detection of the SARS-CoV-2 genome in the respiratory tract. Based on the qPCR test: they were positive for up to two weeks or positive for more than three weeks ( C,D). Metabolite fluctuations were observed in subjects that remained positive for SARS-CoV-2 for up to 2 weeks ( C).
Subjects in the COVID-19 group were significantly older than the non-COVID respiratory symptoms (non-COVID) group, while sex distribution was similar ( ). Most symptoms were more prevalent in the COVID-19 group, with highlights to fever, cough, adynamia, anosmia, and ageusia ( ). Regarding the duration of symptoms in the COVID-19 group, 44.4% had symptoms for up to 15 days and 19% for 30 days or more. The prevalence of comorbidities was similar between groups, and almost half of the subjects in both the COVID-19 and non-COVID groups presented as being overweight or obese. The majority of subjects in both groups were unvaccinated (~71% in the non-COVID and ~63% in the COVID-19 groups).
Representative spectra of T1 and T2 samples indicated differences in metabolites in the aliphatic, amide, and aromatic regions when comparing the acute and post-acute phases, mainly in the COVID-19 group ( B–E). Metabolites with differential signals, such as 2-hydroxybutyrate, (CH 3 ) 3 choline-related metabolites, sugar regions (mainly glucose), fumarate, phenylalanine, and histidine, are indicated in the figures. The heatmap plot confirmed the differences between T1 and T2 in each group and revealed two clusters of changes in metabolites. Cluster 1 comprised metabolites that increased in T2, and cluster 2 comprised metabolites that decreased in T2 compared to T1 levels in the COVID-19 group. In the non-COVID group, there is no variation in the levels of metabolites in cluster 1, and the pattern of metabolite changes in cluster 2 was opposite to what was observed in the COVID-19 group ( F, ). In cluster 1, glucose, (CH 3 ) 3 choline-related metabolites, 2-hydroxybutyrate, and BCAA stood out, and in cluster 2, acetate and lysine contributed to this distinct pattern in T2 compared to T1. Despite the PCA score plot did not indicate a discriminant pattern between T1 and T2 in both groups ( ), the PCA loading factors plot confirmed the heatmap results, where changes in glucose, 2-hydroxybutyrate, BCAA, and lactate contributed to the profile between the acute and post-acute phases of COVID-19 ( ). Univariate statistics were used to gain meaningful insights into the changes associated with the transition from acute to post-acute phases of COVID-19. We selected primary discriminating metabolites based on the PCA loading factors ( ) and heatmap results ( F, ), and their levels were plotted as T2 relative to T1 in the COVID-19 and non-COVID groups ( ). In the COVID-19 group, a significant increase in T2 compared to T1 in the levels of glucose, fumarate (a tricarboxylic cycle acid metabolite), (CH 3 ) 3 choline-related metabolites (choline, glycerophosphocholine), 2-hydroxybutyrate, and the amino acids BCAA and taurine were found ( B). In contrast, creatine/creatinine and lysine/putrescine (considered together due to signal overlap), histidine, phenylalanine, and acetate decreased in T2 relative to T1 ( B). Enrichment pathway analysis indicated that the most affected metabolic pathways in the transition from the acute to post-acute phase in the COVID-19 group were amino acids, lipids, the TCA cycle, and glucose metabolism (considering p < 0.05 values) ( ). Importantly, in the non-COVID group, only lactate was reduced in T2 compared to T1 ( A), which was not altered in the COVID-19 group ( B). Taken together, our data indicate that the clinical evolution to the post-acute phase in COVID-19 subjects involves metabolic changes differing from subjects that present non-COVID respiratory symptoms.
Longitudinal changes in metabolites in the COVID-19 group were also evaluated as a function of the body mass index (BMI), as excessive body weight is a risk factor for COVID-19 severity. COVID-19 subjects were classified as eutrophic (BMI < 25 kg/m 2 ) or overweight/obese (BMI ≥ 25 kg/m 2 ) ( A,B). In eutrophic subjects, no significant differences were found in metabolites when comparing T2 relative to T1 ( A); whereas, in the overweight/obese group, an increase in fumarate, (CH 3 ) 3 choline-related metabolites, 2-hydroxybutyrate, taurine, and BCAA and a decrease in acetate, histidine, and creatine/creatinine were observed ( B). Vaccination status was also considered to evaluate the longitudinal changes in metabolites in the COVID-19 group, and subjects were grouped into unvaccinated and vaccinated (received at least one dose of the vaccine). In the vaccinated group, only BCAA presented a longitudinal increase ( C). Interestingly, in the unvaccinated group, there was a decrease in amino acids and acetate, and an increase in glucose, fumarate, (CH 3 ) 3 choline-related metabolites, and taurine in T2 ( D). Moreover, when stratifying COVID-19 subjects according to biological sex, few longitudinal variations in metabolites were observed ( ).
This study intended to identify metabolic markers in the saliva associated with the clinical evolution of COVID-19 and to explore the contributing factors involved in the metabolic dysregulation caused by SARS-CoV-2 infection. Saliva metabolome reflects systemic and in situ metabolism associated with the disease state, and the contribution of oral microbiota must be considered. We identified two clusters of metabolites in the transition from the acute to post-acute phase that distinguished COVID-19 subjects from those that presented non-COVID respiratory symptoms. These metabolites are enrolled in the metabolism of amino acids, lipids, the TCA cycle, and glucose. Importantly, these longitudinal changes evidenced in our study contribute to the description of a metabolic signature involved in the tissue repair cascade but also unveil some persistent metabolic imbalances associated with late symptoms of COVID-19. A set of changes in metabolites observed in our study may be associated with poor recovery and can be regarded as long COVID responses, including the decrease in histidine and acetate, and the increase in 2-hydroxybutyrate and glucose levels. Histidine is a substrate for the synthesis of histamine by immune cells, regulating physiological and unbalanced immune responses in target cells . The significant reduction in histidine levels may be a metabolic response of persistent inflammation due to an increased histamine synthesis by macrophages, dendritic cells, and T lymphocytes . In agreement, it has been reported that excessive histamine production may decrease plasma histidine levels in COVID-19 subjects, contributing to rashes and inflammation seen during the disease . Indeed, the local release of histamine may regulate cytokine production by different immune cells, associated with inflammation observed in COVID-19 subjects [ , , ]. Likewise, the reduction in acetate in the saliva was associated with hospitalized subjects due to complications in the post-acute phase of COVID-19 . Acetate is produced by the microbiota, and it has been shown to improve B cell metabolism involved in the production of specific antibodies against SARS-CoV-2, playing a crucial role in the control of infection . These results may reflect dysregulations in the gut and oral microbiota observed in the acute phase of SARS-CoV-2 infection [ , , ], which probably persist in long COVID . Interestingly, the reduction in histidine and acetate levels was observed mainly in overweight/obesity subjects, which have been considered a risk factor for the progression to the severe forms of COVID-19 and also in unvaccinated subjects, reinforcing the protective effect of vaccination in the acute and long-lasting inflammatory process in COVID-19 . Increased levels of 2-hydroxybutyrate and glucose in the circulation were also correlated with acute inflammatory responses in COVID-19 subjects [ , , ]. A study by Barreto et al. demonstrated that COVID-19-related hyperglycemia is a result of a PEPCK-dependent gluconeogenic effect in infected hepatocytes . Thus, the persistent hyperglycemic environment evidenced by our study may be indicative of compromised tissue functions, including the liver, and decreased insulin sensitivity [ , , ]. Similar to what we observed, the development of hyperglycemia occurs irrespective of sex, BMI, and other comorbidities, such as diabetes mellitus . Increased glucose levels and other markers of persistent inflammation were observed in subjects with higher viral load and were positive for up to 2 weeks, indicating that hyperglycemia is associated with SARS-CoV-2 replication . In agreement, previous studies have associated SARS-CoV-2 replication with the extension of the induced inflammatory response and disease outcome . In addition, hyperglycemia has also been correlated with higher viral load in COVID-19 subjects and directly impacts viral replication, ACE2 expression, and cytokine production by peripheral blood monocytes infected with SARS-CoV-2 in vitro . Therefore, it can be suggested that the magnitude of the metabolic changes in the post-acute phase was influenced by the degree of the inflammatory response and viral replication in the acute phase of infection [ , , ]. Interestingly, Heald et al. showed that vaccination against COVID-19 had a positive effect on regulating blood glucose levels in subjects with diabetes mellitus . On the other hand, we observed some longitudinal metabolic changes in the post-acute phase of COVID-19 that could be involved in the resolution of inflammation and also the tissue repair cascade. Specifically, the increase in fumarate, BCAA, taurine, and choline and the decrease in creatine/creatinine levels could be potential markers of protection. Fumarate and its derivatives, such as dimethyl fumarate, are potent modulators of anti-antioxidant responses that inhibit SARS-CoV-2 replication and induce inflammation [ , , , ]. In addition, low levels of BCAA have been associated with systemic manifestations and fatal cases of COVID-19 . Indeed, taurine and BCAA supplementation have been proposed as a strategy to attenuate skeletal muscle wasting and mitigate tissue damage in COVID-19 subjects [ , , ]. In our study, BCAA levels were only increased in subjects with low viral load, reinforcing that BCAA are potential markers and protector factors for the clinical evolution of COVID-19. Higher levels of creatine/creatinine were also associated with pro-inflammatory cytokines, such as IL-6, in subjects with severe COVID-19 and also with COVID-19 adrenal dysfunction and mortality . Therefore, the reduction in creatine/creatinine levels suggests some degree of tissue recovery in the post-acute phase. It is also worth highlighting that creatine/creatinine dysregulation is recognized as a facilitator of viral replication , which is consistent with the higher levels of this metabolite during an acute phase. The increase in choline levels in the post-acute phase of COVID-19 subjects can also be considered a protective response. A reduction in choline and its derivatives in the plasma of subjects with severe COVID-19 and in individuals with mild COVID-19 infected with SARS-CoV-2 omicron [ , , ] have been associated with reduced synthesis of phospholipids, liver fibrosis, alterations in the gut microbiota, and thrombotic events . The novelty/strength of our work was to show that there are important changes in these metabolites in the post-acute compared to the acute phase of SARS-CoV-2 infection. It is important to draw attention to the fact that, in general, the metabolic signature linked to tissue repair or inflammation was present in the group with high viral load but not in subjects with persistent detection of SARS-CoV-2. This indicates that the magnitude of the inflammatory response and persistent metabolic changes induced by viral replication are linked to viral clearance and the activation of tissue repair pathways. Thus, it is possible that long COVID symptoms are determined by persistent inflammation but are also a result of activation of the repair cascade. Our work showed that saliva is a potential biofluid to address these aspects. It is not clear whether our findings correlate with the severity of the disease, as only a small proportion of subjects in our study had prolonged symptoms, and additional studies are necessary. In addition, longitudinal studies evaluating plasma metabolites would contribute to differentiating metabolic markers of the oral/respiratory replication site from those that are observed systemically.
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The stability of quetiapine oral suspension compounded from commercially available tablets | 74f07978-1db1-4c5b-8ddb-12d8bb053ea7 | 8354457 | Pharmacology[mh] | Delirium can be defined as acute changes in brain function that can result in sudden, severe confusion and disturbances in attention, developing directly from an underlying illness or its treatment . Intensive care units (ICUs) have been shown to have high rates of delirium, occurring in 50–80% of mechanically ventilated adult patients . ICU delirium is associated with prolonged hospitalizations, increased mortality, long-term cognitive impairment, and increased cost. Although under-recognized due to difficulty in developing appropriate assessment tools, ICU delirium can also occur in critically ill children at a rate of 12–65% . Treatment of ICU delirium is multifactorial and includes both nonpharmacologic and pharmacologic interventions. There are currently no FDA approved medications for this indication; however, there is evidence that antipsychotics may be beneficial. Quetiapine is a dibenzothiazepine atypical antipsychotic indicated for the treatment of schizophrenia and bipolar disorder in adolescents and adults . Although the exact mechanism of action is unclear, quetiapine is an antagonist at multiple neurotransmitter receptors in the brain such as Serotonin 5-HT 1A and 5-HT 2 , dopamine D 1 and D 2 , histamine H 1 , and adrenergic alpha 1 - and alpha 2 -receptors. Quetiapine is commonly used due to its short half-life allowing for rapid titration and favorable adverse effect profile. Case reports and small clinical studies have documented the successful use of quetiapine in the treatment of delirium in critically ill children and infants as young as 2 months of age . Quetiapine is poorly water soluble and thus is marketed as its fumarate salt, Quetiapine Fumarate (QF), to improve its solubility. QF is currently only commercially available in the United States as immediate and extended-release tablets, limiting its application to treat ICU delirium in critically ill children . A liquid dosage form is needed to be able to provide weight-based doses to infants and young children who are unable to swallow tablets. Therefore, the treatment of delirium in critically ill children and infants is relied on the extemporaneous preparation of a compounded oral QF suspension by pharmacists in clinical settings. Although there is a published compounded protocol for 10 and 40 mg/mL compounded suspensions of QF, the formula was made in oil that was sticky, unpalatable, and generally disfavored . Importantly, the stability of these nonaqueous oral QF suspensions was not measured. The extemporaneous preparation of QF compounded oral suspension changes the formulation from a solid form to a liquid form. Thus, the stability of QF compounded oral suspension should be critically evaluated to guide pharmacists for administration and storage of QF compounded oral suspension. The objective of this study was to develop QF compounded oral suspensions at 10 mg/mL using commercially available tablets in two readily available aqueous vehicles (Ora-Sweet and Ora-Blend) and measure their stability at both room temperature and under refrigeration. The stability of QF aqueous suspensions was analyzed by total drug content, the amount of the drug dissolved in selected vehicles, pH, visual appearance and odor in refrigerated and room temperature conditions for 60 days. We developed and validated an HPLC method for QF to study the stability of QF oral suspensions. The FDA drug stability guidelines were used to evaluate the data of stability for conclusion.
Reagents and chemicals QF compounded suspension preparation from tablets HPLC method HPLC method validation Stability study designs Physical tests of QF compounded suspensions during the storage Chemical tests of QF compounded suspensions during the storage Statistical analysis Data are presented as mean ± standard deviation (SD). All experiments were at least repeated for three times, and the data were measured from three different batches.
A single lot of QF 400 mg tablet (Alkem, India) was used to conduct the stability testing. Ora-Blend and Ora-Sweet vehicles used in this study were obtained from Perrigo (Minneapolis, MN). Acetonitrile was purchased from Fisher Scientific Canada. Quetiapine standard, phosphoric acid and methanol was purchased from Sigma-Aldrich (MO, USA). Trimethylamine (TEA) was purchased from Janssen Pharmaceutical (Belgium). Water was purified using a Milli-Q Synthesis A10 system (Millipore, Etobicoke, ON, Canada).
QF compounded suspensions (10 mg/mL) were prepared either in Ora-Blend or in Ora-Sweet. One QF 400 mg tablet was grinded to a fine powder by using a mortar and pestle. Two mL of propylene glycol was added to the mortar and pestle as a solvent and mixed to form a paste. After measuring 40 mL of the vehicle (Ora-Blend or Ora-Sweet) in a graduated cylinder, a small amount (2–5 mL) of the selected vehicle was added into the mortar and pestle and mixed until a uniform paste was formed. The vehicle was continually added, without using more than 50% of the total volume, until a pourable mixture was formed. This pourable mixture was transferred to a graduated plastic amber bottle. Using less than 15% of the total volume of the vehicle each time, the mortar and pestle was washed out into the amber bottle twice. The remaining amount of vehicle was then poured to quantum satis (Q.S.) into the plastic bottle until the final volume of 40 mL was achieved.
The HPLC instrumentation (Waters, Milford, MA, USA) consisted of an Alliance 2695 Separation Module, equipped with a model 2487 wavelength ultraviolet detector. The column used was an Eclipse Plus 18 column (100 x 4.6 mm internal diameter, 3.5 mm particle size, Agilent, USA). The mobile phase A consisted of acetonitrile: methanol at 90:10 (v/v). The mobile phase B contained 200 μL of phosphoric acid and 500 μL TEA in 200 mL water. A flow rate of 0.8 mL/min was used for gradient elution. The elution started from 40% of mobile phase A for 0.5 min, then increased to 80% of mobile phase A within 4 min, maintained for 3 min and then changed to 40% of mobile phase A within 0.5 min and finally maintained at 40% of mobile phase A for 5 min. The injection volume was 20 μL. QF was monitored at a wavelength of 220 nm. QF peak had a retention time of about 5.9 min, and area of the peak was used to perform the quantification.
The HPLC method was validated in terms of linearity, accuracy, precision, limit of detection (LOD), and limit of quantification (LOQ) according to the ICH guideline Q2(R1) . The standard solutions were prepared by using an aliquot method. One hundred milligrams of QF were weighted and dissolved in 100 mL of methanol in a volumetric flask to prepare the stock solution. The stock solution was diluted with methanol to obtain the standard solutions at 1, 5, 10, 25, 50, and 100 μg/mL. The standard samples were injected in triplicate at three different concentrations (10, 25 and 50 μg/mL). The peak areas were used to calculate the mean and the % relative standard deviation (RSD) values. The calibration curve was constructed with six concentrations: 1, 5, 10, 25, 50 and 100 μg/mL. Each level of concentrations was injected in triplicate. LOD and LOQ were determined based on signal-to-noise ratios at 3 and 10 times, respectively. The accuracy was measured by the recovery of QF. The intra-day precision was tested with three injections per day for three days at three concentrations (10, 25, and 50 μg/mL). Force degradation samples prepared from stock solution were completed to evaluate the specificity of HPLC method, as well as predict and monitor the chemical degradation during the storage process. Forced degradation studies included four sets of samples: Set 1 (untreated), Set 2 (treated with 0.1 N HCl), Set 3 (treated with 0.1 N NaOH), and Set 4 (treated with 3% H 2 O 2 ), which was conducted at 50 μg/mL of QF.
The stability of QF suspensions was tested for day 0, 7, 14, 30, and 60. After measured the stability at day 0, the preparations of the QF compounded suspensions (10 mg/mL) were stored in 30 amber PET bottles. The bottles were randomly divided to two even portions. Fifteen of the bottles was stored at monitored room temperature (22°C) and the other 15 were stored in a refrigerator (2°C). Three bottles were randomly pulled out and labeled #1, #2, and #3 to obtain three batches for each testing day and each temperature. On the testing day, the three batches that needed to be measured were taken back to room temperature, if stored in a refrigerator, and shaken very well before withdrew samples to obtain triplicate data. This design set the stability samples in three separate containers, which provided the independent triplicated measurement and storage.
The physical tests were measured by changes in visual appearance, re-dispersibility, and odor. The changes in visual appearance included color, precipitation and solution clarity on the assigned testing days. The re-dispersibility was evaluated by homogeneousness of suspensions after manually shaking. The changes in odor were monitored by nose for the presence of unpleasant smell. The results were scored positive for changes and negative for no changes.
The chemical stability was evaluated in terms of pH, total QF drug content, the amount of the drug dissolved in the vehicle, and degradation. The pH of each sample was evaluated by a pH meter. To prepare samples for the measurement of total QF drug content, 1 mL of compounded oral suspension was drawn out from each plastic bottle and dissolved in 25 mL of a methanol/water (1:1, v/v) solution. After the mixture was vigorously vortexed and sonicated for 10 minutes, 1.5 mL of the mixture were transferred into a centrifuge tube and centrifuged at 14000 rpm for 5 min at 25°C. Then, 1 mL of the supernatant was collected and diluted with 10 mL methanol, and the solution waso measure the amount of drug dissolved in the suspending vehicle, 1.5 mL of the compounded oral suspension were transferred into a centrifuge tube and centrifuged at 14,000 rpm for 5 min at 25°C. The supernatant was taken and centrifuged again to obtain the solution that contained the dissolved QF in the vehicle. The solution was diluted with 25 mL of a methanol/water (1:1, v/v) solution. Then, 1 mL of the mixture was further diluted with 10 mL of methanol and filtered with a 0.22 mm filter. The first 700 μL of the solution were discarded, and 180 μl of the solution were transferred into the HPLC vials for analysis. The degradation of QF suspensions was studied by monitoring the extra peaks in the HPLC chromatograms at the predetermined time points.
HPLC method development and validation Forced degradation study Stability of QF compounded suspensions using Ora-Sweet and Ora-Blend Limited by tablet sizes, the minimum dose of QF that can accurately be provided is 6.25 mg, which is one-quarter of a 25 mg QF tablet. The reported dosage range for QF in pediatric ICU delirium was 0.43 to 2.8 mg/kg/dose every 8 hours . Therefore, 10 mg/mL oral suspensions were prepared in this study to provide a more accurate measurement to be used in the setting of pediatrics, especially in young infants and children. Ora-Sweet and Ora-Blend are commonly used aqueous suspending vehicles in pharmacy because they are commercially ready-to-use. Both of them contains flavorants and preservatives. Thus, microbiological attributes are not needed to test for compounded suspensions when using these vehicles within their expiration dates. Physical stability was monitored based on appearance and odor. The compounded samples were remained milky white (the appearance in Day 0 over 60 days without any abnormal colors. Additionally, the samples did not develop any unpleasant smell over 60 days. All samples were re-dispersible by manually shaking in the testing period of 60 days. The data indicate the physical stability of the tested QF nonaqueous suspensions over 60 days. The Ora-Sweet QF suspension tended to settle more over time than the Ora-Blend QF suspension, which could lead to a lack of dose uniformity in practice. This could be attributed to the formulation of the flavoring agents, as Ora-Sweet does not contain suspending agents in comparison to Ora-Blend. If using Ora-Sweet as the vehicle, vigorous shaking would be required to disperse the drug in the solution, making this a difficult formulation to use in clinical setting. Chemical stability was monitored based on pH, degradation, drug content and dissolved QF. In clinical setting, QF compounded suspensions could be stored at room temperature (22°C) and in a refrigerator (2°C). Thus, both storage conditions were tested. The pHs of Ora-Sweet and Ora-Blend preparations were acidic with pH = 4.83±0.02 at Day 0. Over 60 days, the pH of both QF Ora-Sweet and QF Ora-Blend compounded suspensions rarely changed within a range of 4.78–4.89 in both storage conditions. The targeted concentration in the QF compounded suspensions was 10 μg/mL. After the preparation, the measured concentrations of QF Ora-Sweet compounded suspension and QF Ora-Blend compounded suspension were 10.16±0.17 and 9.59±0.15, respectively. Thus, the1.6% and 95.9% of the targeted QF concentration, respectively, which met the FDA criteria that the amount of preparation should be within 90–110% of the labeled amount. These results demonstrate that the QF compounding protocol in this study was reliable and able to prepare 10 mg/mL of QF suspension by using broken QF tablets. According to the FDA guidance, significant change of a drug product is defined as a 5% change in drug content from its initial value . In Tables and , the measurements that were over 5% changes from their initial values are highlighted. According to the data, the drug contents for QF Ora-Sweet compounded suspension were less than 5% changes over 30 days at both temperatures and over 60 days at 2°C without degradation. However, the drug content for QF Ora-Sweet compounded suspension decreased over 6.6% and 26.3% for two batches out of three tested batches at room temperature, indicating instability at this condition that could be caused by hydrolysis. The drug contents for QF Ora-Blend compounded suspension were less than 5% changes over 60 days at both temperatures without degradation. Utilizing the goal of +/- 5% change, the data conclude that the QF in Ora-Blend preparation did not experience a significant change in drug content at 60 days in both room temperature and refrigeration. Since the QF tablet was crushed and further mixed into a liquid preparation, QF would be found as both dissolved and as suspended particles. The percentage of dissolved QF in the suspending vehicles could affect the bioavailability of QF. Thus, the amounts of QF dissolved in the compounded suspensions were monitored over 60 days at 22°C and 2°C. Tables and , and indicate that storage conditions affected the amounts of dissolved QF in the suspensions. In refrigerated conditions, the percentage of dissolved QF in Ora-Blend decreased from 52.1% on day 0 to 42.5% by Day 14, and the percentage of dissolved QF in Ora-Sweet decreased from 48.0% on day 0 to 40.5% by Day 7. Room temperature storage also decreased the percentage of dissolved QF in Ora-Sweet from 48.0% on day 0 to 40.7% at Day 7. In contrast, the percentage of dissolved QF in Oral-Blend at 22°C did not have significant changes, as the concentrations remained above 50% through Day 60. Additionally, visually shows that QF in Ora-Blend stored at room temperature had both the highest amount of dissolved QF and also the most consistent level of dissolved QF over the entire 60 day testing period. Although sampling suspensions could generate the deviation on the measurements, it was clear that Oral-Blend at room temperature was better than other tested conditions in terms of keeping constant amounts of the dissolved QF. Thus, room temperature storage was preferable to keep the suspension with the same drug content as well as the dissolved QF from a given bottle over its 60 day storage time.
An accurate and precise analytical method is critical for the measurement of stability of drug formulations. HPLC separates impurities from drug substance through a column before a detector collects the signal of drug substance. Thus, it is widely used as an analytical method to measure drug content and degradations in a drug formulation. In this study, a new HPLC method using an Eclipse Plus 18 column was developed and validated for QF. Fumarate itself has absorption at 220 nm, which could interfere the absorption of quetiapine. Thus, the HPLC conditions need to separate fumarate and quetiapine. To maintain quetiapine at its neutral form and separate from fumarate, TEA and phosphoric acid were tested as a modifier of mobile phase. According to the results, phosphoric acid was selected as the modifier because of better separation and reproductivity compared to TEA. Gradient elution and a flow rate of 5 mL/min were used to obtain an optimal separation with adequate retention time. The HPLC method was accurate over six injections for 10, 25, and 50 μg/mL . The mean percentage of recovery for QF ranged from 102 to 111%. The linearity curve for QF was obtained with the correlation coefficient (R 2 ) of 0.9995 in the concentration range of 1–100 μg/mL . The inter-day and intra-day precision measured the degree of reproducibility or repeatability of the analytical method under normal operating conditions are shown in . The RSD values for inter-day and intra-day precision were less than 2%. The LOD was detected at 0.5 μg/mL while the LOQ was determined at 1 μg/mL. The ingredients in both Ora-Blend and Oral-Sweet did not interfere with QF in the chromatograms. Thus, this HPLC method for QF is precise and accurate.
Force degradation samples are important to evaluate the specificity of HPLC method, predict and monitor the chemical degradation during the storage. It is known that QF mainly degrades under oxidative and hydrolytic conditions. Thus, the force degradation of QF was studied for acidic and basic hydrolyses and oxidation . The results of forced degradation of QF are shown in for acidic hydrolysis, in for basic hydrolysis and in for oxidation. Retention time was a useful parameter to identify degradation products. After 24 hours, 84.9%, 33.1% and 11.5% of QF were degraded in 0.1N HCl, 0.1N NaOH and 3% H 2 O 2 , respectively. After 48 hours, 66.1% of QF were degraded in 0.1N NaOH and 100% of QF were degraded in both 0.1N HCl and 3% H 2 O 2 . The data demonstrate that QF was not stable in these testing conditions and the HPLC method was sensitive enough for detecting the reduced amounts of QF caused by the degradation.
In this study, a reliable aqueous compounding formula for a 10 mg/mL suspension of QF was developed by using commercially available suspending agents, Ora-Sweet and Ora-Blend. An accurate and precise HPLC method was used to evaluate the stability of QF compounded suspensions over 60 days under both refrigerated and room temperature storage. Overall, the QF in Ora-Blend formulation showed better stability and better qualities than the QF in Ora-Sweet formulation. Room temperature storage of QF in Ora-Blend was shown to be the optimal storage condition for maintaining a consistent level of dissolved drug in the vehicle throughout the entire study period. The QF in Ora-Blend formulation demonstrated 60 day stability at both room temperature and refrigeration. However, it should be kept in mind that, although the study demonstrated the in vitro stability of compounded oral QF suspension, the in vivo performance of this compounded QF suspension is unknown. Since the data showed that the compounding process dissolved the parts of QF, it could be possible that the tablet, when taken as a whole, and the compounded suspension could have different oral absorption. Future studies are expected to compare in vivo absorption of tablets and compounded suspensions.
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Randomized Trial of Information for Older Women About Cessation of Breast Cancer Screening Invitations | 8fa406e1-096a-4579-b71e-44a61023d7c3 | 11169431 | Patient Education as Topic[mh] | There is uncertain benefit of screening mammography for older women, as women aged ≥ 74 years have not been included in randomized controlled trials. At a population level, potential harms become more likely with increasing age, including false positives and anxiety due to additional tests following detection of an abnormality, as well as overdiagnosis (diagnosis of a cancer that would never have caused problems) and overtreatment. In one Australian modeling study, if screening were extended to 1000 women aged 70–74 years (compared to inviting women up to 69 years), one additional breast cancer death would be averted, eight breast cancers would be overdiagnosed, and 102 additional tests would include 78 additional false positives. However, a US modeling study indicated that continued screening to 78 or 80 years for women with no comorbidity could have similar benefit-harm ratios as screening women aged 50–74 years at average risk. United States (US) guidelines recommend individualized breast screening decisions beyond 74 years based on overall health, life expectancy, and personal preference. – Screening is not recommended for individual women with limited life expectancy (< 10 years) due to the 10-year lag-time to benefit and greater chance of harm. However, over 50% of women aged ≥ 74 years report recent screening in US national survey data, , and around 38% of women with limited life expectancy continue breast screening. In Australia, the nationally funded screening program (BreastScreen) invites women to participate in mammography screening for breast cancer up to 74 years of age through local health districts. Women aged ≥ 74 years can continue free screening and are encouraged to speak with their primary care clinician (general practitioner [GP]) if they wish. Screening rates by life expectancy are unknown in Australia. However, since BreastScreen extended invitations to women aged 70–74 years in 2013, annual participation rates in this age group increased from 25.9 to 55.8%, and data suggests 7.6–10% of women continue to be screened beyond 74 years. , One reason for this may be the high trust people living in Australia have in the healthcare system. Older women should be supported to make informed breast screening decisions (i.e., decisions with adequate knowledge of potential benefits/harms, consistent with their values/preferences). However, many have limited knowledge of the potential harms of screening and believe that they are no longer invited to screen for reasons they consider are ageist and related to government cost-saving or reduced risk of developing cancer. , GPs also find it challenging to explain these recommendations. Multi-level interventions (i.e., patient, clinician, system) are needed to maximize efforts to improve informed decision-making. – We conducted a randomized controlled trial to test how different messaging about the rationale for breast screening cessation impacts older women’s decision-making. We included intervention arms via printed-text or animation video. Animations are increasingly used for communicating health information and may result in greater knowledge improvements compared to printed-text materials, , particularly for people with low health literacy.
Study Design Participants Procedure Outcomes Statistical Analysis This study was an online, three-arm randomized controlled trial. Wewere randomly assigned to one of three study arms using a balanced allocation ratio of 1:1:1. The protocol for this trial was registered with the Australian New Zealand Clinical Trials Registry (ANZCTRN12623000033640) and ethical approval was obtained from The University of Sydney’s Human Research Ethics Committee (2022/809).
Eligible participants included women aged 70–74 years, living in Australia, who had engaged in mammography screening at least once in the past 5 years. Women who had self-reported a current or previous diagnosis of breast cancer were excluded. This age range was chosen as these women would be approaching the upper age limit for cessation of invitation to the program. They were recruited through Qualtrics, a social research company who partners with many panels to recruit individuals who have already signed up to receive surveys. Upon registration, sample providers verify respondent address, demographic information, and email address. They target eligible participants based on specific parameters including age, gender, and location, but do not guarantee national representation among the invites sent or responses received. They use an incentivization system for participation, for which rewards vary (e.g., cash, gift cards, redeemable points, charitable donations).
Qualtrics’ online survey platform was used to administer the questionnaire and piloting was conducted to test the scenarios and questionnaire. Potential participants were sent an email invitation via Qualtrics with general study information, directed to the study landing webpage where they were able to read the participant information statement, screened for eligibility, and then consented to participation. Participants were presented with a hypothetical scenario in which they imagined they had received a standard BreastScreen letter after a recent mammogram which reported that no breast cancer could be seen. The delivery method of this letter was not specified. This format best represents current Australian BreastScreen practice, reflecting the usual result letters sent to women aged 70–74 years. After reading the letter, participants were randomly allocated to one of three arms: Control : standard letter with no additional information. Intervention—printed-text : standard letter with additional information on the following page of the survey explaining in bullet points why they will not receive reminders to screen after 74 years. Intervention—animation video (1 min 51 s long) : standard letter with additional information in a video on the following page of the survey explaining why they will no longer receive reminders to screen after 74 years. The video included subtitles and narration. A professional animator developed the video based on a script and reference videos provided by the lead researcher. The messaging in both intervention arms was developed by the research team, including older female health consumers and experts in health communication (JS, RD, JJ, KM), breast cancer screening (NH, MS, WV), geriatric medicine (VN), and general practice (KW). The information explained why women will not receive reminders to screen after 74 years, due to both potential benefits and downsides of screening (overdiagnosis and overtreatment), and because with increasing age and other health issues the chance of experiencing the downsides of screening outweighs the chance of benefit (see Supplementary Material). The interventions were designed to be brief and feasible for delivery at scale. We therefore chose to focus on the harm of overdiagnosis as it is important in older age and poorly understood by women. In contrast, the harm of false positives is better understood and tolerated by women. The information also emphasized the decision was up to them but that they could speak with their GP if they were unsure. Iterations were revised based on feedback from the research team, including two consumer experts who advised on acceptability and understandability. Randomization was conducted automatically using the randomizer function within Qualtrics, which uses the Mersenne Twister pseudorandom number generator. Both participants and researchers were blinded to the allocation sequence until after data collection was completed.
Immediately after the intervention, participants completed the primary outcome measure of informed choice, which is a binary composite measure (informed/uninformed). In this hypothetical scenario, participants were considered to have made an informed choice if they had adequate knowledge and reported attitudes regarding screening for themselves that aligned with their intention to stop or continue screening (Box 1). Conceptual knowledge was assessed using 11 true/false questions, adapted from measures used in previous cancer screening decision-making research. , – Adequate knowledge was determined using the median score (i.e., participants had adequate knowledge if their total score was the within-sample median or greater). Personal screening attitudes were assessed using six items scored on a total scale from 6 to 30, with the within-sample median score used to dichotomize into “positive attitudes” and “less than positive attitudes,” as has been done in prior decision aid work. Screening intention was assessed by a single item with a 5-point scale (1 = Definitely will to 5 = Definitely will not). We decided a priori to dichotomize responses to intending to screen (definitely or probably will) or not intending to screen (unsure or definitely or probably will not), similar to prior decision aid work. Secondary outcomes included intention to speak to a GP, perceived risk of developing cancer, cancer worry, and emotional response to the letter they received. See Supplementary Table for further details. *Within-sample median threshold was chosen for adequate knowledge because a typical “pass” (or 50%) cut-off would not be appropriate. Participants could, on average, answer 50% of the questions correctly by guessing the answers. After completing outcome measures, participants answered questions to ascertain sociodemographic and health characteristics, including general health, health literacy, screening history, personal and family cancer history, and mortality risk. – An attention check question and consistency check question were also included to assess the quality of respondent data.
We conducted a sample size calculation assuming a conservative estimate that 50% of participants in one study arm would make an informed choice. We required 110 participants per arm (330 total) to detect a 20% difference in informed choice between the arms, which would have 80% power ( α of 5%, two-sided test). Descriptive statistics were calculated for sociodemographic and health characteristics of the sample, and the primary and secondary outcome measures (mean [SD] for continuous variables and frequency and relative frequency for categorical variables). Statistical significance of study arm differences was tested for using chi-squared tests for binary categorical outcomes, ordinal regression models for ordered categorical outcomes, and linear regression models for continuous outcomes. The assumption of parallel lines for ordinal regression models was satisfied. All statistical models included study arm (control, printed-text information, animation video information) as categorical covariates. Where overall differences were identified, planned simple contrasts (e.g., control vs. text intervention, control vs. animation intervention) were conducted. A Bonferroni-adjusted significance threshold of p = 0.017 was used for multiple pairwise comparisons. We conducted sensitivity analyses where participant responses from Western Australia were removed to test whether the outcomes differ, as BreastScreen Western Australia provides older women with information about the upper age of screening programs through their website and final screening letter. This information states, “There is no scientific evidence that for women over 75 participating in a population breast cancer screening program results in more health benefits than harms.” Other states in Australia do not provide older women with information about why screening invitations stop after 74 years. Sensitivity analysis was also conducted with adjusted thresholds set for primary outcomes (see Supplementary Table ). SPSS (version 28) was used for all analyses.
A total of 938 respondents accessed the first page of the survey from March 8 to 29, 2023, of whom 451 were randomized and 376 were included in the final analysis (Fig. ). Characteristics of the participants are presented by study arm in Table . Most participants were born in Australia (69.4%) and located in the states of New South Wales, Victoria, or Queensland (80.6%). Half of the participants had an education level of high school or less (51.1%), over half had private health insurance (60.4%), and most reported adequate health literacy (94.1%) using the single-item health literacy screener. Results for primary and secondary outcomes are presented in-text below, including pairwise comparisons where overall differences were identified between study arms. Table presents results for the primary outcomes by control and intervention arms, including a breakdown of each item contributing to the composite “informed choice” measure. Secondary outcomes by study arm are presented in Table . Primary Outcomes Secondary Outcomes Sensitivity Analysis Knowledge Screening Attitudes Screening Intention Informed Choice (Composite Outcome) Participants who received either intervention were significantly more likely to report adequate knowledge compared to controls (control 23.8%, reference; text 59.8%, RR = 1.90, 95%CI 1.51–2.39, p < 0.001; animation 68.9%, RR = 2.45, 95%CI 1.85–3.24, p < 0.001). There was no significant difference between intervention arms (RR = 1.29, 95%CI 0.92–1.80, p = 0.135). See Supplementary Table for individual knowledge item results.
Participants who received the video were significantly less likely to report high positive screening attitudes than controls (control 65.6%, reference; animation 40.2%, RR = 0.58, 95%CI 0.43–0.77, p < 0.001). There were no significant differences in screening attitudes between those who received the text information and controls (text 51.5%, RR = 0.71, 95%CI 0.53–0.96, p = 0.023) or between intervention arms (RR = 0.81, 95%CI 0.65–1.02, p = 0.070) with Bonferroni correction.
Compared to controls, both intervention arms significantly reduced their intentions to screen (control 17.2%, reference; text 36.4%, RR = 1.30, 95%CI 1.12–1.52, p < 0.001; animation 49.2%, RR = 1.63, 95%CI 1.34–1.98, p < 0.001). There was no significant difference between intervention arms (RR = 1.25, 95%CI 1.01–1.56, p = 0.039).
Participants who received either the text or animation intervention were significantly more likely to make an informed choice compared to participants in the control arm (control 18.0%, reference; text 32.6%, relative risk [RR] = 1.22, 95% confidence interval [CI] 1.05–1.41, p = 0.008; animation 41.0%, RR = 1.39, 95%CI 1.17–1.65, p < 0.001). There was no significant difference between intervention arms (text vs. animation) (RR = 1.14, 95%CI 0.95–1.38, p = 0.165).
Compared to controls, those who received either intervention were significantly more likely to intend to speak with their GP (control 36.9%, reference; text 55.3%, OR = 2.42, 95%CI 1.51–3.88, p < 0.001; animation 51.6%, OR = 2.09, 95%CI 1.30–3.37, p = 0.002). No significant differences were evident between textual information and animated video intervention arms in intention to speak with GP (OR = 0.87, 95%CI 0.54–1.39, p = 0.686). There was no difference between arms in perceived risk of cancer ( p = 0.111) or cancer worry ( p = 0.659). Regarding emotional response to the BreastScreen letter, those who received the text (mean difference [MD] = − 0.58, 95%CI = − 0.98, − 0.17, p = 0.006) or animation (MD = − 0.48, 95%CI = − 0.89, − 0.07, p = 0.023) were less likely to report feeling relieved compared to control (mean = 5.66). There were no significant differences in the degree to which participants were assured, informed, anxious, worried, or confused in response to the letter across intervention or control arms (all p > 0.05).
All main effects for primary outcomes did not differ in a sensitivity analysis ( n = 343) where all participants who reported being from Western Australia were removed ( n = 33) (Supplementary Table ), as well as in analyses with adjusted thresholds for the primary outcomes (Supplementary Table ).
In this online randomized controlled trial, women aged 70–74 years who received information that explained the reasons for screening invitation cessation were more likely to make an informed choice about screening beyond 74 years than women who did not receive this messaging. Women who received the interventions had greater knowledge, less positive screening attitudes, and reduced screening intentions. The animation intervention increased the proportion of participants making an informed choice by 24% compared to controls, and the text by 15%. Similarly, a breast cancer screening decision aid for women aged 70 years resulted in a 25% increase in informed choice compared to controls. Our findings have significance for implementation given that our developed interventions were brief, which would be relatively easy to scale-up across a population screening program compared to a decision aid which requires time and significant discussion for adequate development and use, presenting considerable challenges for implementation. Decision aids may still be useful and appropriate for older adults who desire more detailed information, as interventions at patient, clinician, and system levels are key to improving decision-making around screening for older people. , Screening providers communicating direct to consumer may be a feasible and effective way to counter the negative beliefs women hold regarding the current recommendations for no longer being invited to screen. , Given how challenging it is for consumers to grasp the concept of screening harms such as overdiagnosis, these findings also have wider implications for other screening programs, highlighting how overdiagnosis can be simply and effectively communicated. However, the context in which recommendations are communicated may have different impacts on how this messaging is received. For example, in the US, different levels of trust in the government-funded health insurance and controversy surrounding breast screening for older women may reduce the impact of this kind of messaging. There are consequences of providing information that should also be considered. Firstly, our interventions led some older women to be more unsure about whether to continue or stop breast screening and more likely to want to speak with their GP. Clinicians may need support and training to navigate these discussions and provide recommendations if requested. , Verbal scripts have been developed in the US that suggest how the idea of stopping cancer screening could be introduced, how a recommendation to stop could be explained with further reasoning, or how the benefits and harms of screening could be discussed in a shared decision-making conversation. These strategies for discussing stopping screening could be further adapted for use in contexts where national screening programs are implemented. Secondly, women who did not receive additional messaging reported greater relief in response to the BreastScreen letter. This may be explained by the relieving nature of receiving a letter from a trusted organization that provides a reassuring screen result without needing to consider additional information, or because information about screening benefits and harms reminds women that they are getting older and therefore feel a lack of control. Given women perceive mammograms as a way of looking after their health, this information could lead to feelings of distress. Health animations have shown promise in communicating complex information to people with low health literacy. There were no statistically significant differences observed between text and animation study arms. However, the format of information delivery is worth exploring in future studies with larger, more diverse samples. Animations may be particularly useful for low health literacy audiences when communicating conceptual knowledge rather than numerical risk information. Regardless, both intervention formats improved informed decision-making which could provide helpful flexibility in allowing for potential individual differences in needs and preferences for text or animation formats. Strengths and Limitations This study was strengthened through randomization to study arms ensuring similar baseline characteristics of participants across arms. In addition, differences in outcomes can be attributable to the intervention. We also utilized survey software techniques to ensure quality responses (attention and consistency checks) and designed the messaging to potentially supplement existing BreastScreen communications and be easily implemented and scaled up. There are limitations to this study. Recruitment through an online panel means that our sample is likely not representative of the women aged 70–74 years who would typically attend to BreastScreen services in Australia (i.e., underrepresentation of culturally and linguistically diverse and low socio-economic groups). Qualtrics randomly selects respondents but does not guarantee national representation. Our sample reported around 30% were not born in Australia, but approximately 40% of women attending BreastScreen are not born in Australia. Furthermore, this study was hypothetical in nature. Older women who receive this information may respond differently in real-life settings. Further research is needed, such as a clinical pilot trial conducted through BreastScreen services or other screening programs to understand the feasibility and impact of these interventions in practice, with more diverse samples. We also cannot guarantee all participants paid attention to the entire intervention content despite restricting ability to proceed in the survey after a reasonable time. Additionally, there was a slight difference in the content provided in the two versions of the intervention. Pre-existing beliefs about why screening invitations stop were explicitly acknowledged in the animation version (i.e., government cost-saving, decreased risk), but not in the text version. Future research should test different elements of the information provided to understand which parts are more effective among different audience types, and whether directly addressing pre-existing beliefs is important in supporting informed choice. Our interventions aimed to align with the BreastScreen Australia upper age limit for screening invitations (74 years), but we recognize there is no consensus on this matter. They were therefore limited in covering the nuanced information likely required for informed choice for some older women, but rather general information as a first step to be further elaborated with a primary care physician if desired. Further support is required for older women and clinicians navigating this decision. Another limitation is that intention rather than actual behavior was measured. However, lower screening intentions have been shown to be associated with lower screening rates. Finally, there are ongoing discussions around what thresholds should be set to define an informed choice. Our sensitivity analyses showed that the overall main effects remained significant with different thresholds. Using a threshold of below 30 on the attitudes scale as “less positive screening attitudes” due to the high within-sample median is justified in the context of this study. The public has well-documented strong positive attitudes toward cancer screening, , which are particularly influential in older adults’ cancer screening decisions , (likely due to a longer period of exposure to persuasive screening messaging). Scoring less than the maximum score on this scale may in fact have clinical significance.
Messaging outlining the rationale for cessation of breast screening invitation to women aged 70–74 years in this online randomized controlled trial improved informed choice and knowledge, reduced positive screening attitudes, and lowered screening intentions. This study is an important first step in improving the messaging provided by national cancer screening programs direct to older adults and supporting more informed choices.
Below is the link to the electronic supplementary material. Supplementary file1 (PDF 351 KB)
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Infectious diseases epidemiology, quantitative methodology, and clinical research in the midst of the COVID-19 pandemic: Perspective from a European country | 02556c36-8560-43eb-827c-efd5819cf05d | 7581408 | Health Communication[mh] | Introduction We have to return to 1918, the time of the H1N1 influenza pandemic, the Spanish flu, to encounter a health crisis that had to be confronted without adequate medicinal products, prior to even the concept of vaccination, poor scientific knowledge (viruses had not been discovered), and with little or no historic registration or surveillance data available . Arguably, such an invasive health crisis has a profound transformational impact on virtually all aspects of society. Spinney , in a chronicle of the Spanish flu, asserts that this 1918 pandemic, responsible for a death rate in the order of magnitude of 100 million people (rescaled to today's world population; directly or because of induced comorbidities, in particular also bacterial infections), was at least equally impactful as both world wars for shaping the world as we have known it until the end of 2019. It is interesting from a historic perspective, and crucial in understanding today's evolution, to examine how the Spanish flu impacted society (politics and geopolitics, social relationships, economic power, etc.). Unquestionably, the SARS-CoV-2 induced COVID-19 epidemic holds the same disruptive power. Our focus is on how such a global public health crisis transforms clinical research, and in particular epidemiological, (bio)statistical and clinical trials research. It is insightful to remember that, at the onset of Spanish flu, scientists thought that it was bacterial, before it catalyzed discovery and then the study of viruses and their induced ailments. In more traditional communities around the world, religious, cultural, (e.g., Confucian) or even environmental explanations were given (such as miasma or bad air). Spanish flu was often confused with bacteria-induced typhus, also known as typhus fever. In the absence of proper diagnostic testing, the occurrence of typhus was confirmed as soon as the characteristic rash occurred. Other than that, milder cases of the Spanish flu were hard to set apart based on the symptoms they induced. For severe cases, there was less doubt (e.g., due to partial or full-body dis-coloring), but by that time it was usually too late. While the details are different, the broad-brush similarity between 1918 and 2020 is striking . The post-Spanish flu public health world looked very different from what it was before. The importance of hygiene to fight and prevent disease had been understood since the seminal contributions of Florence Nightingale and the key discoveries of Louis Pasteur regarding bacteria. A key factor had been the discovery of penicillium and eventually antibiotics. This brings to the fore two important ways to confront infections: hygiene as an archetypical non-pharmaceutical intervention (NPI) and antibiotics as an essential example of a pharmaceutical intervention (PI). But, for the Spanish flu, antibiotic development was in its infancy (arsphenamine, discovered by German physician Ehrlich in 1909, was used for syphylis at the time), which remained the case until the discovery and mass production of penicillin during WWII. More importantly, antibiotics do not work for viral infections. While a century ago the world was less globalized than it is now, the mass movement of people due to WWI, but also transatlantic vessels, offered transmission opportunities to H1N1; our hyper-interconnected world did the same for SARS-CoV-2. This meant that Spanish flu had to be tackled with a variety of NPIs and some PIs, including social distancing, facial masks, quarantining after improvements in diagnosis, and more adequate treatment for H1N1-induced pneumonia. Eventually, likely already in 1919, the virus mutated to a less lethal strain, a typical competitive advantage for a virus, even though the mutation between Spring and Fall 1918 was uncharacteristic: the virus became more lethal over the summer, causing a horrendous second wave of infections . It was clear in 1918 that little or no records, apart from anecdotal evidence, were kept about past influenza epidemics. Fast forwarding to 2020, arguably influenza is properly understood, from a viral, epidemiological, epidemic modeling, and vaccination standpoint. National and international surveillance is well developed, e.g., to determine the components of the upcoming season's influenza vaccine and to monitor the emergence of strains with pandemic potency . We have to admit, though that, even though SARS-CoV-1 and MERS-CoV provided a wakeup call, data on coronavirus induced pathology, in contrast, are rare. Vijgen et al. suggest that the Russian flu of 1890 was the birth of hCoV-OC43, rather than H2N2, the 1957–1958 influenza pandemic in East Asia. Like H1N1, also SARS-CoV-2 induced a sense of urgency and mobilized societal, political, and research forces that are in non-pandemic periods unheard of, except in wartime and in the face of catastrophes such as a financial meltdown. On March 10, 2020, Tomas Pueyo, a product and marketing leader at Course Hero, addressing politicians, wrote on medium.com : “The coronavirus is coming to you. It's coming at an exponential speed: gradually, and then suddenly. It's a matter of days. Maybe a week or two. When it does, your healthcare system will be overwhelmed. Your fellow citizens will be treated in the hallways. Exhausted health care workers will break down. Some will die. They will have to decide which patient gets the oxygen and which one dies.” While it sounded alarmist to some, it has proven an accurate vision for countries and regions in all continents except Oceania. The combined fields of biostatistics, epidemiology, survey science, and clinical trials research, in close collaboration and at the service of virology, immunology, and infectiology, contribute towards the following broad areas: understanding the virus and its dynamics by extending and reformulating existing mathematical and statistical models and used to estimate key epidemiological parameters (e.g., basic reproduction number, incubation period, serial interval, generation interval, etc.); studying the immunological response to SARS-CoV-2 exposure, including the determination of the (sero-)prevalence in the population, T-cell mediated and humoral immunity responses, and potential cross-immunity; monitoring the global pandemic and its epidemics (country-wide, regional, city specific) by observing a set of characteristics and using a variety of modeling tools; gathering additional information by way of (longitudinal) survey sampling to gauge the epidemiological effect as well as the societal side effects (social and economic) of NPIs; making short-, medium-, and longer-term predictions – in view of monitoring health care capacity in early phases, NPI exit strategies, and the building of lines of defense towards surveillance in the post-peak period; contributing to clinical research for the development of diagnostic tools, antiviral medicinal products, and vaccines. Every one of these areas has seen tremendous and rigorous scientific development in the pre-pandemic era, both theoretical and applied. In that sense, the body of knowledge in 2020 cannot possibly be compared with the fragmented knowledge in 1918, and still, the knowledge about key aspects (seasonality, immunity, prevalence) is partial and speculative at best. The field of mathematical and statistical modeling of infectious diseases is well established as is, of course, epidemiology and clinical trial methodology. When a pandemic suddenly breaks out, all of these areas are strongly forced to collaborate, whereas scientific areas, even within medicine, tend to be compartmentalized. Researchers in the same field across the globe should work together. In addition, time is of the essence, so that certain principles need to be relaxed out of necessity, while others stand like a rock. A natural consequence for the need and willingness to collaborate is making available all potentially relevant data and an uncompromised commitment to an open access policy. We return to this key lesson in Section 11. Note that the need for open access to data should be paralleled by an open access to code in order to harness the power of the Internet and make research efficient on a worldwide scale. A sobering thought is that, in spite of all of this knowledge, at the outset, all one can do is enlist the key questions that emerge and quickly report early but key findings An epidemic, or even pandemic, of a different nature was the HIV-induced AIDS epidemic . Confronted with a lethal viral infection that affected predominantly younger people and hence led to a considerable number of life years lost, a massive response ensued, with large academic and collaborative AIDS research groups formed around the globe, predominantly in the United States (e.g., the AIDS Clinical Trials Group). It led to the acceptance of placebo controlled trials with frequent interim analyses overseen by an Independent Committee . Also, coerced by the ‘fourth player’ (i.e., the patients and their advocacy groups, next to the three other players: regulators, industry, academia), co-enrolment in several trials simultaneously was grudgingly accepted, but arguably led to the development of highly active anti-retroviral therapy (HAART; ). Undoubtedly, it dynamized the clinical research community and arguably paved the way for dynamic treatment regimes and a new emphasis on personalized medicine. It is evident that any deviation from standard practice poses methodological challenges that may be partially addressed during a crisis. A WWII example thereof is Wald's development of the sequential trial framework: there simply was no time for the established rigorous but slow industrial quality control processes . The new paradigm proposed by Wald led to further developments that continue to influence clinical research today . The current pandemic, just like the earlier ones, shows the need to trust the good faith of experts, and the good intentions of health professionals, rather than build onerous and time-consuming systems that are premised on the possibility of fraud and misbehavior. It is instructive to point out that the position of science and scientists was questioned in 1918 as well as today. Some referred to a “totalitarian system of science” . It is natural that the position of biomedical science and its biostatistics and epidemiological counterparts is debated because seldom are they so prominently present in the public debate. To understand this, note that an epidemic is somewhat archaically referred to as a “crowd disease” . It is natural to consult a physician for an ailment and, the more severe the condition, the more a patient is willing to accept side effects, as long as there is a sufficiently strong therapeutic effect. In fact, this is not different in a crowd disease. In the absence of PIs, the NPIs are society's only therapeutic class. Prescription, dosing, and monitoring of side effects then becomes a societal responsibility, where expert advice is blended with policymaking by mandated politicians, and with input from advocacy groups.
Epidemiological background The biomedical and public health community, as well as the world population, have quickly been learning crucial lessons about SARS-CoV-2 and COVID-19. To date, several aspects, though, remain uncertain or simply unknown. It is useful to briefly review some basic concepts of infectious disease modeling. While more complex models for COVID-19 are undoubtedly more appropriate to account for heterogeneity related to gender and age (in relation to social contact behavior, acquisition of infection, infectivity per average person, symptomatology of infected individuals and corresponding risks of hospitalization as well as subsequent mortality risks), spatial heterogeneity, and/or variation in risks due to societal position, the so-called basic Susceptible-Infected-Recovered (SIR) compartmental model provides a reasonable starting point (see,e.g., ). Although simplistic in the face of the current epidemic, the SIR model does contain the essential ingredients. Abrams et al. (2020) have developed a much more elaborate model, i.e., an age-structured, stochastic model, tailored to the dynamics of SARS-CoV-2 transmission and the subsequent human response upon contracting the disease, both at the level of the symptomatology as well as in terms of humoral immunity responses within hosts . In the basic SIR model, at any time t ≥ 0, the population is divided into three fractions or compartments: S ( t ) represents the susceptible fraction, I ( t ) is the infected (and infectious) fraction, and R ( t ) is the recovered fraction (immune survivors and potentially deaths). The initial states are S (0), I (0), and R (0). Flows of individuals between these states can be described using (ordinary) differential equations. The model is further influenced by two critical numbers: the recovery rate k , and the basic reproduction number R 0 . While R 0 is an implicit model parameter, the force of infection, i.e., the instantaneous rate at which susceptible individuals become infected, determines the basic and time-varying effective reproduction numbers, together with the recovery rate, through the so-called next generation matrix, providing information about the next generation of infected individuals resulting from a single typical infected individual. This basic model is rigid in that it assumes homogeneous (random) mixing within the population, and requires the population to be a closed system. In reality, as in Abrams' model, a population consists of various subgroups, or silos, with different behaviors (such as different levels of social contacts), and borders in a country like Belgium are merely administrative lines between neighboring countries . Also, the three-fraction system is often too simple. For SARS-CoV-2, we need to add an exposed state in which exposed individuals are not yet infectious while viral load is gradually building up, a pre-symptomatic compartment in which individuals are able to infect others even though they do no have symptoms yet, and compartments including asymptomatic individuals and individuals with mild symptoms, severely ill, hospitalised, and intensive care unit (ICU) admitted persons. Recovery and death are ideally kept separate as well. Such an elaborate model is essential if it is to be used against the background of the hospital capacity available, and to gauge the death toll. Consider now the reproduction number R 0 , defined as the average number of susceptible individuals infected by a single typical infected individual during his/her entire infectious period, at least in a fully susceptible population. There is a whole world “not” captured by a single R 0 value. First, it may depend on the initial population characteristics (age distribution, geographical spread], etc.). Second, as time evolves and S ( t ) depletes, the effective reproduction number R e is more relevant. The basic reproduction number as a measure of transmissibility of a pathogen is very different for seasonal influenza, where it is usually around 1.5, as compared to COVID-19, where it is estimated around 2.5 without medication or vaccines, and without NPIs. For an overview of COVID-19 related R 0 estimates, see Abrams et al. (2020) [1]. An early estimate for COVID-19, based on the Wuhan outbreak, can be found in Zhou et al. . As is now everyday knowledge, R 0 < 1 (and R e < 1) implies dampening of the epidemic, whereas with R 0 > 1 (and R e > 1) it picks up until immunity is sufficiently widespread or the susceptible reservoir is depleted. Depending on R 0 and the generation interval (time between infection events in an infector-infectee pair, see ) building up immunity can be a lengthy process, even if no interventions are implemented, which we seem to see with the current pandemic. Moreover, uncertainty surrounding the nature and extent of immunity is considerable, because humoral immunity seems to wane over time and the role of T-cell immunity is yet to be studied in more detail. At the onset of the Wuhan outbreak, there was considerable uncertainty regarding key epidemiological parameters, in particular R 0 , but also the associated (case and infection) fatality rate. We now know that both R 0 and the infection fatality rate (IFR) are relatively high, the latter being highly variable with age. Although highly dependent on the population under study, some additional examples of R 0 values from other infections: measles ( R 0 ≃ 15), mumps ( R 0 ≃ 5), SARS ( R 0 ≃ 2.5). See Riccardo et al. , Chowell et al. , and He et al. . Several other quantities are of epidemiological interest: infectious period (roughly about a week, versus a few days for influenza); age-specific contact rate (the typical number of social contacts of a certain type a member of the population has, see ); mode of transmission (for COVID-19, the mode of transmission was established early as airborne droplets, but this mode was later supplemented with others); probability of transmission upon a contact between a susceptible and infectious individual; shedding of viral load depending on the severity of symptoms; contribution of children to the infection process; high-risk contacts and their influence on disease dynamics (e.g., superspreading events). The infectiousness is strongly person-dependent (cf. the so-called superspreaders) and here secondary transmission via the airborne route is key, i.e., via aerosols . A key population characteristic is the contact rate, i.e., the frequency and intensity of physical social contacts between population members. The number and intensity of social contacts is not a constant. There are group- and individual-specific aspects to the contact rate and, importantly, it can be modified. During the time frame without PIs, modifying the contact rate and intensity (briefly or for a very extended period of time) is essentially the only option available. For a variety of reasons, describing and predicting a real-life epidemic curve may be very difficult. As the pandemic has been unfolding, the country and state specific epidemic curves take about any possible non-linear shape ( https://coronavirus.jhu.edu/data/new-cases-50-states ), underlining the importance and extent of heterogeneity in infectious disease dynamics. 2.1 The non-pharmaceutical intervention period Let us turn to the three possible strategies to modify the aforementioned contact rate, because when the house is on fire, and neither medicinal products nor vaccines are yet available, NPIs are all that one has got. The first strategy is suppression. It essentially means that the reproduction number is forced below one by imposing severe contact restrictions at the population level, as was done in China outside of Hubei (in Hubei, where the Chinese authorities were taken by surprise, this was at first not possible). Of course, a large fraction of the population is then kept in the susceptible state and hence they do not contribute to the build-up of herd immunity. As a consequence, measures should be put in place to avoid the epidemic from flaring up after measures are relaxed, while monitoring very effectively so that, if it does, suppression measures can be enacted again. Clearly, China is in this situation, and will be until vaccines and medication are available. Cheap, widespread, sensitive and specific diagnostic tools help maintain control, potentially supported by electronic means such as smartphone apps, as well as contact tracing and isolation . Needless to say, international travel in and out of susceptible regions is and remains problematic. Suppression is only possible when the viral spread is radically suppressed at an early stage, however, SARS-CoV-2 has stealth characteristics. Its incubation period is relatively long, with a very infectious period near the end of the incubation period . To aggravate matters, there is a large fraction of pre-symptomatic and asymptomatic but infectious cases (possibly 40–50%, although estimates vary widely and could be even higher). These characteristics, combined with a high reproduction number, make the epidemic resemble a bush fire: one match is sufficient to ignite it, after which the fire starts to spread at ground level, invisible to the naked eye until it suddenly evolves in an all-out fire. For this reason, in Europe, suppression was not a viable option after the initial outbreak in Northern Italy. The second strategy is mitigation . This was practiced by about all European countries during their first wave, to more (Italy, Spain) or lesser (Sweden) degrees. Here, measures are taken to bring the reproduction number down, such as reducing the number and nature of social contacts to a pre-specified level, so that the epidemic is slowed sufficiently and the number of critically ill cases at any time can be handled by the health care system. The difference between suppression and mitigation is that the latter aims to build up herd immunity, in such a way that the health care system is able to cope. It can be supplemented by a temporary capacity increase of the system (e.g., field hospitals, annexes to existing hospitals). The third strategy, or perhaps absence thereof, is counting solely on herd immunity . Generally, it will typically produce a shorter epidemic than with mitigation, and afterwards the population will be immune at population level. That is, the fraction of recovered people (immune for a certain time, e.g., the rest of the season) will be large enough, i.e. above the critical threshold, so that the re-emerging virus will not find enough susceptible population members to push the reproduction number above one, and the epidemic will soon decrease and become seasonal (where transmission is typically increased during winter months, as it is for influenza virus and other, more benign, betacoronaviruses, such as hCoV-OC43). It was anticipated, early March 2020, that mitigation in a country or region would lead to a population with roughly 30% of recovered, hence immune, members, whereas herd immunity could lead to 60–70% immunity, at least in the absence of clusters . The latter is sufficient to prevent further outbreaks, or to ensure that they would be short-lived. That is, provided that immunity is sufficiently strong and sufficiently long-lasting. Unfortunately, none of this has played out as anticipated. Sero-prevalence has been building up depressingly slowly . In Belgium, sero-prevalence was estimated at roughly 3% by the end of March, 6% mid-April, 7% mid-May, and back down to 6% and 5% in early June and July, respectively. This points to waning of IgG antibodies, after their discovery has been ridden between a long delay in onset of detectability and relatively poor sensitivity. At the time of writing, this suggests that other aspects of immunity, such as T-cell immunity, need to be scrutinized . A key limitation to herd immunity strategies is the high fraction of critically ill patients, leading to overburdening of the health care system, and the high IFR . In the Belgian non-nursing home population, the IFR is about 0.4%, but this figure masks the strong age gradient, with an IFR close to 0% in the population under 25, but rising to 2.5% in the 85+ population outside of nursing homes, and to 35% for the 85+ in nursing homes. Not surprisingly, the death toll in nursing homes is very large (two thirds of the nearly 10,000 COVID-19 related deaths in Belgium are among nursing home residents). This has been observed in a large number of countries around the globe . The death toll has been quoted as an argument for why lockdowns and other NPIs are unavoidable. In Europe, an estimated 3 million deaths have been avoided by lockdown measures . For Belgium, this boils down to a figure between 50,000 (with a coping health care system) and 250,000 (for a strongly overwhelmed health care system). How to proceed with the mitigation strategy when the peaks in the relevant curves lie in the past? Given the large reproduction number (super-spreading virus combined with a long infectious period), relaxation of NPIs needs to be done with utmost care. Re -emergence of the epidemic is likely as the virus will have built up reservoirs already. Reservoirs take the form of animal species that harbor the virus during time periods when there is no human epidemic(e.g., geese and pigs in the case of influenza). Changing tactic and opting for herd immunity is extremely difficult because it would undo the effects of NPIs, including the hardships they will have induced. It is only a viable strategy if supplemented with sufficiently promising PIs (antiviral medication and vaccines). While pharmaceutical breakthroughs are happening at an unprecedented speed, it is unrealistic to expect major relief from this end in less than a year. It is more realistic to move towards suppression, or a combination of mitigation and suppression when the epidemic is sufficiently under control, i.e., when the number of new infections falls below a certain level. At that point, contact tracing and quarantine measures, needed for suppression, become a viable strategy, supported by increased reliability and capacity of diagnostic testing, the use of electronic tracing (e.g., based on apps) in addition to human tracing (by infectiologists and health inspectors). A final but extremely important aspect is whether or not contact between populations will be possible in periods when there are no peaks or outbreaks. The answer is that this could well be detrimental. Not only is travel itself a risk factor, as is clear from the early introductions around the globe, but contact between populations in different epidemic stages is complex. China's cautious protection of its borders after its initial peak, as well as Europe's initially prudent but now complex international travel situation, even within the Schengen zone of the European Union, are cases in point. Inevitably, new outbreaks will keep emerging until immunity is sufficiently widespread or adequate vaccines are available. Antivirals will not stop this but may prove important in turning mitigation strategies into a success . Note that this provides an interesting link between NPIs and PIs, between mathematical modeling and the outcome of successful clinical trials. The seasonality of COVID-19 (and its successors in subsequent years, i.e., COVID-20, etc.) is poorly understood at this point, although Kissler et al. provide useful predictions, based on knowledge from coronaviruses OC43 and HKU1. Corona virus-induced diseases (typically but not exclusively, common cold) are seasonal, but less so than, for example, influenza. Kissler et al. report that outbreaks are possible at any time of year, with more acute outbreaks in autumn and winter. Depending on the extent of (non-permanent) immunity, either annual or biennial outbreaks are more likely. Other scenarios would be possible if immunity is lifelong (i.e., outbreaks in cycles of 5 years or more). Also, cross-immunity with other betacoronaviruses HCoV-OC43 and HCoV-HKU1 will play an important role in temporal SARS-CoV-2 dynamics.
Modeling and monitoring the epidemic 3.1 3.2 Nowcasting and early warning Modeling the event history of COVID-19 is important for public health policy, especially towards critically ill patients . Event history analysis includes studying the timing (or delay) between different events: infection, symptom onset, confirmed case, hospitalization, recovery and death. First, due to the incubation period and the delay of reporting and/or hospitalization, the impact of intervention measures is only observed after several days. For example, if the sum of the incubation period and delay of reporting is 10 days, then we expect to see an impact of the interventions on the number of confirmed cases after 10 days. However, as the delay time varies from individual to individual, the effect of the intervention is spread over several days. The delay distribution of the incubation period and the time between symptom onset and hospital admission ] is therefore crucial, as is understanding the heterogeneity in the delay times among individuals. Good knowledge of such delay distributions allows one to back-calculate the number of newly (symptomatic) infected cases, known as nowcasting, from either the number of confirmed cases or hospitalised cases, and assess the impact of intervention measures. Second, the length of stay in hospital is important, which varies among individuals and among countries due to different health systems. Information about the length of stay in hospital is important to predict the number of required hospital beds, both for beds in general hospital and beds in the ICU, and to track the burden on hospitals . Individual-specific characteristics, such as, for example, sex, age, comorbidity, and frailty of the individual, can explain differences in length of stay in the hospital and are therefore important to correct for. The estimation of the length of stay is complicated by the truncated and interval-censored nature of the data collected during the unfolding epidemic . Third, the time delay from infection and illness onset to death is important for the estimation of the case fatality ratio . A naïve case fatality ratio based on the proportion of reported deaths to reported cases during an outbreak is generally biased upwards, due to both the delay between case and death incidence and underreporting of cases. An early warning system to monitor COVID-19 trends and forecast increases of the hospital burden are essential in times of a pandemic . Multiple data streams are used as predictors of increases at the national and provincial level in Belgium. The mobility of individuals (tracked via mobile phone data), absenteeism at work, the number of patients with respiratory diseases visiting their general practitioner and the proportion of positive tested cases are important predictors for the immediately following two-week period . This is especially relevant at crucial times during an epidemic with multiple waves. Nowcasting is essential at the onset of the epidemic and when the curve begins to flatten and a peak is reached. It is also relevant when the rate of decrease slows, an often missed signal. While a decreasing curve is qualitatively a favorable evolution, it is important to constantly monitor the rate of decrease: if the decrease slows down while the curve is still at a relatively high level, it might be an early sign that it might eventually stop and then, unfortunately, start to increase again.
Modeling Jewell underscores the importance of high-quality mathematical and statistical models for epidemics. Using such models, key epidemiological quantities are estimated: numbers of infected cases, hospitalizations, people in ICU, and deaths. Some models also permit short-term, medium-range, and long-term predictions, and allow to examine how such quantities change with changing human behavior and measures taken, such as social distancing, face masks, hygiene and, eventually, vaccination programs. It is useful to cast predictions according to a variety of scenarios, to inform policy makers, other scientists, and the public opinion. Each model has its strengths and pitfalls, and simultaneously considering various models strengthens prediction. Some models operate at macro level (e.g., to study the number of cases in the population of an entire country), while others operate regionally or locally. Models are informed by data, mathematical infectious disease theory, and assumptions. Each model provides a piece of the jigsaw puzzle, and it requires a good amount of expertise and skills in infectious disease modeling to lay the entire puzzle. Model uncertainty and sensitivity analysis must accompany every modeling effort. Models, no matter how refined, will never be able to capture every detail of human behavior. In fact, there are striking examples of models that were poorly predictive because they ignored behavioral aspects, such as the need for college students to gather and party . Also, important epidemiological quantities, such as the ones referred to earlier, are (typically) fully unknown at the onset of a pandemic. Over the first half year of the crisis, several quantities have been estimated with increasing precision, though sometimes with hiccoughs (e.g., the length of the pre-symptomatic period). Others, such as seasonality, remain hazy. The determination of immunity has been a roller coaster of progressing insight (see Section 5). In a growth model, such as a logistic or Richards model , hospital admissions, number of tests, etc. are used to compute how the spread of the virus evolves over time. This approach lends itself naturally to estimating how the growth factor of the epidemic changes according to measures taken, or under the influence of a changing testing strategy. Transmission trees aim at mapping the chain of infections among people . One examines the genetic similarity of the virus among people or one makes use of contact tracing. For COVID-19, contact tracing was applied at the onset of the epidemic to find out in which region a person could have been infected. As the epidemic in March 2020 gained strength and the number of infected people increased, contact tracing was no longer feasible in Belgium. However, it is considered a vital component of a suppression strategy for second and later waves. Transmission trees are helpful to estimate key characteristics of SARS-CoV-2, such as the basic and effective reproduction number, and the generation interval, i.e., the time lapse in a so-called infector-infectee pair, the serial interval, i.e., the time between symptom onset in the infector and in the infectee, and the incubation period. Based on COVID-19 data from China and Singapore, Ganyani et al. were able to show that R 0 is larger when estimated from the generation interval as compared to the serial interval, pointing for the first time to pre-symptomatic infections, its associated risks, and implications for an exit strategy A meta-population model is a robust, large-scale model, that allows to incorporate people's mobility. It divides the population into groups based on age category, residence, etc. Each of these groups follow an underlying mathematical model for the spread of the epidemic. Such a model assigns people to the various compartments (susceptible, exposed, infected, recovered). By mimicking interaction between such groups according to various scenarios (e.g., little or a lot of contacts with people outside the household, little or more mobility between towns, etc.), it is possible to predict how the number of infected people changes over time, in the short run as well as over longer time intervals. Important sources of information are the number and the nature of social contacts of people in various age categories, the mobility patterns of people in different regions, etc. An individual-based model, based on the number of hospitalizations, performs well in terms of (1) describing the spread of the disease, and (2) examining the consequences of relaxing the measures taken, i.e., candidate exit strategies. In such a model, each individual is assigned to a family, a school category, type of workplace environment, and the population at large. This assignment is guided by data available from school registries as well as employment data. The model mimics behavior of individuals on a day-by-day basis. It accounts for changes in behavior on weekend days relative to weekdays, during holiday periods and, importantly, also as a result of measures taken, such as school closure and reduced social contacts. When investigating the consequences of exit strategies (e.g., reopening of schools and certain workplaces), the team also examines the added value of household bubbles (i.e., a combination of members from multiple households, matched to have a similar structure in terms of age, composition, etc.), allowing repeated contacts within bubbles and lensuring a reduction in community-level mixing, and contact tracing to monitor and avoid new infections. Although simple deterministic compartmental models, such as the basic SIR model introduced previously, have been used in the initial phase of the pandemic, making an abstraction of some of the important properties of both the pathogen as well as the infected host, an additional layer of complexity is of utmost importance to incorporate in order to adequately describe the dynamics of COVID-19 and to make reliable predictions of the future course of the epidemic. As more and more evidence has been accumulating throughout the progression of the pandemic, it became clear that age-specific differences exist in susceptibility to infection, infectiousness upon infection, probability of being symptomatic and disease severity, thereby leading to large differences in hospitalization and mortality risks upon contracting SARS-CoV-2. Research groups with ample of experience in infectious disease modeling were well equipped to expand and refine existing models for disease spread to account for such complexities. In a Belgian context, an individual-based model (STRIDE) previously developed for influenza was adapted to describe COVID-19 dynamics in the Belgian population, a meta-population model accounting for mobility patterns was adapted to study the impact of exit strategies in the aforementioned setting and a stochastic age-structured compartmental model was designed and specifically tailored to the spread of SARS-CoV-2 following earlier though related work on asymptomatic infections and their role in disease spread . On top of that, preparedness after previous epidemics, such as but not limited to the Ebola virus epidemic in West Africa (2013–2016), and experience in modeling infectious disease dynamics under pressure allows one to go beyond the application of simple models. Complexities imposed by intervention measures taken, such as stringent lockdown measures, and their impact on social contact behavior, pose additional challenges for modeling. Consequently, there is a need to directly relate the spread of the disease to social contact behavior and to inform transmission rates using social contact data. All of the approaches mentioned before (Individual-based, meta-population and stochastic models) rely on such social contact data, besides other sources of information, to calibrate and relate these models to the given epidemiological situation. Needless to say, model outputs and predictions require continuous fine-tuning and validation. Long-term predictions, while very useful should be seen as plausible scenarios at best, that demonstrate the impact of assumptions and variations in behavior. A collection of the aforementioned statistical and mathematical models developed by the team at the Universities of Hasselt, Antwerp and Leuven in Belgium can be found at www.simid.be and https://www.uhasselt.be/dsi-covid19-en . While not always obvious, there are clear links between statistical and mathematical modeling of the epidemic, and COVID-19 clinical trials research. A convincing illustration is found in Torneri et al. , who establish the vital role of antiviral medication in local outbreak control, in other words, the impact of non-pharmaceutical and pharmaceutical interventions can form a virtuous couple. In retrospect, when a number of predictions have been cast, under a variety of scenarios, at most one of these will come close to what actually happened, at least for the country or region for which it was intended. But, in a pandemic, countries and regions around the globe, with varying characteristics, all exhibit their own curves. For example, the Southern and Western states in the US exhibit a very different curve than the Northeastern states (cf. https://coronavirus.jhu.edu/map.html ). While care needs to be taken when comparing an observed curve with a prediction intended for a different geographical entity (or subpopulation), it is useful information for epidemic monitoring as well as for future model refinement and calibration, not only for future pandemics, but also for subsequent peaks of the ongoing one.
rtality reporting Mortality among COVID-19 patients is relatively high when measured by IFRs . The overall IFR is estimated around 0.6% in many countires, but is very strongly age dependent, and the risk is higher for males than for females. This was clear even from early reports . In a pandemic like the current one, it is not uncommon to have (at least) double mortality reporting. For example, in Belgium, Statistics Belgium reports overall mortality, while the Belgian health institute Sciensano reports COVID-19 mortality. Excess mortality can be deduced from overall mortality, providing an alternative estimate for, and perhaps a better one, than COVID-19 mortality . Hence, this is a place where official statistics, epidemiology, and demography meet. Bustos Sierra et al. and Molenberghs et al. from a Belgian perspective, and Aron et al. from an international standpoint, reported that Belgium's excess mortality agrees very closely with COVID-19 mortality. This is because Belgium reports not only confirmed COVID-19 deaths in hospitals, but also suspected deaths regardless of the place of occurrence. In contrast, these authors found that in the Netherlands the reported COVID-19 mortality accounts for only 62% of excess mortality. Arguably, excess mortality, when carefully teased out from overall mortality, is a better estimate of COVID-19 mortality, than reported COVID-19 mortality itself. For example, the number of deaths per million on July 4, 2020, was 843 in Belgium, 650 in the UK, 607 in Spain, 576 in Italy, 458 in France, and 357 in the Netherlands. But, after correction for underreporting, these figures become 1012 for Spain, 860 for Italy, 813 for the UK, 766 for Belgium, 575 for the Netherlands, and 472 for France ( https://github.com/owid/covid-19-data/tree/master/public/data ). Something that has become saliently clear is the very steep IFR curve as a function of age . This, combined with the superspreading context that nursing homes provide has led, in many countries, to a huge death toll in such settings , which has in turn triggered dedicated epidemiological research.
Prevalence determination and other surveys Unlike testing and tracing, which is aimed at finding as many new cases as possible, prevalence determination is aimed at reliably estimating what fraction of the population is recovered and hopefully immune. Apart from the viral and immunological issues related to prevalence determination, it should be done based on representative samples. Hence, sample survey methods can be used, although often alternative methods are used. Prevalence determination is important to gauge IFRs and to assess whether or not herd immunity is building up. 5.1 5.2 Prevalence determination An obvious way of prevalence determination is by means of the sero-prevalence, based on the detection of antibodies in blood serum samples. Herzog et al. proceeded via a nationwide cross-sectional survey of residual blood samples tested for the presence of Immunoglobulin G (IgG) antibodies against SARS-CoV-2. This method, as we know now, is ridden with a number of issues, such as time to IgG seroconversion, detectability, and waning . In Belgium, sero-prevalence around April 1, 2020, was around 3%, three weeks later it was 6%, rose to nearly 7% mid-May, and then started to drop to 5.5% (around June 10) and even 4.5% around July 1. In other words, as mentioned in the literature , waning of IgG antibody concentrations is also observed in this sero-epidemiological study, and the primary route for immunity may not be these antibodies but rather T-cell mediated immunity or other antibodies not measured so far. As a consequence, IFR determination and the status of a population's immunity are referred back to the drawing board and the interpretation of (serial) sero-prevalence studies have to be reconsidered. Evidently, the decrease in seroprevalence implies that the status but also extent of immunity may be very different when based on T-cell mediated and humoral immunity responses. Also, cross-immunity with endemic coronaviruses, especially beta-coronaviruses such as hCoV-OC43, is a relevant study subject, but one about which there is little or no knowledge available . Note that different survey sampling methods and different sub-populations considered (e.g., blood donors, or people spontaneously reporting at hospitals) may well yield different estimates. Apart from immunological issues with prevalence determination, the quality of the representative sampling method used influences the reliability of the findings.
The role of public opinion surveys It is important to keep the finger on the pulse of the public opinion, for various reasons. Well-conducted surveys are vital to get a feel for how the population perceives risk, the impact of measures taken, acceptance and compliance to NPIs, etc. At the same time, it can be a component of an early warning system (Section 3.2) if the occurrence of symptoms is queried. One such example is the “Big Corona Study” (see also ), an online survey that can be filled in by all members of the public on every Tuesday since March 17, 2020; from June 2, 2020 onwards, the survey shifted to a bi-weekly frequency. It collects data about public adherence to measures taken by the government, contact behavior, mental and socio-economic distress, and spatio-temporal dynamics of COVID-19 symptoms' incidences. While public participation is useful as a low-cost method to collect timely information within the context of a pandemic, caution should be exercised at the analysis stage; online surveys, based on self-reporting, often do not reach every societal group equally . It typically causes response rates to vary among citizens of different ages, genders, cultures and economic statuses. This is particularly the case in 2020, where the perception of the seriousness of the COVID-19 pandemic varies considerably between individuals and has become politically coloured. This then translates to increased difficulties to correct for unrepresentative samples, even after standardization methods such as inverse probability weighting are performed. In essence, these problems all relate to non-random missingness patterns , where the absence of information is driven by complex processes. These processes do not lie far from opportunistic sampling phenomena that often occur in biodiversity studies that make use of citizens to collect data . For example, using such surveys to pinpoint areas of increased disease incidence necessitates careful investigation, since response rates' spatial dynamics may be stochastically dependent on the underlying spatial process that generates heterogeneity in the symptoms' incidences. If present, this opportunistic sampling phenomenon, termed preferential sampling , invalidates statistical inference on the spatial dynamics of COVID-19 symptoms. This can be accommodated by using a shared latent process approach where a geostatistical binomial model for the proportion of participants of each Belgian municipality that experiences COVID-19 symptoms shares a spatial random effect with a model for the response rates. The result of this approach is shown in , which depicts predicted symptoms' incidence, corrected for preferential sampling, using data of 397,529 individuals collected during the third round of the “Big Corona Study” (March 31, 2020). The above is an example of how survey sampling methods, citizen science, and spatial statistics come together to gauge the public opinion regarding COVID-19 and, in turn, to inform policy makers. Unsurprisingly, several suveys are undertaken simultaneously. For example, the Belgian health institute Sciensano has conducted several waves of a COVID-19 Health Interview Survey . This study has a longitudinal component; participants can indicate whether or not they are willing to have their responses linked across waves. Smaller scale (longitudinal) surveys towards the public's perceived vulnerability and acceptance of measures are undertaken too . A general perspective on the role of social and behavioral science in the response to COVID-19 research can be found in Van Bavel et al. . In many countries, all such surveys take place in an ad hoc fashion. It can be beneficial, though, to make use of a permanent (online) representative panel for public opinion research. Such a panel exists in the Netherlands . Catalyzed by the current pandemic, a panel of this type is likely to be initiated in Belgium as well.
Diagnostic and serological testing The battle against a novel emerging pathogen such as COVID-19 requires the development of a rigorous screening strategy to detect the virus, with the objective to mitigate its public health impact and to bring the pandemic under control. Aiming to achieve a rapid scale-up of diagnostic testing capacity has rarely, if ever, been attempted at the current pace . Testing is not merely an instrument to diagnose a given individual and to determine individual-level risk factors, it is also a prerequisite to a proper disease surveillance system, serving in monitoring and managing the epidemic. Testing allows unraveling a number of key uncertainties concerning the epidemic, such as the number of infected people, or the proportion of the population that is effectively immune against the virus. Early literature , i.e., from the first quarter of 2020, is a testimony that at first, diagnostic instruments for SARS-CoV-2 were lacking and needed to be developed in a speedy fashion. The SARS-CoV-2 tests that were developed since the start of the COVID-19 outbreak can broadly be categorized in so-called real-time (diagnostic) reverse-transcriptase PCR (RT-PCR) and serological tests. Patients with symptoms are often diagnosed based on RT-PCR tests allowing the detection of viral nucleic acid in oropharyngeal or nasopharyngeal swabs. Such tests identify whether someone has the virus. Serological tests on the other hand, determine the presence of antibodies. With the advent of COVID-19, new serological tests have been emerging, creating new opportunities for an assessment of the SARS-CoV-2 epidemic. Serologic tests are most of the time ineffective at detecting early stages of the infection, since antibody titers only gradually increase days or weeks after infection, but are able to detect past infections providing, in theory, an indication of the proportion of the population that has been infected with the virus, at least when lifelong humoral immunity is conferred. Serological analysis may be useful to actively identify close contacts, define clusters of cases and linking clusters of cases retrospectively to delineate transmission chains and ascertain how long transmission has been ongoing or to estimate the proportion of asymptomatic individuals in the population . Serological tests help to understand the epidemiology and to evaluate vaccine responses, but the reliability for diagnosis in the acute phase of illness and the assumption of protective immunity have been questioned . Detection capabilities of tests may further depend on the delay since the onset of the infection or symptoms . Furthermore, higher antibody levels not necessarily correlate well with an increase in protection against reinfection. Despite their value, serological tests do not allow, given the many current unknowns and uncertainties, to confirm whether or not a person is contagious or if he/she is protected against the virus, unless a correlate of protection is well-established, and do not allow, in other words, the delivery of an “immunity passport”. In the initial phase of an epidemic, knowledge on diagnostic test performance is scarce and not fully reliable. Samples are usually collected from a limited number of patients, and negative controls are not always present. A correct assessment of the limitations and performance of each of these tests is nevertheless crucial to demonstrate their accuracy and clinical utility and to design a correct testing strategy. The performance of a diagnostic test is typically characterized by its sensitivity and specificity. RT-PCR tests are considered reliable for detecting the presence of the virus, and are considered the standard by some, despite a non-negligible rate of false negative results, i.e., a low sensitivity - in some circumstances (see, for example, ). False negatives can complicate governmental decisions to lift confinement restrictions. False-negative results have an impact on the manner in which serological testing might be used to support non-pharmaceutical interventions, as well as implications for the development of large-scale testing pathways . The current evidence about the diagnostic accuracy of COVID-19 serology tests is characterized by high risks of bias and heterogeneity, with limited generalizability to outpatient populations . A full comparison of the performance of serological tests has not yet been conducted on a large set of identical samples. The duration of antibody rises is currently unknown, and the utility of these tests for public health management purposes has been reported as uncertain . Variation in performance characteristics between assays indicates the urgent need for evaluation of the large number of SARS-CoV-2 serology tests that have become rapidly available . Evaluating the performance of diagnostic tests is usually based on comparing test results with a gold standard, but such a “perfect test” is often unavailable. Moreover, even if the diagnostic sensitivity and specificity are considered fixed values, intrinsic to the diagnostic test (i.e., constant and universally applicable), many examples illustrate that these values can fluctuate depending on the context . Estimations of test characteristics are often obtained from studies under well-controlled conditions. The sensitivity of RT-PCR tests used for the diagnosis of COVID-19 may, for example, depend on factors such as the type of specimen, the timing of sampling and the sampling technique . Yet, quantifying the performance of a given test in real-world conditions is essential when interpreting test results, measuring its predictive value, or when choosing a test for a specific use case: screen asymptomatic patients, monitor contacts, identify clusters, support contact tracing, and as a preventive measure. Hitchings et al. explain how the so-called test positive fraction correlates with the incidence in a given population, turning this into a useful surveillance tool. In hospital settings, sensitive and specific diagnostic tests for active infection with SARS-CoV-2, allow guiding the care for individual patients, but a fast and repeated testing strategy at the expense of e.g. a lower test sensitivity may be more effective as a public health strategy . A public health strategy – with the goal to reduce transmission - may indeed ask for the use of rapid tests, removing the focus from the usual dogma of high sensitivity and specificity towards a test to be practically useful, also accounting for factors such as costs, speed, and logistical constraints. A proper evaluation of diagnostic performance in the absence of a gold standard can be done by using latent class models, which do not require a priori knowledge of the infection status. Umemneku Chikere et al. provide an overview of these and other models that allow using the combined information of multiple different tests applied on the same samples and Kostoulas et al. present standards for the reporting of such diagnostic accuracy studies. Models used to analyze the results of multiple diagnostic tests assume that there is an unknown prevalence, sometimes referred to as a latent class, and that the sensitivity and specificity of the diagnostic tests are unknown. This “latent prevalence” can then be linked to the apparent prevalence (i.e., the observed proportion of positive results of the diagnostic tests) through a set of equations allowing estimating all parameters at stake (i.e., prevalence, sensitivities and specificities of each of the tests used) . Further context on issues surrounding diagnostic tests is given in Tang et al. . Once diagnostic tools are available and properly evaluated, their use may be hampered by constraints such as a lack of reagents, limited laboratory capacity, and personnel. Pooling samples may be used to addresss this concern, increasing the number of individuals tested with an available number of tests and providing a cost-effective alternative to individual testing. Over the years, an entire body of research has indeed been developed around group testing in a diagnostic context, for example when resources are scarce and/or under time pressure . This is precisely the situation we are confronted with the current pandemic, creating an opportunity to roll out and test reliable and new methodologies (see, e.g., ). It is another example where existing and seasoned methodology can be tailored to differing circumstances, such as the need for repeated testing, as described by Augenblick et al. . Test results can be compared with the results from non-pharmaceutical components of early warning systems (Section 3.2). Knowledge on the test characteristics can be used and integrated when interpreting survey results (Section 5.2).
Vaccine development While a number of effective vaccines have been developed over the last half century, such as for measles, rubella, smallpox, hepatitis B, Ebola, etc., vaccine development remains a challenging area. For example, no succesful vaccine has been found so far for HIV . Even the determination of the seasonal influenza vaccine, a yearly exercise, is one of hits and misses, due to the volatile nature of the influenza virus. Of particular importance to us is that traditionally coronaviruses (hCoV-229E, hCoV-NL63, and hCoV-OC43) have received little or no attention from a vaccine development standpoint. This changed for SARS-CoV-1 and MERS-CoV but in these cases there was no opportunity to put potential vaccines to the test. While existing vaccine-constructs (e.g., adeno-based, adjuvants, etc.), in particular for influenza and the aforementioned coronaviruses, can provide a step-up for SARS-CoV-2, success is not automatically guaranteed. Because the general consensus is that the global population will be able to return to normalcy only after the development of effective vaccines and the implementation of large vaccination programmes, the challenge is to develop a vaccine at unprecedented speed. Evidently, global collaboration is essential. A candidate vaccine developed in one part of the world may have to be put to the test in another, depending on the succession of epidemic waves. The state of urgency poses ethical questions, such as whether one can, besides the traditional phase 3 efficacy studies, set up controlled human infection model (CHIM) studies where healthy subjects are infected to test a vaccine, while effective treatment may not yet be available. A further challenge is that vaccines need to be developed while the immunology associated with SARS-CoV-2 is still unclear, and knowledge is accumulating, with trial and error. Several pharmaceutical companies have taken the unprecedented step to plan and build production capacity in parallel with candidate vaccine development and testing. A fascinating new chapter is currently being written to bring future vaccines to market; many lessons will be learned that fall beyond the scope of the present paper.
Clinical trials for COVID-19 patients The amount of clinical research generated by the COVID-19 pandemic is mind-boggling: on June 15, 2020, a search of the ClinicalTrials.gov website with the keywords “COVID”, returned more than 600 interventional studies currently recruiting patients . For a more complete coverage of trials worldwide, the ReDO database listed 1144 interventional trials for the treatment of COVID-19 infected patients on June 26, 2020 . Reassuringly, 825 (80%) of these trials were controlled and taking place in a hospital setting (because testing capacity was lacking outside of the hospitals at the start of the pandemic). It is beyond the scope of this paper to cover the various treatment approaches that are being tested against COVID-19, whether using repurposed drugs already in use for other indications, new drugs specifically developed against the virus, or non-drug treatments. The World Health Organization (WHO) published a useful classification of treatment types . Here we focus on key features of some of the clinical trials that were designed and conducted in record time in the early days of the epidemic in Belgium. 8.1 8.2 8.3 8.4 8.5 Outcome measures The natural history of most diseases is well established, and a consensus has in most cases been reached on outcomes that appropriately capture how a patient feels, functions or survives. COVID-19 infections were, at least initially, largely unknown, hence it was challenging to choose outcome measures that would be clinically relevant as well as statistically sensitive to treatment benefits. The best outcomes to use will undoubtedly emerge as the results of clinical trials begin to appear and clinicians have built experience on how to measure these outcomes. In large randomized trials for hospitalised patients such as RECOVERY (Randomized Evaluation of COVid-19 thERapY), all-cause mortality within 28 days was the primary outcome of interest . While all-cause mortality is unquestionably the ultimate clinical outcome most therapies are trying to impact, cause-specific mortality could be more sensitive and also more relevant if (and only if) the treatments had no impact on deaths due to other causes. In practice both all-cause and cause-specific mortality are typically required to assess all treatment effects, and the designation of either one as the primary outcome may depend on the importance of competing risks of death. Other outcome measures of interest are time to invasive mechanical ventilation, and time to discharge. Besides time to clinically important events, the need to quantify the severity of the COVID-19 infection led to the definition of clinical progression scales. shows one such ordinal scale with scores ranging from 0 to 10 . Less granular ordinal scales have been used (e.g., with scores ranging from 1 to 5) with a similar intent. Various outcome measures can be defined using these scales, e.g. time to a score change (improvement or deterioration) of at least 2 points on the chosen scale, cumulative score or area under the score curve up to day 15, etc. Time will tell which scale and outcome measure are simple enough to be used effectively and sensitive enough to detect treatment benefits. Last but not least, inclusion of patient-reported outcomes (PRO) should be considered in trials of COVID-19 patients with prolonged follow-up .
Multi-arm designs The main challenges when conducting clinical trials in the COVID-19 context are (a) the multitude of potential treatments, (b) the lack of patients in some regions to conduct several trials in parallel, (c) the pace at which new scientific insights become available, and (d) the push to use treatments based on incomplete preclinical development and unreliable clinical data. Hydroxychloroquine, for instance, made it into preliminary COVID-19 treatment guidelines without proper supporting evidence, thus undermining the use of untreated controls in clinical trials. This has forced statisticians and clinicians to search for flexible designs which allow including additional promising therapies or removing therapies which have shown not to be effective, while simultaneously allowing for optimal use of the limited available patients and drugs. When two treatments A and B are to be compared to standard of care (SOC), a natural choice would be a randomized multi-arm study comparing A, B and SOC (leaving aside the potential difficulties of access to A and B at once). The advantage is that a single SOC group can be used rather than two SOC groups which would be needed in two separate trials comparing A with SOC and B with SOC. However, classical multi-arm studies require all patients enrolled to be eligible for all treatments. In the COVID-19 context, a research treatment often has contraindications which do not allow patients to be randomized to that particular treatment, but allowing patients to be randomized to some of the other treatments under consideration. A possible solution is selective exclusion. While such designs with selective exclusion have been described in the statistical and medical literature , the statistical analysis of such studies has not received much attention. As an example, consider a scenario in which patients are randomized to treatment A, B, or SOC in a (1:2:1) ratio. Interest is in comparing A with SOC, and B with SOC. Further assume that 10% of the population eligible for A and/or B is eligible for A only (subpopulation 1), while 30% is eligible for B only (subpopulation 2). The remaining 60% is eligible for both A and B (subpopulation 3). This situation is graphically shown in . Out of 100 patients eligible for A or B, we expect 10, 30, and 60 subjects in subpopulations 1, 2, and 3, respectively. In each subpopulation, randomization is performed according to the appropriate ratios, i.e., (1:1), (2:1), and (1:2:1), in subpopulations 1, 2, and 3, respectively. When analyzing the effect of treatment A versus SOC, only concurrent controls can be included. Hence the SOC patients from subpopulations 1 and 3, will be compared to all A patients from the same two subpopulations. However, in subpopulation 3, 50% of the patients received B, implying that subpopulation 3 is underrepresented in the comparison of A versus SOC. If the objective is to estimate the marginal effect of A versus SOC, i.e., the effect one would estimate in a placebo controlled trial of A versus SOC, the patients from subpopulation 3 need to be reweighted by a factor 2, in order to restore the balance between subpopulations 1 and 3. The final analysis of A versus SOC is then a weighted analysis of the 2 × 20 patients from the subpopulations 1 and 3 who received either A or SOC, however, the patients from subpopulation 3 get each a weight of 2. Likewise, the marginal effect of B versus SOC can be estimated using a weighted analysis of the 25 SOC patients and the 50 B patients from subpopulations 2 and 3, but the patients from subpopulation 3 need to be reweighted by a factor 4/3 in order to correct for the imbalance due to the removal of the 25% patients on treatment A in subpopulation 3. Note that the gain of the design in is that an expected 15 SOC patients, i.e., 25% of 60% of the study population, can be used twice, once in the comparison with A and once in the comparison with B. The gain obviously highly depends on the eligibility criteria and on the randomization ratios used. Note also that the methodology can easily be extended to trials with more than two research treatments and to trials with adaptive designs allowing for adding new treatments or removing non-promising treatments.
Factorial designs Factorial designs, a rare exception in trials sponsored by pharmaceutical companies who prefer to focus on a single therapeutic question, were suggested for situations in which more than one treatment could be tested simultaneously in the same patients. As an example of such a design, the COV-AID trial (Treatment of COVID-19 patients with Anti-Interleukin Drugs) simultaneously tested blockade of the Interleukin-1 pathway with Anakinra, and blockade of the Interleukin-6 pathway with either Siltuximab or Tocizilumab, in hospitalised adult patients with COVID-19 infection, acute hypoxia and signs of cytokine release syndrome. The factorial design is premised on the effectiveness of interleukin blockade to prevent hyperinflammation or auto-inflammatory syndromes in COVID-19 infected patients. Interestingly, in such a design, only 2 out of every 9 patients receive usual care while 7 receive usual care plus at least one experimental drug (see ).
Interim analyses and multi-stage designs In view of the huge uncertainties associated with anticipated clinical outcomes as well as treatment effects, it was generally considered appropriate to include one or more interim analyses for safety and/or futility and/or efficacy in the trial designs. Group sequential trial methodology provides a well-known framework for incorporating as many interim analyses as deemed necessary while adequately controlling the probability of a type I error. Any substantial trial benefits from being monitored by an experienced IDMC (Independent Data Monitoring Committee), and in particular trials with interim analyses of efficacy; however IDMCs are in high demand and short supply, and the flurry of COVID-19 trials will not ease the current shortage. Adaptive design methodology was also considered, though its most common applications (choice of an optimal dose, increase in sample size, or enrichment in specific patient subsets) did not address the most acute need in COVID-19 trials, which was to allow seamless addition or dropping of treatment arms to reflect a changing therapeutic landscape. This is the objective of platform trials, such as the multi-arm multi-stage (MAMS) trials . The PRINCIPLE trial (University of Oxford ), served as a model for the design of a similar trial in Belgium, the DAWN (Direct Antivirals Working against nCov) Ambulatory Care Platform trial. Logistical challenges in the setting of COVID-19 include the timely identification of eligible subjects, obtaining informed consent when isolation at home is needed, as well as the delivery of study medication. Initially, this trial will compare Camostat with standard of care in community dwelling adult patients who are at least 50 years old presenting with signs and symptoms compatible with COVID-19. The aim of this large pragmatic trial is to avoid hospitalization by using a well tolerated antiviral to rapidly treat patients at risk who have first symptoms of COVID-19. Like in PRINCIPLE, the DAWN trial will use Bayesian posterior probabilities to add or drop treatment arms while the study is ongoing, but unlike in PRINCIPLE, randomization will not use adaptive randomization, for there is neither a statistical advantage nor an ethical imperative to do so (see , with discussion). Instead, minimization can be used to allocate treatments in a constant ratio while allowing for several prognostic factors to be balanced across the treatment arms.
Pragmatism in trial conduct Perhaps the most impressive aspect of clinical trial activities during the pandemic was the collaborative pragmatism that naturally evolved in response to the crisis. Statisticians from academia, the public and the private sectors voluntarily contributed ideas and resources to come up with optimal trial designs to address the most critical clinical questions. Some of these collaborations pre-dated the pandemic, but many were improvised to respond efficiently to the most pressing needs. When it came to launching the trials, the usual delays and bureaucratic hurdles evaporated, and the trials could all be launched within a couple of weeks - instead of the several months usually required to fulfil all administrative requirements. While excessive speed may create challenges, as discussed in Section 10, on balance it may be preferable to unnecessary delays whenever the health of patients is at stake – and this is the case for many non-COVID-19 related health issues. While an overarching priority was given to rigorous trial designs, implementation details were kept as simple as possible. As was already argued prior to the pandemic, simplicity is a virtue in clinical research , but one that does not align with the commercial interests of the clinical research organizations that implement clinical trials for pharmaceutical companies . Many have argued that the absurdly high costs of pivotal clinical trials are due to inefficiencies in the current clinical research process . Examples of inefficiencies include the collection of data of marginal interest, including details of medical history and concomitant medications, complex procedures to measure outcomes, including central reviews and outcome adjudications, strict visit schedules and examinations that do not reflect clinical routine, and so on. Although some of these inefficiencies may be justified for pivotal trials of new drugs, they should generally be avoided in trials of approved drugs or other non-drug treatments. A clear distinction between pragmatic and explanatory approaches to clinical trials was proposed nearly fifty years ago, yet most trials conducted today adopt the explanatory approach, which is unnecessarily onerous . provides a comparison of trial characteristics under the explanatory and pragmatic approaches . The COVID-19 pandemic provided empirical evidence that inefficiencies in clinical research can easily be overcome in pragmatic trials in times of emergency. Will this lesson survive the end of the pandemic?
Impact of COVID-19 on ongoing clinical trials The COVID-19 pandemic has had, and will continue to have, a major impact on the conduct of almost all ongoing clinical trials, in particular on the treatment of patients and the schedule of their planned protocol visits. Regulatory agencies worldwide have promptly issued guidance on measures to be taken to minimize the impact of COVID-19 on ongoing trials . Given the huge uncertainty associated with the current situation, and the lack of historical precedents, the guidance documents recommend to capture as much information as possible on protocol deviations and other unexpected events, so as to be able to conduct various analyses when the trial is completed. Meyer et al. give an excellent overview of statistical issues and recommendations for clinical trials during the COVID-19 pandemic. From a statistical inference perspective, despite the dramatic health care disruptions caused by the COVID-19 pandemic, intention-to-treat (ITT) analyses of randomized clinical trials remain valid, if (as will generally be the case) protocol deviations impact all randomized treatment groups equally. However, such deviations may induce a dilution of the treatment effect, and as such are likely to result in more conservative estimates of treatment effects (with the exception of non-inferiority trials). In other words, the ITT estimates of treatment effects will in general not be biased by systematic differences between the randomized treatment groups, but they may well underestimate treatment effects that would have been estimated in ‘normal’ circumstances. 9.1 9.2 9.3 9.4 Protocol amendments Because the results of randomized clinical trials are, by nature, protected against changes in the environment that affect all randomized groups equally, there will generally be no good reason to amend the statistical sections of the protocols of ongoing studies, except for sample size calculations and the provision of descriptive statistics on the impact of COVID-19 (number of patients with COVID-19 infections and COVID-19 deaths). Some trials will have to stop as a result of the pandemic with a lower sample size than initially planned. For trials that can continue throughout the pandemic, major protocol deviations may result in a lower treatment effect than anticipated, which might justify a sample size increase to compensate the loss in statistical power. Such sample size increases do not affect the type I error.
Missing data Missing visits, missing clinical assessments, missing scans or laboratory values, and all such like that result from the COVID-19 pandemic will in general be missing at random (MAR), since the pandemic is an external cause of missingness that bears no relationship to the disease or treatment under investigation. Hence COVID-19 related missing data can be appropriately dealt with by using likelihood based methods or multiple imputation under the MAR assumption. To give a few typical examples: (1) hazard ratios estimated using proportional hazards regression models, e.g., survival times, remain valid under independent censoring (and proportional hazards); (2) treatment effects estimated using mixed models for repeated data, e.g., for longitudinal measurements of visual acuity, remain valid if the outcome data are MAR; (3) generalized estimating equations for longitudinal measurements of responses remain valid if missing data are imputed under the assumption of MAR. Multiple imputation may be feasible when the amount of missing data is limited; however, the potential for multiple imputation is limited when large volumes of data are missing, especially when few patients have observed data that can be used to impute the data for patients with missing values. In multinational or multiregional trials, the COVID-19 pandemic may take a different course in different regions; in addition, regional differences such as distance traveled to health care centers may create very different patterns of missingness across regions. This variability may not create a systematic bias if it affects all treatment arms equally. It does offer an opportunity to perform sensitivity analyses using region as a potential modulator of treatment effect. Other sensitivity analyses (such as shift imputation and tipping-point analyses) will likely play a more prominent role due to the larger than usual volume of missing data. Finally, it will be important to rule out situations of differential drop-out rate between the randomized treatment groups. This could happen, for instance, in open-label trials if patients in the control arm are more likely to miss their planned visits than patients who receive an experimental therapy. Conversely, some trials had to stop the experimental treatment (e.g., immunotherapy in cancer) for fear of an interaction with COVID-19.
Outcome assessments Missing visits have a direct impact on outcome assessments. For instance, in oncology trials, tumor response and time to progressive disease are assessed through CT-scans performed according to a fixed schedule. Some conventions that are sometimes applied, e.g., to censor patients if they have missed too many visits, become wholly inappropriate when deviations from the intended schedules are systematic and unavoidable. In such cases, these conventions should be used, if at all, only in sensitivity analyses. The proper primary analysis of time to progression should remain an ITT analysis, in which all patients are followed up as thoroughly as possible, regardless of how long it takes to obtain CT-scans, until they have objective confirmation of disease progression. Because in some patients such confirmation may come with considerable delay, interval-censoring analyses may be helpful to complement or even replace the traditional analyses with right censoring only. Some patients may prefer to avoid hospital or office visits during the COVID-19 pandemic. If outcome assessments were due to take place at the hospital or doctor's office (e.g., a 6-minute walk test), it may be preferable to replace these assessments by their home-based equivalent assessments, when available. In most situations, some data are better than no data at all, under the assumption that data taken in less than ideal situations are not grossly erroneous or misleading. In fact, even if assessments taken at home in poorly controlled conditions are less reliable than those taken at the hospital in the most rigorous conditions, the loss in efficiency in detecting a treatment effect may be surprisingly small, assuming no systematic bias between the randomized treatment groups . It is sometimes believed, wrongly, that patients who have symptomatic COVID-19 infections should be removed from trials of other indications. This is unjustified and should not be done unless it is mandated by the patient's safety or personal choice.
COVID-19 related events It is conceivable that in some cases a randomized trial has its treatment arms differentially affected by the pandemic if the intervention under study is a risk factor for COVID-19. As an example, in oncology, chemotherapy is felt to increase the risk of infection among cancer patients, and some authors have cautioned the medical community about this risk. In a trial comparing chemotherapy with a non-cytotoxic intervention, the incidence of COVID-19 may therefore be higher in the chemotherapy arm. If the infection is a risk factor for one or more of the outcomes of interest (e.g., survival), an association may be created between the exposure (treatment) and the outcome (in this case, survival) through the infection, thus confounding the analysis of such outcome(s), unless cause-specific mortality is used. The reporting of causes of death is generally unreliable and variable from center to center, but COVID-19 related deaths are likely to be reported reliably (respiratory diseases being an exception). It may also be useful to perform competing risks analyses for the outcome of primary interest in the trial (such as disease progression) and COVID-19 infection. It is conceivable that patients with COVID-19 infection will receive treatments that interact with their treatments for other indications. Such interactions would only create a potential bias in randomized trials if they were different for the treatments being compared, an unlikely situation but one that may on occasion occur. A related issue is that most clinical trials forbid the inclusion of patients in other trials of investigational drugs. AIDS advocacy groups argued long ago that co-enrollment in multiple trials was both ethically and scientifically desirable, a view that still prevails today and should be pro-actively implemented in trials .
The Price of speed: methodological sloppiness 10.1 10.2 Uncontrolled trials In times of great pressure, such as when the COVID-19 pandemic erupted, it is very tempting to take shortcuts and experiment with potentially effective treatments in an uncontrolled way, with the hope that some of the treatments tested will be so effective as to constitute real breakthroughs in the management of the disease. Two additional factors may mitigate against conducting properly controlled experiments: the number of patients available, and the severity of their condition (patients admitted to ICU often having a fatal outcome). Yet, despite ethical dilemmas with control arms, randomization was widely considered during the COVID-19 outbreak as the only way to generate reliable, practice-changing evidence . Claims made on the basis of supposedly impressive clinical outcomes of COVID-19 infected patients treated with Chloroquine and Hydroxychloroquine were viewed with skepticism, and the contradictory data that were later published about these drugs, including some that had to be retracted , confirmed that skepticism was indeed in order, and that scientific standards could not be lowered as a result of the pandemic . Observational studies, even when conducted with care, can be so misleading that some authors have argued a moratorium should be placed on reporting them . And indeed, to counteract exaggerated claims based on uncontrolled data, some wide-ranging national or international collaborations were quickly put in place for the conduct of large-scale trials . The SOLIDARITY trial, conducted in 35 countries under the auspices of the WHO, is an example of a large simple trial for hospitalised patients with COVID-19 treated with local standard of care (SOC) against Remdesivir, Lopinavir and Ritonavir, or SOC plus Lopinavir and Ritonavir and Interferon β -1a . Despite the best of intentions, the SOLIDARITY trial ran into contractual and legal difficulties that made its adoption in many countries slow and inefficient. Furthermore, the trial prioritized antiviral agents and other PIs over NPIs, which may have diverted resources away from trials of simple supportive care interventions. Finally, finding international consensus to select or change trial interventions is far more challenging than at the national level. The right balance between national and international efforts will have to be addressed going forward, with the overarching goal of maximizing the efficiency of clinical research. Trial implementation is definitely more efficient at the national level; however the number of patients in a small country like Belgium is insufficient to size the trials properly. Most of the trials started during the early phase of the COVID-19 epidemic will be too small to provide reliable estimates of treatment effects, and it would therefore be advisable to plan prospective meta-analyses of all such trials as soon as possible. Such prospective meta-analyses should be based on patient-level data (see Section 11). In the UK, the large RECOVERY trial tested standard of care against low-dose Dexamethasone, Azithromycin, Tocilizumab or convalescent plasma. This trial accrued 11,303 patients between March and June 2020 and, in this short period of time, was already able to show a highly significant benefit of dexamethasone on mortality , which immediately led to the use of glucocorticoids as standard of care for hospitalised COVID-19 patients.
Methodological errors Methodological issues have arisen in a number of studies dedicated to prediction models for diagnosis and prognosis (mortality risk, progression to severe disease, length of hospital stay) of patients with COVID-19. Wynants et al. conducted a systematic review and detected 51 studies with methodological issues and errors among a collection of 4909 titles screened. In clinical trials conducted in COVID-19 patients, the statistical methods commonly used are based on the standard Cox proportional hazards model and the Kaplan-Meier estimator (see, for example, , and ). When time to death due to COVID-19 is the outcome of interest, these methods implicitly treat discharged or recovered patients as right censored. Doing so is incorrect, however, as right censoring means that the unobserved time to death can be any time point larger than the observed one, whereas patients who recover may in fact never die from COVID-19. A correct way of analyzing this type of data is through the use of competing risk models, such as the model proposed by Fine and Gray which is based on the subdistribution hazard, or on cure models. To study the impact of incorrectly classifying recovered patients as right censored, Oulhaj et al. simulated data from a fictive clinical trial on COVID-19. Six scenarios representing different situations of the effect of treatment on death and its competing event recovery were considered. The hazard ratio of death and the 28-day absolute risk reduction were estimated using the Cox model and the Fine and Gray model. The Cox model estimated the hazard ratio of death due to COVID-19 and the 28-day absolute risk reduction incorrectly in almost all cases. The magnitude of the estimation bias increased when the process of recovery was faster and/or the chance of recovery was higher. In some cases, the estimates obtained from the Cox model also incorrectly showed a harmful effect of treatment when it was in fact beneficial. The simulation study therefore shows that there is a substantial risk of misleading results in COVID-19 research if recovery and death due to COVID-19 are not considered as competing events, and the assumption of non-informative censoring is violated. This issue, and others related to intercurrent events, is best addressed using the estimand framework, which has now become a regulatory requirement for trials aimed at new drug registration . Another well-known issue with the Cox model is the presence of strongly non proportional hazards. Much literature has recently focused on alternatives to the Cox model, especially for situations where deviations from proportionality are expected or have been observed, e.g., in trials of immunotherapy for cancer patients. Accelerated failure time models and the restricted mean survival time have been advocated in such cases, as have approaches based on generalized pairwise comparisons such as the win ratio and the net benefit . Further experience is needed with these alternative approaches, which might advantageously be considered in COVID-19 trials.
The need for data sharing During the pandemic, one of the key needs was and remains the collection of personal and medical data at an individual and group level. This need provided impetus for contact tracing and opened possible avenues of research for understanding the spread of the virus throughout the population and specific subgroups. In the discussion regarding the use of existing medical data, the collection of new data and in particular the collection of contact tracing data, some policy makers argued that there was a conflict between the rights guaranteed by the European Union's General Data Protection Regulation (GDPR), and this need for data sharing. This paradoxical dichotomy potentially inhibits the use of valuable data for research purposes within a country, and jeopardises cross-border scientific cooperation in the case of different interpretations of the same regulation within EU-member states. Several authors have argued that there is ample room within the GDPR for a framework allowing for the scientific use of existing and newly collected data to support the international effort to curb the pandemic . These views are echoed by the European Data Protection Board , and confirmed by the Belgian Data Protection Authority. Specifically, one can invoke article 9(i) of GDPR if ‘… processing is necessary for reasons of public interest in the area of public health …’ and 9(j) if ‘… processing is necessary for archiving purposes in the public interest, scientific or historical research purposes or statistical purposes in accordance with Article 89(1)’. These provisions, together with the European Clinical Trials Regulation and the corresponding Belgian law, provide a solid base for the scientific use and data sharing of medical and personal data . As argued by other authors the COVID-19 pandemic is not a free pass to use these data without any safeguards. The pandemic actually has been an opportunity to show that the principle underlying GDPR can actually be an advantage for data-driven research. The confrontation with a new situation, may also require reflection time, i.e., for a debate, but this time was not available, and hence public interest should prevail, within limits defined by the ethical committees. The availability of granular data from differing sources like individual medical files, or data held by mutual health organizations would provide unique opportunities to support health policy making and develop successful strategies for the current and future pandemics. The adoption of standards for data citation and referencing would also promote data sharing in an international, interdisciplinary, and interdependent research community. Guidelines have been developed by DataCite (https: https://datacite.org/cite-your-data.html ) and DataVerse ( http://best-practices.dataverse.org/data-citation/ ). As far as clinical research is concerned, there has been a remarkable push towards sharing of individual patient data for a number of years, both from publicly-funded trials but also from the pharmaceutical industry . The goal is to share individual patient data from all completed trials within reasonable time after their completion so as to allow for further analyses of these patient data, as well as to help the design of other trials. Such maximization of the use of patient data is certainly in line with greater patient involvement in clinical research, and would pave the way to truly patient-centric research. For the sharing to be maximally useful, the data should be made available as early as possible (without infringing intellectual property rights or publication in full by the trial principal investigators). The COVID-19 pandemic has also made it clear that data should be shared even earlier, albeit confidentially, among the Independent Data Monitoring Committees of trials investigating similar treatments in order to inform decisions about amending or stopping ongoing trials after careful review of all relevant data .
Reflections, concluding remarks, and outlook In this section, we suggest some specific lessons learned for both the modeling and prediction as well as for clinical research. First and foremost, there is a huge need for international collaboration through formal and informal scientific networks during pandemics. While there are local, country specific aspects to the epidemic (culture, population density, demography, health care system), there is commonality from an infectious diseases perspective. The statistical and methodological teams in academia, industry, and government need to connect with each other, nationally and internationally. Steady research capacity is needed, that can quickly scale up in pandemic times in order to respond to pandemics efficiently. For example, statisticians working in other areas (exact sciences, economy, humanities) can be converted quickly to COVID-19 response work provided they are sufficiently broadly trained, and there are pre-existing communication lines (e.g., university wide statistics research centers, learned societies, etc.). More than ever statisticians, modelers, and epidemiologists must be able to communicate and collaborate with research teams from other key fields, such as virologists, health economists, but also economists, social and behavioral scientists, etc. Effective communication lines need to be established between statisticians and other scientific experts, policy makers and international, national, and local policy makers, the public opinion, and the press. A number of statisticians must have received media training and ideally have built up experience in clearly communicating potentially complex statistical matters. An exceptional pandemic situation makes it clear that out-of-the-box thinking is needed. Inevitably, inaccurate or incorrect judgements will be made at some level during the pandemic. It must be acknowledged, and accepted, that knowledge is being built while the response to the crisis is being rolled out. Mutual trust between the parties involved and honest communication towards the public opinion is essential. In this sense, it is fine and even healthy that researchers not automatically agree with one another. Critical reflection and peer review, formally and informally, externally and internally within research groups, is of crucial importance to avoid serious mistakes. A gradually, orderly, and naturally built consensus, can help avoid misguided policies. When the process works, public opinion is ready to accept NPIs, for example, before they are formally announced. A key problem with COVID-19 is the pressure that it can induce on the health care system. It is therefore important to have sufficient reserve capacity (in terms of hospital, staff, supplies). This is difficult because of the cost involved. Statisticians can contribute to planning, health economic evaluation, and, during pandemic times, by monitoring and forecasting hospital load and other capacity. 12.1 12.2 Clinical research The COVID-19 crisis has provided an exceptional opportunity to question the way in which clinical research is conducted, not just for the treatment of COVID-19 patients but also for all other diseases. One of the priorities today should be to streamline clinical research in diseases with high morbidity and mortality (cancer, cardiovascular disease, etc.) This could entail drastic simplifications of trial set-up (protocol review committees, ethical approval, regulatory submissions, access to drugs from competing drug companies for comparative effectiveness trials, etc.) as well as trial conduct (pragmatic trials comparing standards of care using ultra-simple protocols, real-time electronic data capture, central statistical monitoring, common resources for Independent Data Monitoring Committees, etc.) These ideas are by no means new (see, e.g., https://moretrials.net/ ) but with the lessons learned during the COVID-19 pandemic, they may get more traction than ever before. The need for a strengthened international collaboration in epidemiology should be accompanied by a corresponding international preparedness for clinical research, in order to quickly deploy large simple trials simultaneously in as many countries as possible. If the urgency to carry out clinical trials of treatments against COVID-19 could now be expanded to all other diseases, it would be a revolution in using statistical methodology to improve global health.
Modeling, prediction, prevention To avoid methodological errors, even when research is done at very high speed, and to ensure that models built and data analyses undertaken are as stable, broadly valid, and unbiased as possible, it is imperative to share data at the finest granular level possible, including individual patient data in clinical and epidemiological studies, and spatial data used to monitor the epidemic, to deter or alleviate post-wave outbreaks, etc. As is well-known throughout statistics, a well-fitting model (curve) does not automatically imply good prediction qualities. In meteorology, various weather models are juxtaposed to come to a calibrated weather forecast. Good models imply a subtle interplay between epidemiological theory, sophisticated modeling, and the use of real-world data: data about infections, hospitalization, and mortality on the one hand, and non-pharmaceutical interventions taken as well as their gradual relaxation on the other. In a pandemic epoch, a large number of national, regional, and city-wide epidemics can be compared. To date, excellent international resources are available, such as from the European Centre for Disease Prevention and Control (; https://www.ecdc.europa.eu/en/covid-19-pandemic ), Johns Hopkins University ( https://coronavirus.jhu.edu/map.html ), and Our World in Data ( https://ourworldindata.org/coronavirus ). These offer valuable resources on how the epidemic is playing out elsewhere. Especially in contiguous and highly connected areas, such as in the United States and the European Union (especially the Schengen Zone), the epidemic's evolution cannot be seen in isolation, except at the rare times where state or international borders are virtually closed.
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Plant electrophysiology with conformable organic electronics: Deciphering the propagation of Venus flytrap action potentials | 2c09d511-a605-45fd-8f60-8df0d686a22e | 10371018 | Physiology[mh] | Electrical signals in plants are nowadays well-recognized information carriers with direct functional relevance. These signals are often correlated to thigmonastic movements and responses to stress, including touch, (herbivore-induced) wounding , and root nematode attack . Specifically, severe wounds induce slow wave potentials, which are varying amplitude depolarizations that usually last for few minutes . In carnivorous plants, stimulation of mechanosensitive cells (e.g., sensory hairs) induces fast electrical transients called action potentials (APs) that are usually correlated with rapid movements. A key example of plant AP occurs within the carnivorous plant Venus flytrap (VFT; Dionaea muscipula ), and it is considered a model system for fast electrical signaling in plants . Despite the importance of these signals, our understanding of their properties, underlying mechanisms, and precise relationship with plant function remains incomplete. A major barrier to addressing these issues is that plant electrophysiology has thus far mainly relied on cumbersome intracellular recordings or noninvasive surface recordings with bulky inorganic (Ag/AgCl) electrodes, neither of which enable high-resolution mapping of the signal . The signal characteristics of the VFT have been mainly studied with noninvasive surface Ag/AgCl electrodes. AP in VFT is generated after the stimulation of one of the six sensory hairs present in the inner side of the trap . The AP has a diphasic shape, with a fast depolarization phase followed by a more variable hyperpolarization , a signal amplitude between 10 and 150 mV, and a signal duration of 1 to 3 s . Several studies have attempted to estimate the propagation speed of the AP in VFT with the use of electrode pairs, and, so far, a wide range of values have been reported in the literature ranging between 2 and 80 cm s −1 , suggesting a lack of consensus approach to such measurements. If any two of the hairs of the VFT are stimulated within an interval of 30 s, then the trap closes, showcasing a highly regulated biological phenomenon, designed to optimize yield of nutrition for the plant. However, the causal mechanisms triggering trap closure are unclear. A long-lasting hypothesis was that the AP induces a transient increase in calcium ions (Ca 2+ ) concentration in the trap and when the Ca 2+ concentration reaches a putative threshold the trap closure is triggered. Recently, a major milestone has been achieved with the generation of VFT transgenic plants that express the fluorescent Ca 2+ indicator GCaMP6f, enabling visualization of the Ca 2+ dynamics in intact traps . The hypothesis was confirmed since the mechanical stimulation of the sensitive hairs led to an increase in cytosolic Ca 2+ at the base of the hair. The Ca 2+ transient then spread throughout the trap with a time constant of 33 s , and, when a second stimulation was applied within 30 s, a second higher Ca 2+ transient was generated and the trap closed. The cytosolic Ca 2+ concentration level is probably the main “memory” component of the trap, while its proper decoding seems to be necessary to elicit closure . While the transgenic VFT enables the visualization of the Ca 2+ wave propagation, the AP is an integrated signal involving the activity of more than one type of ion channel with different permeabilities for different ions such as Ca 2+ , K + , Cl − , and H + , each being a component of the AP . However, up to now, the different ionic components of the AP have not been fully decoupled. Furthermore, how the AP propagates along the trap and how its propagation correlates with Ca 2+ wave propagation or other ions and mobile signaling molecules or with the trap closure per se remain unknown. Advancements in bioelectronics have resulted in the development of noninvasive electrophysiological recording devices for mammalian systems, enabling unprecedented spatiotemporal resolution and critical insight into neural circuit mechanisms . Systems based on conformable, low-impedance multielectrode arrays (MEAs) permitted recording and classification of individual neural APs from the surface of human and rodent brains . To the best of our knowledge, there are only three studies that used MEA technology interfaced with plant tissues: root slices and leaves of intact plants . In a recent study, a small-scale rigid MEA was used in VFT; however, the study mainly focused on the calculation of conduction velocity . Other recent examples in literature also demonstrate AP recordings with conformable tattoo-like single electrodes based on organic electronic materials or nanomaterials and with single organic electrochemical transistors that enable signal amplification . However, these studies did not provide any new insight on the AP but focused on the device development. In this work, we developed a conformable MEA based on organic electronics for large-scale and high-resolution plant electrophysiology. With these devices, we performed precise spatiotemporal mapping of the VFT AP and demonstrated that it is actively propagating through the tissue in nonclosure- and closure-inducing events. We correlate the electrical signals to trap movement and resolve the origin of spontaneously generated APs. We also found that cells other than the sensory hairs could generate AP that can also lead to trap closure. Last, with pharmacological treatments, we investigate the role of ions on the AP propagation. Our work establishes the capacity of organic bioelectronic devices for high-resolution and large-scale plant electrophysiology that can contribute to the mechanistic understanding of electrical signaling in plants. We developed a NeuroGrid probe with 20 mm by 25 mm spatial coverage using 120 uniformly distributed conducting polymer-based electrodes (500 μm by 500 μm; ). The conformability of the NeuroGrid is an essential feature for large-scale electrophysiology recordings as it allows the MEA to follow the trap curvature, both in open and closed states, without displacement . To record the AP propagation in the VFT trap, we simply laminated the NeuroGrid to the outer surface of the lobe, to avoid triggering of the mechanosensitive hairs, with a small amount of electrolyte between the recording electrodes and plant tissue . Stimulating of one hair resulted in AP generation and propagation that we could track across the lobe . A clear signal delay between the recordings of the different electrodes was observed, indicating that our setup has sufficient spatial and temporal resolution to resolve the signal propagation. The time delay was then quantified as the time difference between the signal of each electrode and the first signal recorded, allowing for the generation of the time delay AP propagation map . As the relative position of the sensitive hairs in relation to the electrodes can be visually estimated, this analysis enabled us to independently confirm that the AP originates at the vicinity of the stimulated hair. These conclusions were then corroborated by phase shift analysis. The phase of each signal was estimated by a Morlet wavelet centered on 1.14 Hz, representing the main frequency content of the AP signal (figs. S1 and S2). The phase shift of the waveform mapping matched the one of time delay, indicating that the waveform of the AP travels without losing its frequency content. . Since the AP originates at the hair, it is important to investigate whether the signal delay recorded by the other electrodes is not due to volume conduction (i.e., recording of a localized signal through an electrolytic medium) but a result of active propagation of the signal through the tissue. To test this, we applied an electrical stimulus on the trap using an external Ag/AgCl electrode and recorded the signal with the NeuroGrid . In this case, the stimulation artifact was recorded by all electrodes without notable time delay or phase shift, confirming that the electrical stimulation does not propagate but can be captured from all electrodes simultaneously due to sufficient ionic conductivity of the epidermal tissue . In contrast, the time delay of the AP propagation and the phase shift mapping suggest that the AP travels through compartmentalized pathways, however without dampening of the signal indicating an active propagation. To map the signal propagation across the whole lobe, we repeated the same experimental protocol with different grid placements with respect to the sensory hairs (H1, H3, and H6) . We found that the signal traveled with constant speed for all the hairs with similar average speed (table S1 and fig. S3). The propagation maps were then calculated with isochronal lines and show that the signal is generated at the hair location, and it radially spreads to the rest of the trap without strong directionality . As H6 is on the opposite lobe from the grid, the signal origin on the map corresponds to the area just above the midrib location closer to H6, clearly showing that the signal propagates to both lobes. A second stimulation of the trigger hair was then performed, with at least 1 min of interval to prevent trap closure . In all cases, the AP propagated more quickly after the second stimulation (preexcited state) compared to the first stimulation (nonexcited state), 11.22 ± 0.82 cm s −1 versus 41.14 ± 2.39 cm s −1 , respectively ( and table S1). Several studies have attempted to estimate the propagation speed of this signal, with most works suggesting a speed of 10 to 80 cm s −1 . Other studies present conflicting propagation speeds, from 2 cm s −1 to 10 m s −1 . However, previous studies relied on electrode pairs without the possibility to extract information about the speed dependence with time and the directionality of the signal. Here, we provide a better estimation for the propagation speed and show that the propagation speed is constant, with no strong directionality. Together with the propagation of the AP, a slower Ca 2+ transient spreads throughout the trap, with a speed of c. 2 cm s −1 , as it was visualized in VFT expressing the GCaMP6f genetically encoded Ca 2+ indicator. They found that the Ca 2+ wave travels faster (5 cm s −1 ) toward the midrib than in the other directions (2 cm s −1 ) and that, similarly to the AP propagation, the Ca 2+ wave speed following the second stimulation increases two- to threefold . In a following study, it was also suggested that the speed of the AP in nonexcited traps is the same as the one of the Ca 2+ transient . According to our results, the AP propagates much faster than the Ca 2+ transient and without strong directionality. Our results therefore suggest that the AP precedes the spread of the Ca 2+ transient. After establishing the propagation of the signal, we analyzed in more detail the AP waveforms, considering the signals recorded by the electrode closest to the stimulated hairs. We observed that the waveforms of H1 and H3 have very similar characteristics, while, in the one recorded from H6, a small hyperpolarization is present . This difference is likely related to the position of H6, in which it is on the opposite lobe of the recording electrodes. Furthermore, when comparing the first and second stimulations of the same trigger hair, there is an increase in amplitude in the latter that, although not statistically significant, had already been previously noted . In addition, we observe that the time interval between the mechanical stimulation of the hair and the onset of the AP decreases for the second stimulation . However, while the AP occurs faster, the depolarization time remains unchanged between the first and the second stimulations . Together, these data indicate that the second AP is facilitated by the first AP, leading to a faster electrical response while maintaining its signal characteristics. The relationship between electrical activity and trap movement was then explored by simultaneously recording electrical responses and acquiring video . In these experiments, a second stimulation was provided within 30 s of the first stimulation to trigger trap closure. Despite having significant natural deformation and movement of the lobe during trap closure, no motion-induced artifacts were observed (fig. S4). We found that the time difference between the AP onset and initiation of trap movement was 472 ± 24.7 ms (means ± SEM, n = 19) and that the closing movement lasted 1408 ± 157.1 ms (means ± SEM, n = 27), both are in line with previous reports . We also found that the trap movement onset is delayed by 12 ms/s of interval between the first and the second stimulations (fig. S5), which is likely related to the decay of the Ca 2+ transient that accompanies the AP propagation . The recorded signals during the second stimulation were diverse , having either simple (only one AP) or complex features where spontaneous AP (one or more) was generated after the mechanostimulated AP. The complexity of the recorded signal is significantly correlated with the interval between the first and the second stimulations, with complex responses being associated with larger intervals and simple responses being associated with smaller ones . Unexpectedly, we found that the spontaneous AP typically originated from an unstimulated hair ( n = 5; , and fig. S6). In c. 60% of the complex responses, we observed that the trap was slowly closing before the occurrence of spontaneous AP and that the propagation of this spontaneous AP subsequently accelerated trap closure . One speculation is that the spontaneous AP is generated to induce more Ca 2+ release to pass the putative threshold that will lead to trap closure. So far, we monitored the AP propagation originating from mechanosensitive hairs. Previous studies have shown that wounding the VFT lobe also generates an AP, although without elucidating the origin of these APs . To investigate the origin and properties of wound-induced AP, we established a wounding assay using a low-power laser focused on a specific location on the inner part of the lobe, far from the mechanosensitive hairs . The mechanostimulation of the hair induced an AP at the hair location , whereas wounding induced an AP originating at the wound site ( n = 4; and fig. S7). Furthermore, when stimulating twice with the laser, the trap closed and, on some occasions, a spontaneous AP arose, originating from a different location, probably a hair location ( n = 2; and fig. S8). These experiments indicate that the activation of the VFT trap circuitry is not restricted to the sensory hairs, but it is extended to other cells where excitations can also be integrated and lead to movement. To examine the roles of the specific ions involved in the AP generation and propagation, we used a pharmacological approach with ion channel blockers. In line with previously described protocols , we immobilized a trap and excised one lobe to apply the blockers at the wounded site. In this configuration, it is necessary to record the AP from the inside of the trap. Therefore, we first verified that recordings from the inside and the outside of a trap are equivalent . We then used a smaller, commercially available high-resolution MEA (FlexMEA) to map the AP from the inside of the trap. In this case, we also verified that the AP propagation speed is similar for recordings with FlexMEA and NeuroGrid in untreated traps . After trap excision, the immobilized lobe was incubated with different concentrations of the Ca 2+ channel blocker LaCl 3 or the K + channel blocker TEACl, and AP was recorded every hour using single Ag/AgCl electrodes. Notably, LaCl 3 is not specific to Ca 2+ -permeable channels, and it can affect other cation channels as well. Control traps were incubated for the same amount of time with deionized (DI) water. As shown in , incubation with LaCl 3 leads to a decrease in AP amplitude in a concentration-dependent manner, with no impact on the waveform FWHM (full width at half maximum). On the other hand, when traps were incubated with TEACl, the AP amplitude was maintained at control levels, but the FWHM increased markedly in a concentration-dependent manner. These modulations of the AP waveform are in line with previous work , while we additionally perform detailed characterization of the AP over time and in response to different ion channel blocker concentrations. Considering the time course of the tested ion channel blockers, an incubation of 3 hours with 10 mM LaCl 3 and that of 4 hours with 100 mM TEACl were chosen to map the signal propagation with the FlexMEA . When Ca 2+ fluxes were perturbed , there was a decrease in AP amplitude compared to the AP baseline, but no significant differences in propagation speed (compared to control traps) were found. In contrast, when K + fluxes were impaired , there was a significant increase in FWHM compared to the AP baseline, but no significant differences in propagation speed were found. There was a generalized decrease in propagation speed under all tested conditions compared to that measured in intact traps. This decrease likely arises from metabolic changes in the excised traps rather than effects of the ion channel blockers, as incubation with DI water induced a similar decrement in propagation speed. In summary, while the data recorded with the FlexMEA corroborates that acquired with the Ag/AgCl electrodes in terms of waveform distortion, we were unable to find any impact of the disruption of Ca 2+ and K + fluxes on the spatial propagation speed of the AP. In this work, we demonstrate the ability of conformable MEAs to elucidate properties of electrical signaling in plants. We mapped mechanostimulated AP propagation in the VFT and found that the signal travels with constant speed in both lobes and spreads radially from the hair with no strong directionality. The AP travels faster than the Ca 2+ transient and does not follow its anisotropy [as compared with data from Suda et al. ], suggesting that the AP precedes the Ca 2+ wave propagation. In preexcited traps, the AP has higher amplitude, travels faster, and occurs with less delay from the mechanostimulation event in comparison with nonexcited traps, indicating that the trap state affects the AP propagation. Furthermore, we found that in some closing events, spontaneous APs are generated that mostly originate from nonstimulated hairs. The likelihood of spontaneous AP generation is highly correlated with a longer interval between stimulations of the sensory hairs and, on some occasions, was associated with the onset of a faster trap movement during closure. The physiological role of the spontaneous AP is not clear; however, it might be generated to induce additional Ca 2+ release that will facilitate complete closure of the trap. What defines the speed of the AP and which tissues are involved in the AP propagation are still major questions. We demonstrate that the AP actively propagates throughout the trap, and it is not just a localized signal that can be probed from various distances through an electrolytic medium. We show that AP propagation is not directional, and, therefore, it cannot be mediated via tissues that are not homogenously distributed across the trap. The hypothesis that AP propagation occurs via the phloem vasculature is not supported by our work because, in this case, AP propagation speed should be increased in directions aligned with phloem anatomy. In addition, pharmacological disruption of Ca 2+ and K + fluxes lead to waveform distortion but did not affect the propagation speed of the AP. Wounding assays revealed that other tissues apart from the mechanosensitive hairs are excitable. These wounded locations could generate APs that were sufficient to subsequently induce trap closure. The circuitry of the VFT trap can be therefore activated not only through the stimulation of the sensory hairs but also via disruption of other cells. In the transgenic VFT expressing the GCaMP6f Ca 2+ indicator, it was demonstrated that cytosolic Ca 2+ increases at the wound site that then propagates across the lobe extending the correlation between AP generation and Ca 2+ release in cells others than the sensory ones. However, what is not clear is whether the Ca 2+ levels of the lobe are associated with the AP propagation? A second stimulation of a sensory hair will lead to trap closure only when the concentration of cytosolic Ca 2+ before the stimulation is high enough. We observed that the propagation speed of the AP during the second stimulus is faster independent whether it will lead to trap closure, signifying that it does not depend on the cytosolic Ca 2+ concentration of the trap. This is corroborated by the results of the pharmacological treatment where blocking plasma membrane Ca 2+ -permeable channels had no impact on the AP propagation speed. Nevertheless, in the latter case, we cannot exclude the contribution of internal stores in the increase of cytosolic Ca 2+ concentration. In addition, any combination of hair stimulations induces faster AP propagation during the second stimulation, indicating that the excitability information must be encoded across the entire trap, rather than coupled with the stimulated hair alone. The nature of this information encoding remains unclear. Recently, in Arabidopsis, it has been shown that upon wounding glucohydrolase, enzymes generate mobile signaling molecules (e.g., Ricca’s factors) and that, together with hydraulic pressure changes, they are involved in the electrical signal and Ca 2+ wave propagation . We can only speculate that, in the VFT, similar mechanisms might be triggered upon AP generation and therefore be involved in the excitability of the trap. The existence of mobile elicitors was suggested by Ricca in studies performed in the sensitive plant Mimosa . Our work leverages the conformability, biocompatibility, and high spatiotemporal resolution of organic electronics to shed light onto the mechanistic underpinnings of electrical signaling in plants. We resolve key properties of AP propagation across the VFT lobes, pinpoint the origin of spontaneous AP, and demonstrate that the VFT circuitry can be activated by cells other than the sensory hairs. This work also establishes the foundation for translating methods validated in mammalian electrophysiology to plant electrophysiology. The ability to spatially resolve electrophysiological signals in plants in a quantitative manner opens a range of new research possibilities. We have validated the technology for fast electrical signaling, and similar approaches can be promptly applied to resolve slower signals. Currently, there are no commercially available solutions for high-resolution or large-scale electrophysiology recording for plants. Although, in this study, we also performed recordings with a smaller commercial MEA that enabled us to record from the inside of the trap, it became apparent that this device was limited for plant recordings due to features optimized for interfacing with the rodent brain. These experiments further highlighted the need for developing customized electrophysiology platforms for plant science. High-resolution electrophysiology can be combined with genetics, and we foresee that it will enable precise quantification of even subtle changes in mutants with minor phenotypic defects, thus representing a fundamental contribution to the mechanistic understanding of long-distance signaling in plants. Plant materialNeuroGrid fabricationElectrophysiology recordingVideo acquisitionWound assayPharmacological treatmentData analysis Raw traces obtained from electrophysiology recordings were smoothed with a Savitzky-Golay filter (200 ms window), and their baseline was brought to zero. The timing of the AP at each electrode was determined on the basis of the point of maximum deflection of the signal (local maximum of the derivative). The relative time delay was then calculated as the time difference between each electrode and the first timestamp. Time maps corresponding to the same condition (triggered hair and stimulation number combination) were then spatially aligned by the electrode of origin and averaged, generating the average propagation maps. For these maps, data between electrodes were generated by a cubic interpolation method. Propagation speed was calculated as the slope of a linear fit of the data of all recording electrodes. Regarding phase shift analysis of the AP propagation, phase angle time series of the APs were created using a complex Morlet wavelet with a center frequency of 1.14 Hz, which represents the main frequency content of the AP signal. Following the generation of the phase angle time series, the phase angle difference relative to the origin electrode (first timestamp) was calculated at the FWHM time point. For statistical analysis, at least three independent experiments were performed in different plants. Data were analyzed with custom MATLAB scripts and GraphPad Prism 9. Movement data were extracted from the videos using the software Tracker. VFT ( D. muscipula ) plants were purchased from Plantagen (Sweden), repotted in sphagnum moss:perlite (2:1) soil mixture, and kept under greenhouse conditions (22°/18°C light/dark, 12 hour photoperiod, and 60% relative humidity). Plants were watered with DI water. One hundred–millimeter 550 μm ± 10 μm P-doped wafers were coated with 2 μm of parylene-C using an SCS Labcoater 2 to form the base layer of the NeuroGrid. Metallization was patterned using a liftoff approach. AZ nLOF 2020 photoresist was spin-coated at 3000 rpm with soft bake and postexposure baked at 110°C for 90 s each. Ultraviolet exposure was performed using a Suss MA6 DUV Mask Aligner, and samples were developed in a bath of AZ 300 MIF. An adhesion layer of 10 nm titanium was evaporated, followed by an evaporated 150 nm gold layer using a ultrahigh vacuum angstrom electron beam evaporator. Liftoff was performed using a bath of remover PG. Subsequently, a second layer of parylene-C (insulation layer), followed by an additional sacrificial layer of parylene-C (for the subsequent peel-off process), was deposited. The adhesion between the first and the second layers of parylene-C was enhanced by Silane A-174 (chemical vapor deposited), while an antiadhesion agent (5 wt % of Micro-90 diluted in DI water) reduced the adhesion between second and third layers. The stacked layers were patterned with a layer of AZ9260-positive photoresist and dry-etched with a plasma reactive ion etching (RIE) process [Oxford Plasmalab 80+; 180 W, 50 sccm (standard cubic centimeters per minute) O2, and 2 sccm SF6] to shape the electrodes and contacts. Specifically, AZ9260 was spin-coated at 5000 rpm, baked at 115°C for 90 s, exposed using a Suss MA6 Mask Aligner, and developed with AZ400K developer (1:4 with DI water). An extra layer of AZnLOF2020 (3000 rpm; the guard photoresist) was added between the metal layer and the later parylene layers to protect Au contacts from direct ion bombardment during RIE process.The contact area of the electrodes were realized by spin-coating and baking poly(3,4-ethylenedioxythiophene):polystyrene sulfonate (PEDOT:PSS) (600 rpm) and patterned by peeling off the sacrificial parylene layer. After patterning, the wafer is cleaned thoroughly with acetone and isopropanol to remove guard photoresist and released in a water bath. A multilayer polychlorinated biphenyl (PCB) was designed using Autodesk Eagle software to interface 32 NeuroGrid electrodes with the ME2100-System (Multichannel Systems, Germany). The NeuroGrid was attached to the 350 μm PCB connections using a mixed conducting particulate composite material anisotropic film. Last, the PCB was equipped with a 36 pin connector (1.27 mm pitch, Preci-dip, Switzerland) to be compatible with the used headstage (ME2100-HS32-M, Multichannel Systems). VFT plants were placed inside a Faraday cage and allowed to acclimate for at least 10 min before the experiments. For NeuroGrid recordings, the NeuroGrid was simply laminated to the outer part of one of the lobes, using small amount of phosphate-buffered saline electrolyte. A Ag/AgCl electrode in the soil was used as the reference electrode. Baseline recordings (no activity) were captured for at least 20 s before stimulation. Different trigger hairs were then mechanically stimulated using a thin metal wire. For nonclosing experiments, each stimulation was performed with at least 1 min interval. For closing experiments, two stimulations were performed within 30 s. For experiments with ionic blockers, either a Ag/AgCl or a FlexMEA72 (Multichannel Systems, Germany) was placed on the inner side of the immobilized trap, being interfaced with Signa gel or in-house fabricated PSSNa hydrogel (4:1:1 mix of PSSNa, d-sorbitol, and glycerol) , respectively. All experiments were acquired using a ME2100-System (Multichannel Systems, Germany) in DC-coupled mode, with sampling rates from 10 to 50 kHz. Video recordings of trap closure were acquired with an area scan camera (acA1280-60gc, Basler AG, Ahrensburg, Germany) with a 6.0 mm lens (FL-CC0614A-2 M - F1.4/6 mm, Ricoh, Tokyo, Japan) at 30 frames/s. Frame acquisition was controlled by a signal generator (InfiniiVision 3000A X-Series, Keysight Technologies, CA, USA), sending a pulse wave of 5 V and a frequency of 30 Hz. This signal was also used to synchronize the video with the electrophysiology setup. For wound-induced APs, a millisecond-pulsed laser (OFL365-5-TTL3, OdicForce) beam was directed toward the inner side of the trap in a location away from the hairs. The laser beam (wavelength, 450 nm) was operated at a 0.5 W output power using Pulse Width Modulation (PWM), with a pulse width of 1 ms. For optimal wounding, the laser beam of smallest spot size was applied at the region of interest, by properly adjusting the focus. The position of the laser module was fixed with a rigid clamp. The ON duration of the laser beam application was set at 500 ms and controlled by a push button. The laser ON/OFF power switch and the PWM-modulated laser power controller was custom-designed using an Arduino microcontroller and basic electronic components. The wound size of 1 to 2 mm diameter disk was realized by fixing the height of the laser to 10 cm from the target spot. Thel NeuroGrid was placed on the outer part of the trap to record the wound generated APs. Traps were immobilized with double-sided tape on a glass carrier, and the free lobe was excised by the midrib. The exposed lobes were then incubated with different ion channel blocker solutions. For the impairment of Ca 2+ signaling, plants were incubated with different concentrations of LaCl 3 in DI water (10, 50, and 100 mM). For the impairment of K + signaling, plants were incubated with different concentrations of TEACl in DI water (10, 50, and 100 mM). Control plants were incubated for the same time with DI water. For the determination of the time-dependent action of the ion channel blockers, APs were recorded every hour with a Ag/AgCl electrode. For the high-resolution mapping, traps were exposed to 10 mM LaCl 3 for 3 hours or 10 mM TEACl for 4 hours; at which points, APs were recorded with the FlexMEA72 purchased from Multichannel Systems. |
A Highly Sensitive Method to Detect Avocado Sunblotch Viroid for the Maintenance of Infection-Free Avocado Germplasm Collections | 117911e9-0d2e-4fdb-92be-1d78deb775a5 | 6630647 | Pathology[mh] | Avocado sunblotch viroid (ASBVd) is a single-stranded circular RNA molecule of 247 nucleotides (reference sequence variant GenBank J02020.1) that is confined in nature to avocado ( Persea americana ) . Symptomatic and asymptomatic strains are recognized, although even the apparently asymptomatic strains are associated with yield loss . The most characteristic symptom of sunblotch disease is sunken scars on the fruit surface, which may be pink or yellow depending on the color of the skin at maturity. ASBVd is transmissible through seed and mechanical damage, but has no insect vectors. Precautions need to be taken during propagation to prevent infection as rates of seed transmission are high at 86%–100%, and ASBVd can also be introduced through the use of infected budwood . Rates of spread in the field are low and based on patterns of infection, it is suspected that ASBVd spreads in the field by root grafting. Pruning could potentially transmit the viroid but the efficiency of this mode of transmission is thought to be low . Pollen transmission does occur, but this only results in infection of the seed and not the mother plant . There are no known methods to cure trees of infection and even micrografting or somatic embryogenesis does not eliminate ASBVd . Trees must therefore be rogued to prevent infection of neighboring trees. The USDA-ARS Subtropical Horticultural Research Station (SHRS) in Miami, Miami-Dade County, Florida is a subtropical germplasm repository. As part of the National Germplasm Repository, SHRS distributes germplasm material through the GRINGlobal ( www.grin-global.org ). The SHRS repository is a living collection of subtropical and tropical species that have recalcitrant seeds that cannot be stored. Included in the collection are at least 270 unique accessions of avocado. Detection of ASBVd in the SHRS avocado collection ranged from 18%–21% of the trees sampled . Avocado is the second most important fruit tree crop in Florida after citrus, with the majority produced in Miami-Dade County. Laurel wilt, a lethal fungal disease caused by Raffaelea lauricola and vectored by the red bay ambrosia beetle ( Xyleborus glabratus ), has been a problem in orchards in Miami-Dade since 2011 , but has not yet been observed at the SHRS. Due to the serious threat posed by this disease, a backup collection of the SHRS avocado germplasm is needed at an alternate site that is free of laurel wilt. However, ASBVd-infected accessions cannot be moved in order to prevent the spread of the viroid pathogen. In addition, for some SHRS germplasm trees, results of a reverse transcriptase PCR (RT-PCR) assay were not consistent over years, suggesting that a more sensitive assay was needed to provide certainty that a tree was not infected prior to distribution to or inclusion in the backup collection. The most sensitive assays for ASBVd are based on RT-PCR. Previously, the detection of the amplified fragment was by polyacrylamide gel electrophoresis or capillary electrophoresis . Recently, a qPCR assay using real-time PCR of the reverse-transcribed viroid RNA has been developed using SYBR-Green. . The SYBR-Green assay is reported to be ~100x more sensitive than previous RT-PCR methods, but is, therefore, also more sensitive to cross contamination during sampling. We describe here a method using nested primers for specific pre-amplification of the ASBVd viroid, specific detection of the viroid using a TaqMan gene expression assay, and quantitation by real-time PCR. This assay is sensitive enough to accurately detect a 1:10 7 dilution of viroid RNA from a single infected tree and allows for confidence in detecting a single infected tree in pools of samples of eight trees. Using this assay, we identified and removed all infected trees from SHRS, which has allowed us to confidently create a backup collection and distribute viroid-free material.
2.1. Avocado Trees 2.2. Viroid Indexing 2.3. RNA Extraction 2.4. Primers and Probes 2.5. Reverse Transcription and Pre-amplification 2.6. TaqMan Real-Time PCR Assays 2.7. Roguing Infected Trees 2.8. Creation of a Backup Germplasm Collection Accession details for avocado trees used in experiments are described in . A full list of cultivar names is provided in .
2.2.1. Sampling 2.2.2. Tissue Disruption Samples were processed within 48 h after collection by either manually dicing interveinal leaf tissue into 1 mm wide squares (~100 mg) using disposable blades and cleaning surfaces with 20% bleach between samples or by taking a leaf disc using a PlantTrak Lx Benchtop Plant Sampling & Barcoding by Brooks Life Sciences with a 3 mm punch head. The punch head was sterilized between samples by punching a Whatman PlantSaver FTA Card (WB120065, Whatman, GE Life Sciences, Pittsburgh, PA, USA) soaked in 20% bleach ten times and wiping the punch head and PlantTrak surface with a disposable paper towel with 20% bleach. The leaf tissue and a one 1/52’’ metal grinding bead were then added to individual wells of a 96 deep well plate in an arrangement where no samples were placed next to each other to avoid cross contamination. Samples were stored at −80 °C if RNA extractions were not done immediately.
For routine viroid indexing, one leaf from each of six positions on the tree (north, south, east, west, top north and top south) was collected and placed into a pre-labeled bag. Leaves from eight trees were combined in a single bag when pooling samples. To avoid cross-contamination of the viroid, tasks were divided, with one person collecting the samples and a second responsible for record-keeping and opening the bag to receive the leaves. The person sampling the leaves had gloved hands that were wiped with a 20% solution of commercial bleach after each tree and cutting tools were decontaminated in the same way. Leaves that were too old, damaged, or too young and thin were avoided. The decontamination process was repeated between each leaf sample when examining the distribution of the viroid within a tree at six positions (north, south, east, west, top north, and top south). A detailed leaf collection protocol is available in . One negative control sample, from a tree that had never tested positive over years of testing , was collected every 17 samples and was included on each extraction plate. Samples were stored in an ice cooler or at 4 °C until extraction.
RNA was extracted using a modified method of Ainsworth . Five hundred μL of extraction buffer with 0.16% ( w / v ) dithiothreitol was added to each 1.2 mL well, which was capped and wrapped in parafilm to avoid leakage. Each 96 well plate was ground in a Genogrinder 2000 (SPEX SamplePrep, Metuchen, NJ, USA) for 2 min at 2000 strokes per minute, centrifuged at 1000× g for 30 s, and the process repeated at least one more time until no large leaf pieces were visible. Plates were then centrifuged at 6100× g for 10 min at room temperature and the supernatant transferred to a new 1.5 mL tube, removing caps one sample at a time and washing gloves with bleach between samples. After adding a 1/3 volume of 8 M lithium chloride (LiCl), the samples were vortexed and left to incubate in an ice water bath placed in a 4 °C refrigerator overnight. After centrifugation at 10,000× g for 10 min at 4 °C, the supernatant was removed and pellets were resuspended in 100 µL of 2 M LiCl by flicking and pulse vortexing. The centrifugation and resuspension steps were repeated twice more, but after the final centrifugation, the pellets were resuspended in 50 µL of deionized, diethyl pyrocarbonate (DEPC)-treated water and the samples sat at room temperature for 10 min. One-tenth of a volume of 3 M sodium acetate and 2.5 volumes of 100% ethanol were added and samples were left overnight −20 °C. Samples were then centrifuged at 15,000× g for 10 min at 4 °C and the supernatant was removed. Pellets were rinsed with 100 µL cold 70% ethanol, mixed by flicking, and the centrifuge and 70% ethanol extraction were repeated. Samples were then dried for 5 min in a vacuum desiccator. Pellets were resuspended in 20 µL DEPC water. Samples were vortexed and allowed to incubate for at least 10 min at room temperature before quantification with a NanoDrop 2000 spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA). Samples were normalized to 100 ng/μL prior to analysis by RT-PCR.
The primers for the control target, glyceraldehyde phosphate dehydrogenase (GAPDH), were designed across exon junctions to only amplify cDNA . Primer and probe information for all other targets is provided in .
GAPDH was used as the positive internal control for the RNA extraction, reverse transcription (RT), pre-amplification, and TaqMan assay. Reverse transcription reactions were performed either as separate ASBVd and GAPDH reactions, resulting in an ASBVd cDNA sample and a GAPDH cDNA sample for each RNA extraction, or with the ASBVd and GAPDH primers multiplexed in one reaction. Reverse transcription reactions were performed on the avocado leaf samples using the High-Capacity cDNA Archive Kit from Applied Biosystems (ThermoFisher Scientific, 4368813) with modifications. Reactions were done in 20 µL volume with 10 µL master mix and 10 µL RNA sample at 100 ng/µL for a total of 1 µg RNA. Forward and reverse gene-specific primers (SB1-F1 and SB1-R1 for ASBVd, and GAPTM-F1 and GAPTM-R1new for GAPDH, see ) at a final concentration of 0.5 µM were used instead of random primers, one set each for separate RT reactions and both sets for the RT multiplex. A no template control (NTC) was included for each primer pair. Reactions were run at 25 °C for 10 min, 37 °C for 120 min, 85 °C for 5 min, and held at 4 °C. Pre-amplification reactions consisted of 20 µL reactions including 1× buffer (M0270L, New England Biolabs, Ipswich, MA, USA), 0.2 µM dNTPs, 0.3 µM each forward and reverse primers (same gene specific primers as reverse transcription reactions), 0.8 units of Taq polymerase, and 2 µL of reverse transcription product. One NTC was included with each pre-amplification reaction plate. Reactions were run at 94 °C for 2 min, 10 cycles of 94 °C for 30 s, 60 °C for one minute, and 72 °C for one minute, then 49 °C for one minute, 72 °C for 5 min, and a hold at 4 °C.
TaqMan assays were performed on Fluidigm BioMark (Fluidigm, San Francisco, CA, USA) platform on a 192.24 gene expression chip. The assay mix included final concentrations of 0.6× Fluidigm 2× assay loading reagent (Fluidigm 100-76116), 5.4 µM each forward and reverse primers, and 1.2 µM probe in a total of 5 µL. Twenty ASBVd and 4 GAPDH assays were run on each chip, providing 20 ASBVd and 4 GAPDH replicate reactions for each sample, including NTC for each chip. The sample mix included 0.12× TaqMan 2× gene expression master mix (ThermoFisher Scientific, 4369514), 0.12× Fluidigm 20× GE sample loading reagent (Fluidigm, 100-7610), 1 µL ASBVd pre-amplification product, and 0.35 µL GAPDH cDNA in a total of 3 µL. The gene expression chip was prepared following the Fluidigm manufacturer’s instructions. All NTCs from reverse transcription and pre-amplification reactions were also run on both TaqMan assay platforms to be able to determine that each step was free of contamination. A positive signal was determined if the cycle threshold (CT) values for all replications of the sample were lower than the NTC CT value. If all replications did not show a CT value lower than the NTC, or if the CT value was over 30, that sample was rerun. If rerun samples showed consistent values over 30, but lower than the NTC, the CT value of that sample was considered positive.
Trees testing positive for ASBVd were rogued. Infected trees were either knocked over or dug up depending on size. The resulting debris were collected and discarded offsite.
Budwood from viroid-free trees was couriered to the USDA-ARS Foreign Disease-Weed Science Research Unit in Ft. Detrick, MD (FDWSRU) and grafted to viroid-free rootstock. After at least one year’s growth in the greenhouse, two leaves from the grafted scions were retested for ASBVd infection. Budwood from viroid-free trees were shipped to the USDA-ARS Pacific Basin Agricultural Research Center in Hilo, HI (PBARC) for backup collection maintenance. Grafted trees at PBARC were also retested for ASBVd infection before planting.
3.1. Assay Design and Testing 3.2. Sensitivity Determination 3.3. Distribution of ASBVd in A Single Tree 3.4. Pooling Samples and Multiplex of ASBVd and GAPDH 3.5. Application Leaves from grafted trees of previously negative SHRS avocado trees in FDWSRU were re-tested with the current assay. If any of the trees in FDWSRU tested positive, the source tree at SHRS was tested with the TaqMan assay . These trees at the SHRS were also used to test leaves from different locations on the tree . All grafted tree samples at FDWSRU were re-tested with the TaqMan assay before being sent to PBARC. Budwood sent from FDWSRU was grafted at PBARC Hawaii and all trees were tested with the TaqMan ASBVd assay before they were planted in the field. ASBVd positive grafts were detected at FDWSRU from ASBVd negative genotypes at SHRS. All avocado trees in the germplasm collection at the SHRS were tested with the TaqMan ASBVd assay between 2014 and 2016. Trees were first sampled in pools of 8, then all trees in pools that showed positive results were tested separately. If a tree was consistently positive over two or more previous testing years, or if a tree showed phenotypic signs of ASBVd infection, they were considered positive and were not tested again. Out of 435 clonal avocado trees, 113 tested positive (26%, ). A random sample of 50 trees, pooled in groups of eight, was tested from the Florida avocado mapping population. All these trees were negative. The origin of the ASBVd infection was in “Avocado Circle”, a collection of cultivars that showed signs of ASBVd infection since 1992 . Almost all trees located in this circle were infected. The majority of an older avocado collection located in close proximity to Avocado circle were also infected. Infected trees were removed as described in Materials and Methods.
Four sets of primers for detection of the ASBVd molecule have already been designed . Three of these amplify the entire ASBVd molecule, as shown in : the red (SB1-F1 and SB1-R1, Semancik) , the purple (ASB-F1 and ASB-R1, Schnell et al.) , and the green (AVFL1 and AVFL2, Randles et al.) . The blue primers (ASBTM-F1 and ASBTM-R1 amplify a section of the ASBVd molecule which was used to design the TaqMan probe. Initial analysis of the red, purple, and green primer sets revealed that the red primers amplified the positive control tree with the most consistent signal (average CT 16.01, standard deviation 0.6, average tm 76.28, standard deviation 0.1) and never amplified the no-template control. Further assay development used the red primers only. The objective of this study was to increase the sensitivity of the RT-PCR assay by pre-amplifying the entire ASBVd molecule with one set of primers and then assaying the amplified molecule with another set of primers that targeted only a section of the ASBVd molecule amplified by AVLF1 and ASBTM1. We tested detection of the smaller amplicon using a TaqMan assay for a probe complementary to a sequence in the smaller amplicon . The red primers were used for pre-amplification. As the reverse blue primer sequence ASBTM-R1 overlapped the ends of the red primers, the green primer AVFL1 was used with the forward blue (ASBTM-F1) and probe (ASBTM) for the TaqMan assay.
The sensitivity of this assay was tested using serial dilutions from a positive control sample. A tree that had always tested positive for ASBVd (“Aycock Red” No. 19, ) was used as the ASBVd positive control in the absence of a purified source of viroid. A tree that had always tested negative for ASBVd (“Collinred B”, ) was used as a negative control. The following serial dilutions were made from ASBVd cDNA of the entire molecule from the ASBVd positive control (PC-ASBVd cDNA) using water as the diluent: 1:10 3 , 1:10 4 , 1:10 5 , 1:10 6 , 1:10 7 , 1:10 8 , and 1:10 9 . The ASBVd negative control cDNA (N4-ASBVd cDNA) was cDNA from a reverse transcription reaction with ASBVd primers on the negative control tree. The ASBVd negative control cDNA was used for background in the pre-amplification reaction to simulate the normal assay conditions (undiluted cDNA) for each dilution. The pre-amplification product was not diluted. Twelve replicates of each dilution were run on a 192.24 GE Fluidigm chip on the BioMark, with 12 ASBVd assays and four GAPDH assays, providing 144 replicate reactions for each dilution. It was determined that an ASBVd positive signal could be detected reliably in the real-time TaqMan PCR assay on the Fluidigm BioMark at a dilution of 10 7 .
Great variation in viroid titer was observed in experiments to determine the distribution of the viroid in a tree . None of the trees tested for titer distribution showed visual symptoms of the viroid. Average CT values across all sampled trees ranged from 7.3 to no amplification, and from 9 to no amplification within a single tree (Honalindo 1).
Three separate RNA extractions were isolated from three separate pools consisting of 49 leaf discs from the negative control tree and one leaf disc from the positive control tree to investigate assay sensitivity for detecting lower levels of ASBVd contamination in pooled samples. A separate collection was made for each pool making biological replicates. In addition, 1:50 and 1:100 dilutions of samples from FDWSRU were assayed on the Fluidigm BioMark. The 49:1 negative to positive RNA showed clear positive results (CT under 10, ) and the majority of the FDWSRU sample dilutions showed a positive signal where CT values for all replicates were lower than 30. The pooled and diluted FDWSRU samples were also run with the ASBVd and GAPDH multiplexed in the reverse transcription. After determining that using the control (GAPDH) internally in the ASBVd RT reaction resulted in an ~26% decrease in sensitivity , two separate reverse transcription reactions were performed on the avocado leaf samples, and samples were then assayed with separate ASBVd and GAPDH cDNA. Furthermore, the fluorescence for the GAPDH when it was pre-amplified was often so high it would increase the gain or sensitivity of the Fludigm BioMark and mask the ASBVd signal. Therefore, samples were run on the TaqMan assay with ASBVd pre-amplification product and a separate GAPDH cDNA. The number of cycles in pre-amplification was also optimized to minimize background or over-fluorescence, especially in samples with high levels of ASBVd contamination.
The mission of the SHRS includes the maintenance, curation, and distribution of disease-free germplasm through the GRINGlobal system. The SHRS houses many collections of tropical/subtropical species, with avocado and mango being two of the more important collections. As seeds of these trees cannot be stored, nor would they represent the genetically identical cultivars in the collection, we maintain living collections in the field and distribute budwood of the accessions. Curating such collections is complicated by both abiotic (hurricanes) and biotic (disease) stresses. We have been aware, since 1992, that avocado trees in our collection are infected with ASBVd, which prompted our development of an RT-PCR assay to detect the viroid in symptomless trees . Nonetheless, some trees were found to be positive in one year’s assay and negative in another. When it became clear that ASBVd was spreading at the station , we realized the need for a more sensitive assay to confidently identify all infected trees. The issue of infected trees became even more pressing when laurel wilt was discovered in Miami-Dade County in 2011 . To preserve the germplasm collection, prophylactic treatment with fungicide was begun and a backup collection at PBARC was planned. Hawaii has an avocado production industry and no ASBVd has been detected in Hawaii. Thus, to create a backup germplasm collection in Hawaii to avoid infection with laurel wilt, a highly sensitive ASBVd assay was needed to confidently identify viroid-free trees prior to shipping to Hawaii. Our assay is an improvement on previous RT-PCR and real-time RT-PCR assays as it utilizes a pre-amplification process for increased sensitivity and a probe detection of an ASBVd region with increased specificity. Comparison of our assay with other published assays is difficult as each research group has used a different standard ASBVd (plasmid DNA, transcribed plasmid DNA) to estimate sensitivity. Our goal was to develop an assay sensitive enough to guarantee no false negatives and based our sensitivity studies on known infected trees, known uninfected trees, and trees that had shown different results over different years. We also addressed the problem of cross-contamination of samples during collection, processing, and assay. Collection tools and leaf cutting tools are easily and permanently contaminated with viroid RNA that causes many false positives. In addition, as our method became more sensitive, the potential for cross-contamination simply from opening tube caps was also observed and led to the contamination prevention protocols described in Material and Methods. False positives are of major concern in curating a germplasm collection, especially when the only prophylactic method is tree removal. Additionally, ASBVd is transmissible through pollen, and seed propagated ‘Lula’ rootstock from SHRS could have contributed to ASBVd positive results in grafted plants from ASBVd negative budwood. This indicates the need for caution to ensure disease-free rootstocks are used when establishing backup collections for avocado. SHRS has more than 2000 avocado trees including research populations. Attempting to assay so many trees on an annual or biannual basis is beyond our resources. We focused first on removing ASBVd infected trees from the germplasm collection. Then, we improved the efficiency of our method by determining that we could accurately identify viroid infection by sampling four leaves from the cardinal points and two leaves from the top of each tree in the field. Although differences were seen in viroid amount among the six leaves, the assay was sensitive enough to accurately determine infection from this six-leaf assay. We also demonstrated that viroid from a single leaf disk from an infected tree could be detected in the presence of a pool of 49 leaf disks from an uninfected tree. Since we harvested six leaves from each tree, this meant we could pool the leaves from eight trees for each assay. If the pooled assay was positive for viroid, we would then assay each tree in the pool individually. Finally, we are in the process of collecting a random sample of 48 trees (six pools of eight trees) per field at the station using the same method as above. Our initial random samples showed no viroid infection of any of the 1500 trees in our research populations. Assaying random samples from each field makes it possible to assay the entire population of avocado trees on the station each year. It is always difficult to destroy trees in a germplasm collection, especially if this leads to the loss of all representatives of a cultivar. However, having viroid infected trees on the station prevents us from distributing material and in creating a backup collection. Since all the infected trees have been removed, we have completed our backup collection at PBARC and are able to confidently distribute avocado budwood upon request. Any plot that contained an infected avocado tree will not be used to replant an avocado as persistence of viroid RNA in the soil is too great a risk.
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Biochar’s role in improving pakchoi quality and microbial community structure in rhizosphere soil | 31c7784a-a8cc-446a-af1a-205c6a350177 | 10956520 | Microbiology[mh] | Biochar is a highly aromatic and refractory solid substance produced by the pyrolysis and carbonization of plant biomass under complete or partial hypoxias . Due to its porous structure, high surface area, and strong adsorption capacity, it is commonly used as a soil amendment for sustainable agricultural purposes. Numerous scholars have confirmed that adding biochar can improve the physiological characteristics and growth of vegetables, and can increase yield and quality under adverse conditions. Research has shown that the application of biochar and wood vinegar alone or together can improve the yield and nutritional quality of blueberries . Research has displayed that biochar addition can significantly enhance tomato growth, yield, and quality under reduced nitrogen application . The use of biochar and chitosan in heavy metal contaminated soil has improved the yield and quality of cultivated eggplants . The combined use of biochar and arbuscular mycorrhizal fungi has a significant impact on the root morphology and chlorophyll content of Fenugreek . Biochar can improve the performance of the rhizome of ginger and increase the activity of soil enzymes, thereby improving soil nutrient supply . The effect of biochar on improving fruit quality is related to the improvement of acidic soil properties . The impact of biochar on vegetable quality in weakly alkaline soil deserves attention. Applying biochar can change soil’s physical and chemical properties and improve the growth and quality of crops. Soil improvement with biochar can enhance soil stability and fertility because biochar enhances soil properties and nutrient availability . Soil-applied biochar can mitigate nitrate leaching and significantly increase the available levels of nitrogen (N) in the soil . Additionally, the individual or co-application of biochar and wood vinegar increases soil nutrient availability ( e.g. , NH 4 + -N, NO 3 − -N, and Mg 2+ ) . found that the physical activation of biochar decreased the contents of NO3 − -N and P by approximately 55% and 90% (w/w), respectively. Different biochar samples, except for N-rich biochar, exhibited minor N release after successive batch extractions. Ammonium was the primary form of N released from biochar, followed by organic N. Nitrate was in the range of 2%–30% in the leachates, whereas organic N was up to 59%. The release of dissolved organic carbon, N, and P into the soil solution was significantly correlated with biochar volatile matter content and acid functional group density . Adding biochar improved nitrogen utilization by adsorption and release of nitrogen. However, the correlation between nitrate accumulation in vegetables by biochar and the balance of soil nutrients has become an important issue. Biochar as a soil amendment can stimulate microbial processes in organic farming, resulting in better vegetable production and improved soil properties for sustainable farming . Research depicts that the highly porous structure of biochar provides a habitat for microorganisms to settle, allowing them to grow better in the soil environment . Research has demonstrated that biochar with organic–inorganic fertilizer improves soil nutrients, enzymatic activities, and bacterial abundance . Firmicutes , Proteobacteria , Acidobacteria , Bacteroidetes , and Actinobacteria were the dominant phyla in all soil samples, and applying biochar affected the abundance of dominant bacterial groups . Evidence displays that biochar amendment shifts bacterial community structures by altering soil chemical properties . The short-term application of vinasse biochar can change the structure and diversity of the soil bacterial community and control the risk of soil nitrogen leaching by inhibiting ammonia oxidation and nitrification of soil to improve soil nitrogen availability . Biochar application level influences the structure and diversity of the soil microbiome and plant performance . However, there is a gap in the study of the effect of biochar on nitrate content in vegetables and the interactions between soil microbial communities. Biochar can bioremediate organic contaminants by harboring microbial populations, releasing contaminant-degrading enzymes and protecting beneficial microorganisms from the immediate toxicity of surrounding contaminants . The biological mechanism by which biochar improves the quality of pakchoi remains unclear. We aimed to test the following three hypotheses: (1) biochar can improve growth and quality of pakchoi; (2) biochar can enhance soil enzymatic activities and soil available nutrient content; (3) biochar can improve the quality of pakchoi by altering the abundance of soil dominant bacterial communities and altering the soil nutrient environment. The purpose of this study is to evaluate the improvement effect of different concentrations of biochar on the rhizosphere soil of pakchoi, and to analyze the correlation between biochar and soil microbial and quality improvement. To this end, physiological and biochemical methods were used to estimate soil physicochemical properties and tomato nutrient uptake, and Illumina MiSeq sequencing was used to analyze the abundance and composition of soil microbial communities.
Experimental materials Experimental design Experimental methods Data processing In this study, the soil was obtained from the experimental base of the Agronomy College of Heilongjiang Bayi Agricultural University. The soil is weakly alkaline chernozem with the following physical and chemical properties: contained 156 mg kg -1 alkali-hydrolyzable nitrogen, 14.99 mg kg -1 phosphorus, 100 mg kg -1 potassium, and 0.478 mg kg -1 heavy metal cadmium, with 3.78% organic matter, and the soil’s pH was 8.3. This research acquired the pakchoi variety of “ Siji Xiaobaicai” (Year-round plantable pakchoi). The biochar was prepared with corn stalk (preparation temperature was 450 °C, provided by Anhui Xiangzheng Biological Engineering Co., Ltd., pH 8.41, EC 1.07 mS cm −1 , NH 4 + -N 7.61 mg kg −1 , NO 3 − -N 30.82 mg kg −1 , available P 97.12 mg kg −1 , available K 3,012.00 mg kg −1 , bulk density 0.35 g cm −3 .
The experiment was conducted in the Heilongjiang Bayi Agricultural University greenhouse from May to June 2019. CK1, without biochar, was used as a control. Four treatments with 1% (T1), 3% (T2), 5% (T3), and 7% (T4) biochar were repeated thrice in the test facility. The cultivated soils were filled into a plastic basin with a 22 cm height and an 18 cm diameter. The same compound fertilizer and chicken manure were added to each pot. The sowing and thinning management were kept constant for all experiments. Samples were collected after 30 days (seedling stage) of growth to determine the relevant indexes of pakchoi.
When pakchoi samples were collected after 30 days (seedling stage) of growth, plant height and root length were measured using a meter ruler. The aboveground and underground parts of pakchoi were rinsed, leaf moisture was dried using filter paper, and the plant was weighed on a scale to obtain the fresh weight. The plants were wrapped in paper, placed in an oven at 105 °C for 30 min to remove water, and dried at 75 °C to obtain dry weight. This study took 0.1 g from the fifth leaf of fresh pakchoi in a leaching solution (alcohol: acetone = 1:1) for 24 h, and colorimetry was performed at 663 and 645 nm to calculate the contents of chlorophyll a, chlorophyll b, and the overall amount of chlorophylls . A 10 g sample of pakchoi and 5 ml of 2% oxalic acid solution were ground in a mortar. The ground sample was diluted to a 100 ml volumetric flask with 2% oxalic acid, filter, and a 10 ml of the filtrate was placed in an evaporating dish. It was titrated with a calibrated 2,6-dichlorophenol indophenol solution until it turned pink and did not fade within 30 s. The amount of dye used was recorded and the content of vitamin C was calculated . The method established by was used to determine the soluble sugar content. A total of 0.2 g of the sample to 20 ml of distilled water was added and extracted in a water bath for 30 minutes. Then, 1.5 ml of distilled water was added to 0.5 ml of the extraction solution. Then, 0.5 ml of ethyl acetate and 5 ml of concentrated sulfuric acid were introduced into the test tube in sequence. After vigorous shaking, the mixture was subjected to a boiling water bath for 1 min. After cooling, absorbance at 630 nm was measured to calculate the soluble sugar content . A total of 2 g of the sample was ground into a homogenate and extracted in 45 °C water bath for 1 h. A total of 0.2 ml of the extraction solution was added to a test tube containing 5 ml of 5% salicylic acid sulfate solution for 30 min, then 19 ml of NaOH solution was added absorbance was measured at 410 nm, and nitrate content was calculated . A total of 0.5 g of fresh sample was ground into a homogenate with 5 ml of distilled water, then centrifuged at 10,000 r min −1 for 10 min; then, 1.0 ml of the supernatant was taken and placed in a test tube, 5 ml of Coomassie Brilliant Blue G-250 solution was added, mixed and placed for 2 min at 595 nm for colorimetry. The absorbance was measured, and the protein content was calculated . A total of 0.2 g of dry sample was placed into a beaker, 100 ml of 60% H 2 SO 4 was added, shaken well, and filtered with a Buchner funnel into another beaker. A total of 2 ml of the filtrate was taken into a stopper tube, 0.5 ml of 2% anthrone reagent was added, and 5 ml of concentrated H 2 SO 4 was added. The stopper was sealed and shaken well, and let to stand for 12 min. The absorbance was measured at a wavelength of 620 nm and the cellulose content was calculated . For the experimental soil samples in 2019, the soil was sampled by shaking the roots after 30 days of growth. For each sampling, the roots from five tomato seedlings were collected and pooled together as one sample with three samples per treatment ( n = 3), and sieved using a 20 mesh. Some of these soil samples were stored at 4 °C to evaluate soil enzyme activities. The phenol-sodium hypochlorite colorimetric and disodium phenyl phosphate methods were used to determine soil urease and alkaline phosphatase activities. In addition, 3,5-dinitrosalicylic acid colorimetric, TTC staining, and pyrogallol colorimetric determination of sucrase activity, dehydrogenase activity, and soil polyphenol oxidase activity, respectively, were performed. Soil cellulase activity was determined using 3,5-dinitrosalicylic acid . Another portion of these soil samples was naturally air-dried to determine the alkali-hydrolyzable nitrogen by the alkaline hydrolysis-diffusion-titration procedure in a diffusion disk, the available phosphorus by molybdenum blue colorimetry using a spectrophotometer, and the available potassium by ammonium acetate extraction-flame photometry. The pH and EC values were determined using an acidimeter and a conductivity meter, respectively, after leaching and filtering the soil-water mixture (soil: water = 1:5) . Then, 0.5 g of the pulverized dry plant samples were digested with the HNO 3 -HClO 4 (V:V = 4:1) mixed acid system and analyzed using an atomic absorption spectrometer . For the experimental soil samples in 2019, adopting the MolPure ® Soil DNA Kit soil DNA extraction kit (18815ES50) can increase recovery yield by: (1) preheating the eluent at 65 °C; (2) adding the filtrate to the column again, let it sit at room temperature for 3 min, and elute. The sample DNA can be stored at −20 °C for short-term storage and −80 °C for long-term storage. The soil microorganism MiSeq was sequenced by the Shanghai Meiji Biological Company (Shanghai, China). Polymerase chain reaction (PCR) amplification as described by then the bacterial v3–v4 and fungal regions were amplified. The primers employed in this investigation were 338f/518r. The PCR products were analyzed using quantifluor™. Each PCR reaction, with a total volume of 20 µL, consisted of 9 µL of 2 × Real SYBR Mixture, 0.2 µL of each primer (10 µM), 2.5 µL of template DNA, and 8.1 µL of ddH2O. The qPCR reaction conditions are as follows: (1) Annealing: Following denaturation, the reaction proceeded to an annealing step. The annealing temperature was set at 65 °C and lasted for 30 s. During this step, the primers bind to their complementary sequences on the DNA template. (2) Extension: After annealing, the reaction underwent an extension step. The extension temperature was set at 72 °C and lasted for 1 min. During this step, the DNA polymerase extends the primers, synthesizing new DNA strands. (3) Final extension: The amplification process was repeated for a total of 22 cycles, followed by a final step of elongation. The temperature was set at 72 °C and lasted for 10 min. This step ensures that any remaining DNA strands are fully extended. (4) Fragment length: The amplified fragment length obtained in this study was approximately 230 base pairs (bp). This length corresponds to the targeted DNA region amplified by the primers. To analyze the quantitative data, a ST Blue Fluorescence Quantitative System (Promega, Madison, WI, USA) was used. Additionally, the MiSeq library was constructed and sequenced to obtain the sequence of DNA fragments .
All collected data were analyzed using IBM SPSS Statistics ver. 18 (SPSS Inc., Chicago, IL, USA). Duncan’s Multiple Range tests (DMRT) and post hoc tests were used to compare the means of the treatments. The significance level was set at P <0.05. The biological information analysis method statistically and visually analyzed the sequencing data to obtain optimized effective sequence statistics, OTU distribution statistics, diversity index analysis, and sample community composition analysis. Finally, a heatmap diagram was plotted .
Effects of different treatments on the growth of pakchoi Effects of different cultivation modes on the quality of pakchoi Effects of different concentrations of biochar on soil nutrient and enzyme activities Effects of different concentrations of biochar on soil bacterial community structure of pakchoi The results demonstrated that pakchoi root length, shoot dry weight, and root dry weight significantly increased under 3% biochar treatment (T2) compared to CK. Compared with CK and 3% biochar treatment (T2), the fresh and root dry weight of pakchoi significantly increased under 5% (T3) and 7% biochar treatments (T4). Therefore, the growth of pakchoi can be promoted by adding biochar at a concentration of at least 3% .
The experiments proved that the soluble sugar, soluble protein, VC, and cellulose contents in pakchoi leaves were significantly increased after biochar treatment compared with CK. The application of biochar at 3% (T2) and 7% (T4) treatments significantly increased the chlorophyll a, chlorophyll b, and total chlorophyll content in pakchoi leaves compared to the control without biochar application. Nitrate reductase activity in pakchoi leaves was significantly higher than in control and 1% biochar treatments when the amount of biochar added was more than 3%. With an increase in biochar content, nitrate content first decreased and then increased, and it was significantly reduced in the 3% biochar treatment. Therefore, the quality of pakchoi under the 3% biochar treatment was significantly better than that under the other treatments .
The urease, phosphatase, sucrase, catalase activity, and dehydrogenase of pakchoirhizosphere soil increased after adding biochar . The dehydrogenase activity of pakchoi in the rhizosphere soil increased significantly with biochar application rate. Compared with the soil under control conditions, the urease, phosphatase, sucrase, and catalase activities in the soil first increased and then decreased with increased biochar added. These properties peaked in the soiltreated with 3% biochar, which was significantly higher than that of CK. The pH value and contents of organic matter, alkali-hydrolyzable nitrogen, phosphorus, potassium, and EC values in the rhizosphere soil of pakchoi were enhanced after applying biochar . Compared with the soil under control conditions, the contents of phosphorus, potassium, EC value, and organic matter in the rhizosphere soil of pakchoi gradually increased with the increase in biochar content. The highest values were found in the soil treated with 7% biochar and were significantly higher than in the control soil. The alkali-hydrolyzable nitrogen content and pH value were first enhanced and then reduced. Peak values were found in the soil under the 3% biochar treatment and were significantly higher than those without biochar addition.
After adding biochar, the structure of bacterial flora in the rhizosphere soil of pakchoi changed . Among the relatively abundant dominant phyla Anaerolineae , the relative abundances of Gemmatimonadetes and Deltaproteobacteria were reduced, and Alphaproteobacteria , Gammaproteobacteria , Bacteroidia , Acidimicrobiia were increased. The microbiot structure changed significantly when the biochar content exceeded 3%. Adding different biochar impacted the bacterial diversity in the rhizosphere soil of pakchoi significantly , while adding 1% biochar (T1) had no significant change compared with the control, which was clustered together. The structure of the bacterial community in the rhizosphere soil of white pakchoi was significantly changed after adding 3% biochar (T2). The structure of the microbial community in the rhizosphere soil of white pakchoi treated with 3% biochar (T2), 5% biochar (T3), and 7% biochar (T4) was similar and clustered together. Microbial communities in the rhizosphere soil of pakchoi were classified into two groups. The first group was comprised Ilumatobacter , Luteolibacter , Lysobacter , Arthrobacter , Mesorhizobium , Pontibacter , Lactobacillus , Faecalibacterium , and Bifidobacterium , and the relative abundance of these bacteria increased significantly. The second group comprises the sweet and sour bacteria Gemmatimonas , Bacillus , Stenotrophobacter , Ramlibacter , Bryobacter , Actinoplanes , Comamonas , Flavisoib , Megamonas , Steroldobacter , Ohtaekwangia , and Luteitalon . These were clustered together, and the relative abundance of these bacteria was significantly reduced. Adding 3% biochar significantly changed the microbial community structure in the rhizosphere soil of pakchoi plants. Cluster analysis depicted that 1% biochar (T1) clustered together with the control group withsimilar bacterial community structures. A biochar content of >3% (T2) was also clustered. However, the structure of the bacterial community differed from that of the control group. All treatments were grouped into two types: (i) the control (CK) and 1% (T1) biochar, and (ii) the treatment above 3% biochar (T2, T3, and T4), among which the abundance of Gemmatimonas was significantly reduced after treatment with more than 3% biochar (T2) . Analysis of similarities (ANOSIM) compared the difference in beta diversity between samples from different groups. The boxplot results revealed significant differences between the T2 and T3 treatment groups and the control group . Principal component analysis demonstrated that CK and T1 significantly differed from T2, T3, and T4 in the PC1 axis when they were clustered together three times. Adding 1% biochar had no significant effect on the structure of the soil microbial community, and adding more than 3% led to significant changes in the structure of the soil microbial community . There was a significant positive correlation between Gemmatimonas and nitrate content and a significant negative correlation between Gemmatimonas and pH, NR, SDW, and RDW. There was a significant negative correlation between Ilumatobacter and nitrate content and a significant positive correlation between Ilumatobacter and pH, NR, SDW, and RDW. The control, without biochar (CK), was positively correlated with nitrate and VC. Biochar treatment (T2) was positively correlated with soil pH and NR, and biochar treatments (T3 and T4) were also positively correlated with COSS, nr, and urease activities. Therefore, biochar application is closely related to dry weight, nitrate accumulation, and root DW. In addition, COSS, NR, pH, and urease activity were the main influencing factors . The control rhizosphere soil had significantly higher carbohydrate transport and metabolism, amino acid transport and metabolism, general function prediction, energy production and conversion, post-translational modification, protein turnover, and chaperones than T2 treatment. The T2 rhizosphere soil had significantly higher lipid transport and metabolism, transcription, cell motility, signal transduction metabolism, intracellular trafficking, secretion, and vesicular transport than CK . The control rhizosphere soil had significantly higher carbohydrate transport and metabolism,amino acid transport and metabolism, and general function predictions than T3 treatment. The T3 rhizosphere soil had significantly higher intracellular trafficking, secretion, vesicular transport, secondary metabolite biosynthesis, transport and catabolism, cell motility, lipid transport and metabolism, and transcription than the control (CK) . Carbohydrate metabolism, membrane transport, cellular community-prokaryotes, energymetabolism, biosynthesis of other secondary metabolites, xenobiotic biodegradation and metabolism, lipid metabolism, and global and overview map control (CK) were significantly higher than those in treatment T4 (7%). The following metabolic processes were significantly higher in T4 (7%)-treated rhizosphere soil than in control soil (CK): nucleotide metabolism, glycan biosynthesis and metabolism, cell motility, replication and repair, cell growth and death, signal transduction, amino acid metabolism, metabolism of cofactors and vitamins, metabolism of other amino acids, and translation . Therefore, carbohydrate transport and metabolism in the rhizosphere soil of pakchoi decreased, and lipid transport and metabolism increased after applying biochar. These metabolic changes may explain the increase in soil nutrients after applying biochar.
Biochar promotes the growth of pakchoi and improves its quality Biochar enhances soil nutrient availability and reduces nitrate in pakchoi Correlation analysis between biochar changes in soil nutrient metabolism and microbial community structure Several studies have focused on the effect of biochar on vegetable plant growth, yield, andquality. A study demonstrated that the combination of 35 t ha −1 biochar and 200 kg ha −1 N fertilizers significantly increased tomato yield, VC content, sugar-acid ratio, and economic benefits . Wood chip biochar enhanced the growth of ice plants in coastal soil. Adding wood chip biochar improved the nutritional quality of the plants, regardless of whether chemical fertilizer was applied . The results of this study confirmed the hypothesis that the application of 3% biochar enhanced the growth and chlorophyll content of pakchoi, improved the nutritional quality of pakchoi . The positive effect of biochar amendment on pakchoi growth and quality may be attributed to improvements in soil properties , improved soil bulk density and water retention , enhanced nutrient availability , alterations in the soil microbial community . Research has shown that biochar can promote the development of plant roots, and then enhance the photosynthesis of leaves, so that plants can grow healthily, which is conducive to the plant production and the cultivation of soil . The results of this study show that indicate that 3% biochar significantly increased root length and chlorophyll content of pakchoi ( and ), this may be one of the main reasons for promoting the growth and improving the quality of pakchoi.
Biochar has a loose and porous structure, and its addition to the soil can improve the soil structure and enhance soil fertility , fix nutrients in the soil, and increase the content of available nutrients . The high carbon content of biochar can replenish the organic carbon in the soil and increase the soil organic matter content; biochar applied at certain concentrations gradually absorbed the nitrogen released from the N, P, and K compound fertilizers, reducing the nitrogen amount absorbed by plants and decreasing the nitrate content in plants . Because biochar is alkaline, its application to the soil can increase soil pH . Moreover, the relatively large surface area of biochar addition based on biochar in the short term can improve soil quality by neutralizing soil pH, increasing soil nutrient contents , and promoting plant growth. In this study, under weakly alkaline soil conditions, adding 3% biochar to the rhizosphere soil of pakchoi resulted in a peak alkaline nitrogen content, a significant increase in soil pH ( and ), and nutrient balance, which was beneficial for the growth and quality improvement of pakchoi. Biochar can significantly promote the growth of plant roots , and an increase in root biomass can increase the secretion of secretions , thereby altering the abundance of rhizosphere soil microorganisms and increasing soil enzyme activity . In this study, 3% biochar significantly increased soil urease activity and nitrogen availability in the soil . Studies have illustrated the combined action of biochar and nitrogen-fixing bacteria on microbial and enzymatic activities of soil N cycling . Application of biochar accelerates the metabolism of soil biota, turning more nitrogen from fertilizers into organic forms; hence, less mineral nitrogen is left for plant intake, which reduces nitrate levels in red beet . It can increase the utilization rate of nitrogen in the soil and reduce nitrate accumulation in the leaves of pakchoi . However, the improved soil nutrient metabolism and microbial community structure caused by adding biochar require further in-depth research.
Biochar amendments can alter plant rhizosphere microbiome, which profoundly affects plant growth and fitness . Regulation of C/N ratio by adding biochar and N fertilizer affects the composition and diversity of soil bacterial communities . This study demonstrated that the relative abundance of the bacterial community in pakchoi rhizosphere soil was changed by applying more than 3% biochar. Among the relatively abundant dominant phyla, Anaerolineae , Gemmatimonadetes , Deltaproteobacteria , and Verrucomicrobiae were reduced, and 303 Alphaproteobacteria , Gammaproteobacteria , Bacteroidia , and Acidimicrobiia relative abundance increased . Biochar affects the symbiotic pattern of microorganisms, increasing the proportion of positive interactions in the microbial community , the advantages of these changes will be verified in the future. Previous research has revealed that biochar amendment significantly increases the relativeabundance of the potential biocontrol bacteria Bacillus and Lysobacter . Bacteria, including species within the genera Arthrobacter and Bacillus , can solubilize phosphate or mitigate salinity stress . The abundance of some potentially beneficial bacteria, such as Luteolibacter , Glycomyces , Flavobacterium , and Flavihumibacter , increases in organic fertilizer-amended soil . The application of both types of biochar increases the nodule number by 52% under well-watered and drought conditions by improving the symbiotic performance of chickpeas with Mesorhizobium ciceri . Research displays that Gemmatimonas possess unique characteristics for assimilative and dissimilative N processes, with new implications for cultivation strategies to better assess the metabolic abilities of Gemmatimonadetes . In addition, the relative abundance of Gemmatimonas was reduced, whereas the relative abundances of Ilumatobacter , Luteolibacter , Lysobacter , Arthrobacter , Mesorhizobium , and other bacteria in soil supplemented with 3% biochar were significantly increased . The increase in these five ( Ilumatobacter , Luteolibacter , Lysobacter , Arthrobacter , and Mesorhizobium ) beneficial bacteria after adding biochar might be related to increased soil available nutrients and improved pakchoi quality. Gemmatimonas species carry genes that promote mineralization, nitrification, dissimilatory/assimilatory nitrate reduction, denitrification, anammox reactions, and N fixation. The functional microbes Gemmatimonas reduce various N conversion processes at different rates in biochar-added soil, which might reduce nitrate accumulation in pakchoi . The bacterial diversity was significantly improved, growth-promoting bacteria, such as Gemmatimonadetes and Proteobacteria , became more abundant, and Acidobacteria and Actinobacteria became less abundant after adding biochar . demonstrated that under biochar application conditions, the relative abundance of Acidobacteria , Chloroflexi and Gemmatimonadetes decreased, while Proteobacteria , Bacteroidetes and Actinobacteri a increased. The results were not nearly identical and may be related to the soil type and original flora of the test site. Adding modified biochar to the soil changed the relative abundance of dominant bacterialcommunities and increased the relative abundance of some functional bacterial communities . In this study, the nitrate content was positively correlated with the abundance of Gemmatimonadetes , and the reduction in nitrate accumulation by biochar addition was closely related to the decrease in Gemmatimonadetes abundance. Biochar enhances the retention capacity of nitrogen fertilizers and affects the diversity of nitrifying functional microbial communities in soil . However, the role of microorganisms in soil metabolism requires further verification. Ilumatobacter is a new genus of acid microorganisms that have been isolated from the sea by scholars . In this study, the nitrate content was significantly negatively correlated with the relative abundance of Ilumatobacte and positively correlated with aboveground dry matter accumulation and soluble sugar content . This indicates that the increase in the abundance of this genus is closely related to the growth and quality improvement of pakchoi.
Our results provide experimental evidence that biochar application In the production of pakchoi can improve soil available nutrient content and soil enzyme activity, the application biochar affected the abundance of dominant bacterial groups ( e.g. , Gemmatimonadetes and Ilumatobacter ) communities in the soil.The alterations in soil microbial metabolic pathways results in an enhanced soil nutrient environment, thereby facilitating nutrient uptake by crops, augmenting crop productivity, mitigating nitrate accumulation in leaves, and significantly enhancing of pakchoi, the effects of incorporating 3% biochar are particularly pronounced.
10.7717/peerj.16733/supp-1 Supplemental Information 1 The experimental process and results 10.7717/peerj.16733/supp-2 Supplemental Information 2 The first raw data obtained from high-throughput sequencing of the first repeated soil sample compared in the experiment CK, biochar treatment was not applied; 1, 2, and 3 represent three replications. 10.7717/peerj.16733/supp-3 Supplemental Information 3 The second raw data obtained from the first repeated soil sample high-throughput4 Supplemental Information 4 The first raw data obtained from the second repeated soil sample high-throughput sequencing of the control in the experiment 10.7717/peerj.16733/supp-5 Supplemental Information 5 The second6 Supplemental Information 6 The first raw data obtained from the thir7 Supplemental Information 7 The second8 Supplemental Information 8 The first9 Supplemental Information 9 The second raw data obtained from the first repeated soil sample high-throughput sequencing of T1 treatment in the experiment T1, treated with 1% biochar. 1, 2, and 3 represent three replications. 10.7717/peerj.16733/supp-10 Supplemental Information 10 The first raw data obtained from the second1 Supplemental Information 11 The second2 Supplemental Information 12 The first raw data obtained from the thir3 Supplemental Information 13 The second4 Supplemental Information 14 The first raw data obtained from the first3% biochar. 1, 2, and 3 represent three replications. 10.7717/peerj.16733/supp-15 Supplemental Information 15 The second6 Supplemental Information 16 The first raw data obtained from the second repeated soil sample high-throughput sequencing of T2 treatment in the experiment T2, treated with 1% biochar. 1, 2, and 3 represent three replications. 10.7717/peerj.16733/supp-17 Supplemental Information 17 The second8 Supplemental Information 18 The first raw data obtained from the thir9 Supplemental Information 19 The second20 Supplemental Information 20 The first raw data obtained from the first repeated soil sample high-throughput sequencing of T3 treatment in the experiment T3, treated with 5% biochar. 1, 2, and 3 represent three replications. 10.7717/peerj.16733/supp-21 Supplemental Information 21 The second2 Supplemental Information 22 The first raw data obtained from the second3 Supplemental Information 23 The second4 Supplemental Information 24 The first raw data obtained from the thir5 Supplemental Information 25 The second6 Supplemental Information 26 The first raw data obtained from the first10.7717/peerj.16733/supp-27 Supplemental Information 27 The second8 Supplemental Information 28 The first raw data obtained from the secon9 Supplemental Information 29 The second30 Supplemental Information 30 The first1 Supplemental Information 31 The second raw data obtained from the third repeated soil sample high-throughput sequencing of T4 treatment in the experiment T4, treated with 7% biochar. 1, 2, and 3 represent three replications.
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Statistical methods for discrimination of STR genotypes using high resolution melt curve data | b9bc9730-b96c-4148-90ad-54fa10d7bed8 | 11490427 | Forensic Medicine[mh] | Over the past two decades, methods for forensic DNA analysis have greatly increased in efficiency, sensitivity, and accuracy. However, samples with a limited number of DNA copies continue to create challenges for forensic laboratories. The stochastic effects associated with low template (Lt) DNA, such as STR allele drop-out, allele drop-in, and inter-locus/intra-locus peak imbalances, can cause uncertainty in the interpretation process . In addition to the difficulty of interpreting Lt DNA profiles, the presence of DNA from multiple contributors can further complicate the data, increasing uncertainty . Adjustments to procedures that could compensate for these issues and potentially yield more discernable profiles are often not possible, as artifacts and other profile characteristics are not revealed until the end of the DNA workflow. In order to correct these issues, the analyst may need to return to the sample preparation, DNA extraction, or STR amplification stage, which is costly, time-consuming, and may not be possible if the sample was consumed in the initial testing. Thus, having more information earlier on in the analytical workflow would make better use of analyst time and maximize the utility of limited evidentiary samples. Melt curve analysis utilizing qPCR instrumentation has long been explored as a potentially useful tool for a variety of forensic applications including mRNA analysis for body fluid identification, species identification, individualization of twins via methylation pattern analysis, Y STR screening, and human identification via single nucleotide polymorphism (SNP) analysis . Recently, Torres et al. (2023) developed a high-resolution melt (HRM) assay that, when combined with the Quantifiler™ Trio kit (Thermo Fisher Scientific, Waltham, MA) components and machine learning algorithms, was capable of accurately predicting if a forensic sample is a single-source or mixed DNA sample for 79% of samples tested . However, the assay was unable to predict the number of contributors (for mixture samples) nor was it able to determine the contributor genotypes . Introducing a detection assay to the quantification step that could identify contributor genotype and determine the number of contributors present would significantly strengthen this application. For example, having access to minimal STR genotyping information at the DNA quantification step would allow the examiner time to adjust the workflow prior to STR multiplex amplification. Further, this would allow them to provide early exclusionary information to investigators, if genotypes can be properly resolved. Melt curve analysis of STR amplicons has been explored for rapid genotyping of single source DNA samples . For example, one UK study explored amplification of the D18S51, TH01 and D8S1179 loci using HyBecon ® fluorescent probes and non-fluorescent blocker oligonucleotides to enhance melt curve analysis; this study successfully obtained partial STR profiles from buccal swabs . Additionally, Kuehnert et al. developed an optimized procedure for the amplification of D5S818 and D18S51 with subsequent high-resolution melting (HRM) using a Rotor-Gene ® Q (QIAGEN, Hilden, Germany) and an intercalating dye (EvaGreen ® ) . While distinguishable peaks for each STR locus were observed from resulting melt curve data, genotypes were not consistently discernible . Further, this assessment only analyzed 16–20 samples, all having one of only three closely related STR genotypes. Similarly, Nguyen et al. developed a melt curve screening tool for forensically relevant samples utilizing mini-STR primers for CSF1PO and TH01 along with either Taqman ® chemistry or intercalating dyes (SYBR ® Green or LCGreen ® Plus). The study reported accurate STR allele determination from degraded and inhibited samples, but noted inconsistent reproducibility of the assay . Unfortunately, none of these studies reported exploration of statistical software-based models, which could help improve accuracy and remove subjectivity from melt curve analysis. The Qiagen Rotor-Gene ® ScreenClust HRM ® software (Qiagen) incorporates principle component analysis (PCA) as a way to group like-samples using melt curve data . PCA is a correlational technique which transforms data into its main elements; from this transformation and reduction in dimension, a linear combination of variables can create new data. The newly created data can then be assessed for underlying patterns and variation . Alternatively, linear discriminant analysis (LDA) is a classification algorithm that attempts to make a distinction between observations. LDA assesses the data provided and compares it to other previously classified data patterns by determining similarity based on variance between classes and within classes . As of today, there is not a packaged software available for high resolution melt curve analysis which utilizes an LDA-type classification algorithm; however, previous work has generated code in R statistical software (©The R Foundation, Vienna, Austria) to meet this goal . Further exploration of HRM analysis for STR genotyping should include an analysis of a wide range of STR genotypes as well as a quantitative assessment of the melt curve data using statistical prediction modeling in order to determine if HRM could be used to provide reliable STR genotyping information for forensic investigations. Sample collection & initial DNA analysisSTR locus amplification & melt curve detectionGenotype prediction analysis from HRM dataThis study utilized previously collected DNA samples as well as buccal swab samples collected from volunteers in compliance with Virginia Commonwealth University Institutional Research Board protocol number HM20002931 and HM20006066. DNA from newly obtained samples was extracted using a QIAcube liquid handling robot (QIAGEN, Hilden, Germany) and the standard manufacturer’s Buccal Swab Spin QIAcube Protocol using QIAamp ® DNA Blood Mini kit reagents (Qiagen). Extracted samples were quantified using manufacturer’s protocol, but with half-volume reactions using the Investigator ® Quantiplex Kit (Qiagen) on the Rotor-Gene ® Q (Qiagen). Reference STR profiles for each sample were developed by amplifying 1ng of DNA extract with the AmpFLSTR ® Identifiler ® PCR amplification kit (Thermo Fisher Scientific, Waltham, MA) on the GeneAmp 9600 thermal cycler (PerkinElmer, Waltham, MA). The 15 µl reaction consisted of 5.7 µl of PCR Reaction mix, 2 µl of Primer set, 2.1 µl Tris-EDTA (TE), 0.2 µl of AmpliTaq Gold™ Polymerase (5U/µl) (Applied Biosystems, Waltham, MA), and 5 µl of template DNA. Thermal cycling conditions included a pre-denaturing step at 94 °C for 11 min, followed by 28 cycles of: denature 94 °C for 1 min, anneal 59 °C for 1 min, extension 72 °C for 1 min, and final post-extension step of 60 °C for 90 min. Amplified STR products were then separated and detected on the ABI PRISM ® 3130 Genetic Analyzer (Thermo Fisher Scientific) using a 36 cm capillary array with a 10s injection. Each reaction consisted of 0.1 µl of GeneScan™ 500-LIZ™ size standard (Thermo Fisher Scientific) and 12 µl of Hi-Di™ formamide (Thermo Fisher Scientific) diluent. The wells containing an allelic ladder received 1 µl of the ladder; 1.5 µl of amplified DNA was added to all other sample wells. The profiles were analyzed using GeneMapper ID™ software v4.1 (Thermo Fisher Scientific) with an analytical threshold of 75 relative fluorescent units (RFUs). The D5S818 and D18S51 genotypes were documented as the known reference genotypes for comparison in all studies detailed below. Ultimately, 311 samples were obtained and selected for this study. Samples selected were those that were available at the time of testing and had one of seven closely-related D5S818 genotypes [(10,11), (11,11), (11,12), (11,13), (12,12), (12,13), (13,13)] and/or one of six closely-related D18S51 genotypes [(12,14), (12,15), (12,16), (13,14), (13,16), (14,15)]. Samples selected were amplified for each of two STR loci (D5S818 and D18S51) separately on the Rotor-Gene ® Q using the primer and amplification parameters previously established . Each amplification reaction included 1X concentration of AmpliTaq Gold™ Buffer, 3mM MgCl 2 , 250µM dNTPs, 1µM each of forward and reverse primer, 2U AmpliTaq Gold DNA polymerase, 1X concentration of EvaGreen ® intercalating dye (Biotium, Fremont, CA), and 250ng/µl of bovine serum albumin (BSA) in water. Two microliters of template DNA were added to each reaction for a total reaction volume of 40µl. Primer sequences for D5S818 were (F) 5’-GGGTGATTTTCCTCTTTGGT-3’ and (R) 5’-AACATTTGTATCTTTATCTGTATCCTTATTTAT-3’; primer sequences for D18S51 were (F) 5’-CAAACCCGACTACCAGCAAC-3’ and (R) 5’-GAGCCATGTTCATGCCACTG-3’. The amplification cycling for both primer sets consisted of an initial 10 min 95 °C denaturation followed by 45 cycles of: 95 °C denaturation for 5s, 56 °C annealing for 20s, and 65 °C elongation for 30s with fluorescence detection at the 65 °C elongation step in the standard green channel. A cycle of 72 °C for 2 min, 95 °C for 20s, 55 °C for 20s and 56 °C for 2 min followed to transition into the melt phase. The amplicons were melted by 0.1 °C incremental increases in temperature from 60 to 95 °C. Each incremental step was held for 2s with fluorescent detection throughout the melt using the high-resolution melt curve detection channel. For PCA analysis, melt curve data generated from each sample at both STR loci were separately analyzed using the Rotor-Gene ® ScreenClust HRM ® software. For the D5S818 sample set, 56 samples were assigned as the training samples or “standards” based on their known genotypes; similarly, for the D18S51 sample set, 52 samples were assigned as the training samples or “standards”, with 7–10 samples per genotype for both loci. For each locus analyzed, all experimental samples were included as unknowns submitted for prediction analysis; the software placed each unknown into a genotype category based on highest probabilities given acceptable variability from the group mean. From the predicted clusters, confusion matrices were generated and then used to assess the software’s prediction accuracy (given as an overall percentage). From this, the percentage of misclassification for each genotype was determined and trends were identified. Geno-groups were formed based on these patterns of misclassification at each locus tested as well as the similarity of the genotype (and thus, amplicons produced). To subsequently evaluate the prediction accuracy using the identified geno-groups, the standard (training) samples were re-assigned in the software (as belonging to a geno-group, rather than a specific genotype), unknown samples were reanalyzed, and the newly predicted clusters were assessed for accuracy, as indicatPCA-based prediction accuracy. For LDA analysis, the melt curve data generated from each sample at both STR loci were separately analyzed using LDA code in R statistical software. The change in fluorescence (dF) with respect to temperature was exported, melt curves generated, and the primary peaks and shoulders were identified (Figs. and ). The data from each sample were then summarized into its primary peak and shoulder peak(s) temperatures along with their corresponding peak heights. For D5S818 samples, the peak/shoulder temperatures and peak heights for up to three observations were used; if only two peaks/shoulders were observed, the height at 64.95 °C was used as the third data point (required, as samples with disparate numbers of data points cannot be compared). No sample had fewer than two observed peaks. For D18S51 samples, the peak/shoulder temperatures and corresponding peak heights for four observations were used; if only three peaks/shoulders were observed, the peak height at 64.95 °C was used as the fourth data point. No sample had fewer than three observed peaks at this locus. For the D5S818 sample set, the same 56 samples used above were again assigned as the training samples for this analysis based on their known genotypes; similarly, for the D18S51 sample set, the same 52 samples used above were assigned as the training samples for this analysis. Code was generated in R statistical software so that the accuracy of LDA-based predictions could be calculated. Confusion matrices were generated and then used to assess the LDA, trends were identified, and geno-grouping options were created. Geno-groups were formed, training samples were reassigned, and unknown samples reanalyzed, as described above. Several different geno-grouping options were explored in order to determine the best option for the highest LDA-based prediction accuracy. D5S818D18S51When using the Rotor-Gene ® Q ScreenClust HRM ® software to predict D5S818 genotypes from HRM data using a PCA approach, samples were classified correctly only 23.77% of the time (Table ). Overall, samples with known homozygous genotypes were more likely to classify accurately (39.58%) than samples whose known genotypes were heterozygous (20.18%). Most often, misclassified homozygous samples were predicted as having another homozygous genotype, whereas heterozygous sample misclassifications were more evenly split among homozygous and heterozygous genotypes. Conversely, when using LDA in R statistical software to predict D5S818 genotypes from the HRM data, samples were classified correctly at a rate of 58.92% (Table ), which is substantially higher than the PCA-based model and the random chance rate of 14.29% (one in seven). As with the PCA method, samples with known homozygous genotypes were more likely to classify accurately (65.08%) than samples whose known genotypes were heterozygous (55.74%). In an attempt to increase prediction accuracies, 13 different geno-groupings were created (based on the above trends and misclassification rates) and tested. As expected, geno-grouping improved classification accuracies, regardless of which geno-grouping option was used or algorithm tested (data not shown). The three geno-grouping options that produced the highest prediction accuracies for each prediction model used in this study were assessed using the converse method to allow for direct comparison (Table ). Geno-group option A provided the highest LDA-based and highest overall prediction accuracy (81.0%). Alternately, the highest prediction accuracy achieved with the PCA-based method was 46.6% (option F). Regardless of geno-grouping option tested, LDA-based prediction modeling provided higher geno-grouping prediction accuracies. This result may be due to the fact that LDA aims to maximize the separation amongst classes, in order to heighten class discrimination. In order to determine if a more polymorphic STR locus would be better for genotype predictions using the two selected models, the testing was repeated using primers targeting the D18S51 locus. With more common genotypes known and higher levels of heterozygosity reported for this locus , one may expect the melt curve resolution, and thus genotyping predictions, to improve. When using the PCA-based Rotor-Gene ® Q ScreenClust HRM ® software to predict heterozygous D18S51 genotypes from HRM data, samples were classified correctly 40.38% of the time (Table ). Of the 62 samples that misclassified, 40.32% had one allele predicted accurately with the second allele off by only one-repeat unit (one allele value). Additionally, the samples expected to produce heterozygous amplicons with the greatest difference in base pair length (those with 12,16 genotypes) were the most likely to be classified correctly (57.14%). This result is not surprising as the amplicons with the greatest difference in base pair length correspondingly have the greatest difference in melting rates thus producing visually distinct melt curves when compared amongst melt curves with amplicons that are close in base pair length and thus have similar melting rates. When LDA was used to predict D18S51 genotypes from the HRM data, samples were classified at a rate similar to the PCA-based method (45.10%, Table ). However, unlike the PCA model for D18S51, the data obtained using the LDA approach showed no discernible trends when misclassifications were closely examined. As with the D5S818 locus, D18S51 geno-grouping options were created based on observed trends and classification rates; eight different geno-groupings were tested using both prediction models. As described above for D5S818, geno-grouping improved classification accuracies when the PCA algorithm was used; for LDA, however, only half of the geno-grouping options tested resulted in favorable increases in prediction accuraciesE provided the highest PCA-based and highest overall prediction accuracy (65.4%). The highest prediction accuracy achieved with the LDA-based method was very similar (63.5%, option G). This study suggests that PCA-based methods may work better for predicting genotypes of loci that have increased allele diversity, such as that observed with D18S51. This study evaluated the use of PCA- and LDA-based prediction modeling tools for their ability to distinguish between genotypes of two STR loci using HRM data obtained from the Qiagen Rotor-Gene ® Q qPCR platform. When assessing the D5S818 locus, the LDA model substantially outperformed the PCA model for predicting genotypes. This trend held true when like-genotypes were grouped together for prediction analysis into geno-groups with prediction accuracies exceeding 80%. However, when assessing a more polymorphic STR locus (D18S51) with a more heterogeneous sample set, the differences in prediction accuracies between the models tested were far less pronounced suggesting that the LDA-based method may work better for predicting homozygous genotypes. Regardless of method or locus tested, placing samples with closely aligned genotypes into geno-groups for classification results in improved prediction modeling, but fewer classification options would limit the forensic utility of an HRM-based assay, as DNA from different contributors will be less likely to be individualized. Ultimately, the data from this study suggests that the best prediction model for STR genotyping may differ from locus-to-locus, depending on the nature and complexity of the STR locus tested. Further, the inclusion of additional heterozygous genotypes in the training sets used to train the software may improve overall prediction rates, regardless of the testing model employed. Considering additional factors may be helpful when selecting a prediction model to use for genotyping using HRM data. For example, the PCA-based ScreenClust HRM ® software is commercially available, requires no coding, and is easy to use. However, the software is proprietary and the principal components it utilizes for analysis are unknown. Further, the ScreenClust HRM ® software requires that all known (training) standards be run on the instrument at the same time as tested unknown samples to provide the most accurate clustering. This would be impractical for wholescale use in forensic settings and becomes highly impractical when assessing loci with large repeat ranges and many common genotypes. Alternatively, R statistical software is free and training set data is stored for use of classification of unknown samples subsequently and independently tested. However, it requires some initial programming and forensic implementation would require the development of a more user-friendly interface. In conclusion, this study provides foundational data documenting the performance of prediction modeling for STR genotyping based on HRM data. In order to expand the forensic applicability of the HRM assay described herein, it may be useful to test it using more commonly utilized qPCR platforms, such as Thermo Fisher’s QuantStudio™, and potentially incorporate it into the previously described mixture detection assay . Further, exploring other prediction models that use similar classification schemes to those used in this study but are designed to classify larger data sets (e.g., comprehensive melt curve data), such as support vector machines (SVM), may prove useful . |
Analysis of MLKL, RIP1 and RIP3 Immunostaining Markers in Human Liver Tissue from Fatal Yellow Fever Cases: Insights into Necroptosis | e12ca5f0-2abf-4cca-bdea-028e0a87cf89 | 11768900 | Anatomy[mh] | Yellow fever (YF) is an arboviral disease transmitted by hematophagous mosquitoes that can present a spectrum of clinical manifestations ranging from asymptomatic cases to severe forms with hemorrhage. It is a disease transmitted by mosquitoes infected with the Yellow Fever Virus (YFV) belonging to the Flavivirus genus in Africa, and it is maintained in wild cycles by non-human primates (NHP) and hematophagous arthropods of the Haemagogus and Sabethes genera in South America. Its lethality ranges from 20% to 50% . The urban cycle encompasses Aedes aegypti and human interactions, posing the most significant public health concern. The resurgence of this urban cycle in the Americas, coupled with vulnerable human populations, has raised alarms among public health authorities. This is primarily due to inadequate vaccination coverage in both non-endemic and endemic regions . After the virus enters the human body through the bite of the transmitting mosquito, it quickly reaches the lymph nodes and spreads into the bloodstream. Viral replication begins in the lymph nodes. After the release of virions into the bloodstream, a period known as viremia, organs such as the liver, kidneys and spleen are also infected . From the point of view of the pathophysiology of yellow fever, with the triggering of immune evasion strategies adopted by the virus, liver failure becomes a central problem where, in the phase of intense viral replication, the exacerbated immune response of the host leads to the massive propagation of the activation of cell-death mechanisms that culminate in severe hepatocyte necrosis. As a consequence, jaundice, hepatomegaly, the elevation of transaminases, the presence of hemorrhage, hypovolemic shock and renal dysfunction are factors that contribute significantly to acute liver failure in fatal cases. Apoptosis and necrosis have long been considered the main processes of cell death in YF infection, but it is believed that necroptosis may play an important role in the cell death of hepatocytes . Necroptosis, a recently recognized type of programmed cell death, contributes to inflammation and various diseases such as cancer, stroke and kidney disease . When triggered by various stimuli, necroptosis is initiated by the activation of receptor-interacting protein kinase 1 (RIP1). Activated RIP1 interacts with RIP3 through their RIP homotypic interaction motifs, phosphorylates RIP3 and forms an RIP1/RIP3 complex called the necrosome . Then, mixed lineage kinase domain-like protein (MLKL) is recruited and phosphorylated by RIP3 in the necrosome . Phosphorylated MLKL monomers aggregate to form oligomers and translocate to the plasma membrane to execute necroptosis . Although multiple cell death pathways are involved in YF, including apoptosis and necrosis, the effect of necroptosis remains largely unknown . In this study, our aim was to explore the activation of necroptosis in YF and identify the role of RIP1/RIP3/MLKL-mediated necroptosis in human liver tissue. 2.1. Patients, Samples and Diagnosis of Yellow Fever Infection2.2. Ethics Statement2.3. Immunohistochemistry (IHC)2.4. Quantitative Analysis and Photo-Documentation2.5. Statistical AnalysisThis study used 26 human liver biopsies. Among them, 21 samples of fatal cases of YF were confirmed through obtaining positive results for the virus by reverse transcriptase reaction followed by polymerase chain reaction (RT-PCR) and immunohistochemistry (one or both methods). In addition, five control samples from patients with preserved liver architecture tested negative for YF and other flaviviruses circulating in Brazil, according to the death verification service (the Renato Chaves Scientific Expertise Center) in the city of Belém in the state of Pará, Brazil. The confirmation of the diagnosis for the positive cases of YF was based on the studies of Melo et al. and Olimpio et al. , including histopathological, immunohistochemical and real-time quantitative RT-PCR (RT-qPCR) analyses. For RT-qPCR, samples were processed using the 7500 Fast Real-Time PCR System (Applied Biosystems, Waltham, MA, USA) and the AriaMx Real-Time PCR System (Agilent Technologies, Santa Clara, CA, USA) using two RT-qPCR kits: (1) the Superscript III ® Platinum ® One-Step Quantitative RT-PCR System (Invitrogen, Waltham, MA, USA) and (2) the QuantiTect ® Probe RT-PCR (Qiagen, Germantown, MD, USA). For the histopathological diagnosis, the paraffin-embedded biopsies were cut into 5 μm sections and stained with the hematoxylin–eosin method. shows detailed information about the patients included in this study. The patient samples were obtained and processed as part of the response measures to the surveillance of the YFV epidemic in Brazil on an emergency basis, as defined by the Ministry of Health. This study was approved (No. 2.824.592) by the Research Ethics Committee (CEP) of the Evandro Instituto Chagas (IEC). All of the methods were performed in accordance with the relevant guidelines and regulations approved by the CEP/IEC and the Brazilian Ministry of Health rules and regulations for studies with biological samples. Immunostaining of the hepatic tissues with antibodies specific for MLKL (Abnova, Taiwan, TW, H00197259-M02, dilution 1:50), RIP1 (Abcam, Cambridge, UK, ab72139, dilution 1:50) and RIP3 (Abcam, Cambridge, UK, ab152130, dilution 1:50) was performed using the streptavidin–biotin peroxidase immunohistochemical method (SABC) and adapted according to Olímpio et al. . Briefly, the tissue samples were deparaffinized in xylene and hydrated in a decreasing series of ethanol (90%, 80%, 70%). The liver sections were incubated in 3% hydrogen peroxide for 45 min to block endogenous peroxidase. Incubation in citrate buffer, pH 6.0, for 20 min at 90 °C was realized to recover antigens. Non-specific proteins were blocked by incubating the sections in 10% skim milk for 30 min. The histological sections were then incubated overnight with the primary antibodies diluted in 1% bovine serum albumin. The slides were immersed in 1 × PBS and incubated with the biotinylated secondary antibody (LSAB; DakoCytomation, Glostrup, Denmark) in an oven for 30 min at 37 °C. The slides were immersed in 1 × PBS and incubated with streptavidin peroxidase (LSAB; DakoCytomation) for 30 min at 37 °C. For visualization, the specimens were treated with a chromogenic solution (0.03% diaminobenzidine and 3% hydrogen peroxide). Finally, the histological sections were washed in distilled water, counterstained with Harris hematoxylin for 1 min, dehydrated in ethanol (70%, 80%, 90%) and deparaffinized in xylene. The markers that characterize in situ necroptosis were analyzed with the Axio Imager Z1 microscope, where the expression of each marker was quantified from the random selection of 10 fields, each divided into areas smaller than 10 × 10 μm, delimited by a 0.0625 mm 2 grid. The microscopic scanning for each field was established in the hepatic acinus, including the zones (Z3: pericentral zone; Z2: midzonal zone; Z1: periportal zone; PT: portal tract) that comprise the Rappaport space. These zones were analyzed for both the fatal cases that were positive for yellow fever and for those in the control group. The data were stored in a Microsoft Excel 2016 spreadsheet and analyzed using GraphPadPrism 9.0. The numerical variables were expressed as the mean, median, standard deviation and variance. A one-way ANOVA, Tukey’s test and the Pearson correlation were also applied; the results were considered statistically significant at p < 0.05. Expression of RIP1, RIP3 and MLKL in the Hepatic Parenchyma in Fatal Yellow Fever Casesresults obtained from the analyses of the fatal cases that were positive for YFV compared with the control samples revealed significant differences of RIP1, RIP3 and MLKL, with highly significant p values, as observed in and . Furthermore, the immunostaining for each marker demonstrated that, in the zones (Z3, Z2 and Z1), the expression of the markers was predominantly found in hepatocytes , while in the portal tract, it was centralized in the inflammatory infiltrates composed mainly of lymphomononuclear cells . The current study provides crucial insights into the expression and correlation of necroptosis markers (MLKL, RIP1 and RIP3) in liver tissue from fatal yellow fever (YF) cases, shedding light on the underlying mechanisms of YF pathology. The immunostaining results confirmed the presence of MLKL, RIP1 and RIP3 in hepatocytes and inflammatory infiltrates ( , and ). The preservation of the hepatic parenchyma and the minimal expression of these markers in the control cases further support their specific involvement in YF pathology. The quantitative analysis revealed a significant upregulation of these markers in YF cases, particularly in the midzonal zone, followed by the pericentral and periportal zones and the inflammatory infiltrates in smaller expressions ( , and ). This spatial variation suggests the involvement of necroptosis in liver damage and hepatocyte loss as having distinct roles in necroptosis induction and execution during YF infection. The Pearson correlation analysis revealed significant correlations among MLKL, RIP1 and RIP3 in YF cases . The positive correlations between MLKL and RIP1, as well as between MLKL and RIP3, indicate their cooperative role in necroptosis signaling. Moreover, the negative correlations observed among MLKL, RIP1 and RIP3 in certain liver zones and portal tracts imply the existence of complex regulatory mechanisms that vary across different tissue microenvironments . Such scenarios indicate the dichotomy of responses of the association of markers, which can influence the cellular response to tissue damage that can contribute to the deterioration of liver tissue as well as aggravate inflammation and liver injury. This can cause a vicious cycle in which the virus, through immune evasion strategies, can modulate cellular injury in different aspects that impact cell swelling and membrane rupture, which is a hallmark of necroptosis. Supporting these findings, Vandenabeele et al. demonstrated the crucial role of RIP1 and RIP3 in necroptosis signaling pathways. The study identified RIP1 and RIP3 as key players in the formation of the necrosome complex, which leads to the execution of necroptosis. In our study, we observed significantly higher expression levels of RIP1 and RIP3 markers in liver tissue from fatal YF cases compared to controls. This finding supports the involvement of necroptosis in YF pathogenesis, suggesting that RIP1 and RIP3 may contribute to hepatocellular necroptosis and tissue damage. Furthermore, Wu et al. highlighted the significance of MLKL in mediating necroptosis-associated inflammation. MLKL is a downstream effector molecule activated by RIP3, leading to plasma membrane disruption and the release of intracellular contents, triggering inflammation. During necroptosis, the formation of RIPK1/3 heterodimers gives rise to a complex that triggers the NF-kB-mediated pro-inflammatory response, facilitating the release of cytokines/chemokines . Additionally, the secretion of damage-associated molecular patterns (DAMPs), such as ATP and HMGB1, is notably heightened during necroptosis . As a consequence of these signaling cascades, morphological changes distinct from apoptosis become evident in necroptosis, including organelle swelling and cellular lysis . This specific morphology is discernible in (black arrows), indicating the occurrence of necroptosis in a severe yellow fever infection. This is further supported by elevated levels of MLKL expression in the hepatic tissue of fatal yellow fever cases as well as by the positive correlation between MLKL and RIP1/3 expression levels . Review work by Verburg et al. discussed the contribution of RIP3 and MLKL to immunopathology in viral hepatitis. The study demonstrated that the activation of RIP3 and MLKL promotes liver inflammation and tissue damage. Our results are consistent with these findings, as we observed upregulated expression levels of RIP1, RIP3 and MLKL in liver tissue from fatal YF cases. The positive correlations observed among these markers suggest their involvement in promoting hepatocellular necroptosis and inflammation in YF. While our study focused on YF, this research provides insights into the crosstalk between different forms of regulated cell death. Necroptosis and pyroptosis share common signaling pathways and may contribute to tissue damage and inflammation . Although our study did not investigate pyroptosis specifically, the findings highlight the complexity of cell death mechanisms in viral infections and their potential impact on disease progression. Overall, the literature supports our results, indicating the involvement of necroptosis and the upregulation of MLKL, RIP1 and RIP3 markers in liver tissue from fatal YF cases. The positive correlations observed among these markers further reinforce their interplay and potential contribution to hepatocellular necroptosis and tissue damage. Understanding the molecular mechanisms of necroptosis and its association with YF pathogenesis may pave the way for the development of targeted therapies to mitigate liver injury and improve patient outcomes in YF cases. Our study provides quantitative evidence for the upregulation of the necroptosis markers MLKL, RIP1 and RIP3 in human liver tissue from fatal YFV cases. The differential expression patterns of these markers in different liver zones and portal tracts suggest a spatially regulated involvement of necroptosis in the pathogenesis of yellow fever. Furthermore, the observed correlations among MLKL, RIP1 and RIP3 expression levels in the YFV cases support their cooperative role in necroptosis induction during YFV infection. Further research is needed to elucidate the underlying molecular mechanisms and the potential therapeutic implications of targeting necroptosis in yellow fever. |
Predictors of weight reduction in a Nigerian family practice setting | ec616260-6b5c-499b-bca9-cc644894cb84 | 10336632 | Family Medicine[mh] | Obesity, a risk factor for Non-Communicable Diseases, is a global public health concern in both developed and developing countries because of increasing westernisation and the adoption of foreign lifestyles. , Obesity has several health risks with multi-systemic complications, including type 2 diabetes mellitus, dyslipidaemia, hypertension, increased depression and lower self-esteem. It is estimated that over one billion adults are overweight, out of which at least 300 million have obesity. WHO also reported a 50% increase in the number of adults with obesity worldwide between 1995 and 2000. From the NHANES, 2013–2014, in the U.S.A., obesity was reported to have risen to 38% from the 30.5% documented in 1999–2000. In 2015, in the U.K., the obesity rate was 26.2%, and in Turkey, obesity prevalence was documented as 22.3%. Countries in economic transition from developing to developed, such as China, Brazil and South Africa, are particularly affected and have an increased rate of obesity across all economic levels and age groups. In South Africa, the self-reported prevalence of obesity was documented as 26.5%. Obesity rates in SSA were documented in 2016 as 10% in rural Uganda, 14 % in peri-urban Uganda, 31 % in Nigeria, 40 % in Tanzania and 54% in South Africa. Obesity prevalence has a geographical variation in Nigeria, West Africa, with 41.8% in the South-west, 47.3% in South-south, 33.3% in the East and 8.1% in the North-central region. In Nigeria, even with a poor Gross Domestic Product (GDP) and a high poverty index, there are documented increasing levels of obesity rising from about 20% between 2002 and 2010 to 33.3%. , The healthcare cost of complications of obesity is large, consisting of direct and indirect costs. The various factors driving weight reduction in obesity worldwide are non-modifiable factors, including genetic susceptibility, Leptin and gender, 14,15 and modifiable factors that comprise lifestyle variables such as physical activity and diet. Questions (Qs) 3 and 4 raised by the Obesity Expert Panel are critical to the issue of weight reduction. Q1 was: What are the Cerebrovascular Disease (CVD) health benefits associated with weight reduction? Q2 was: What are the health risks of weight reduction relative to CVD, and secondly, are the limits for Body Mass Index (BMI) and Waist Circumference enough for population subcategories? Q3 was: For weight reduction, what dietary plan can be achieved? Q4 was: Is a package for lifestyle consisting of diet, exercise and behaviour therapy enough? Q5 was: What type of patients would benefit from obesity-related surgery? The efficacy of behavioural weight reduction treatments is well-known among healthcare professionals in developed countries. Predictions of treatment compliance and weight reduction have been widely proven elusive because obesity varies in different subgroups of patients. This present study is different because it was aimed at determining the predictors of weight reduction among patients with obesity both in a developing country and in an urban setting in Ibadan, South-west Nigeria, as compared to previous research in rural Nigeria, which determined weight change and influencing factors. , A secondary objective of this study was to develop a statistical model that can predict individual weight reduction. It will be useful in health facilities with a weight loss program or in community-based programs.
The study was designed as a prospective cohort study among adult patients with obesity who were above 18 years attending the General Outpatients' (G.O.P.) clinic, a Family Practice setting in the University College Hospital, Ibadan, Oyo State, South-Western Nigeria. The study population was adult patients aged 18 years and above who presented consecutively between 14th August 2013 to 5th May 2016. The sample size was calculated using the formula for a single estimate, , with the average mean weight change (0.39kg per year) among adult Nigerians. The inclusion criteria were obesity (BMI ≥ 30kg/m 2 ) and informed consent. The exclusion criteria were those with acute illness, pregnant women, diabetes mellitus, physical deformities affecting the spine and limbs, and prescription weight loss medication. Institutional ethical approval was obtained from the University of Ibadan/ University College Hospital Ethical Committee, and the registration number was NHREC/05/01/2008a. An interviewer-administered semi-structured questionnaire was designed and used to obtain relevant information such as socio-demographic characteristics, history of obesity, previous treatment for obesity, with family and lifestyle history. Validated instruments used included the Multidimensional Scale of Perceived Social Support (MSPSS), a 12-item tool that measures support with aggregate scores ranging from 7–84. High acuity/support was 69–84, moderate acuity (49–68) and low acuity (12–48). MSPSS has a Cronbach's alpha coefficient of 0.86 to 0.90. Depression was assessed with the Zung Depression Scale (ZDS), a 20-item questionnaire with a maximum score of 80. Severe depression was scored as ≥70, moderate depression (60 - 69), minimal depression (50 – 59), and < 50 is normal. ZDS, when administered in Yoruba (the local dialect) and English languages, has an index reliability of 0.64 – 0.79. , The Rosenberg Self-Esteem (RSE) scale is a 10-item tool that measures self-esteem. Each item gives a maximum score of 4 points, on a scale of 10 to 40 points, with higher scores reflecting higher self-esteem. RSE has a reliability and validity of and a Cronbach's alpha of 0.87. The Garner's Eating Attitude Test-26 (EAT-26) has 26 items used in a West African population. , and has a Likert scale with scores above 20 indicating concerns about body weight, body shape and eating. The 24-hour dietary recall assesses diet and validated face-and content, with cultural adaptations in a previous study. Food portion sizes were estimated using household measures or the hand guide. The Total Dietary Assessment (TDA) software was used to analyse the 24-hour dietary recall data of consumed food and drinks. A human nutritionist/statistician checked this dietary data inside the Nigerian food composition table. Foods and drinks consumed were converted to a weight equivalent and then inserted into the TDA, which then converted it to caloric intake. The International Physical Activity Questionnaire (IPAQ)-short form (IPAQ-SF) was used to assess physical activity. The IPAQ-SF was categorised into inactive/sedentary, minimally active and Health Enhancing Physical Activity (HEPA) active. The inactive/sedentary group were further re-categorized into the inactive category, while the minimally active and Health Enhancing Physical Activity (HEPA) active group were categorised into the active group. Clinic visits and follow-up Data analysis participants were observed over 3 months at enrollment (first visit), one month later (second visit) and two months after (third visit). At the first visit, every participant was formally identified with a unique number and had their details, such as their telephone numbers, addresses, and initials, written in a logbook. The interviewer administered the questionnaire, and anthropometric and blood pressure measurements were done. For the Fasting Lipid Profile tests, the participants were told to fast overnight for 12–14 hours and have their blood samples collected the following day. An EDTA bottle was used to collect the blood samples. Centrifuging of the blood was performed for 5 minutes at 3000 revolutions per minute after collecting the sample by a qualified Laboratory Scientist. If the analysis was not done the same day, the plasma sample was kept overnight in the fridge at 4°C or frozen at -20 o C in plain plastic bottles. The colourimetric method was used for the estimation of Total Cholesterol (TC), High-Density Lipoprotein (HDL)-Cholesterol and Tri-glycerides (TG) using an auto-analyzer Hitachi 902. The cholesterol oxidase phenol 4- amino antipyrine peroxidase (CHOD-PAP) by Abell-Kendall was used to estimate the TC. In contrast, the triglyceride glycerol-3-phosphate oxidase phenol + aminophenazone (TG GPO-PAP) was used to determine the TG. The HDL was measured using the homogeneous enzymatic colourimetric test, while the Low- Density Lipoprotein (LDL) - cholesterol was calculated using the formula: LDL =Total cholesterol- ( TG/5 +HDL). Patient Information Leaflets (PILs) with a lifestyle modification mnemonic: A-Activity increase, B-Behavioral modification, C- Calorie reduction (ABC); were used to counsel the patients for target weight reduction. The participants were informed and counselled about the investigation results during the second and third visits. The anthropometric and blood pressure measurements were performed. Target weight reduction was noted and used to classify the participants into either target weight reduction or no target weight reduction group. Participants were referred to specialists such as dietitians and physiotherapists for further management as the need arose.
Data entry and analysis were carried out with the Statistical Package for Social Sciences version 20 (SPSS-20). Target weight reduction was defined as 10% (2.5% in 3 months) of their baseline weight. Based on existing literature, age was classified into less than and above or above 40 years. Descriptive statistics, such as measures of central tendency like means and medians, were first used to analyse the data, and inferential statistics using the Chi-square test were used. The TDA software was used to analyse the 24-hour dietary recall by a statistician/ Human Nutritionist. Multiple linear regression analysis was employed to arrive at the statistical model for weight reduction. The repeated measure of Analysis of Variance (ANOVA) was also carried out. The level of significance was set at 5%.
The majority of the 130 participants, 108 (83.1%), were 40 years, with a mean age of 48 (± 8.4) years. shows that target weight reduction was greater in those less than 40 years 14 (63.6%), in females 57 (52.8%), and in those with greater than secondary school education 53 (51.0%). A greater proportion of the participants who were not married, 15 (75.0%), achieved the target weight reduction, which was statistically significant (p=0.02). Of those who had blue-collar jobs, 65(51.2%) and an income below 18,000naira (the minimum standard wage as at the time of the study in Nigeria), 12 (54.5%) were able to achieve more target weight reduction. Most participants (n=99) achieved a mean weight reduction from 97.2±16.4kg at baseline to 95.6±14.2kg at the end of the study. This is seen in the error-bar chart in . The median weight change was -2.3kg (IQR-4, -0.5). Out of all the 130 participants, 99 (76.2%) participants were able to reduce weight by the end of the study, while 66 (66.7%) were able to achieve the target weight reduction of 10% (2.5% in 3 months). This is depicted in . shows that half of the participants who achieved target weight reduction were informed about obesity when they were less than 40 years 49 (56.3%). Less than half of the participants who achieved target weight by weight reduction methods tried before 48 (51.6%) had tried to lose weight through exercise-related techniques before the study 15 (53.6%). About half of the participants who achieved the weight target reduction 53 (52.0%) had a family history of obesity, which was highest in the siblings 6 (64.5%). More of those who achieved target weight reduction had low support 4 (57.1%). shows that of the participants with minimal or mild depression, half 5 (50.0%) were able to attain target weight reduction while more participants with normal self-esteem, 65 (52.0%), achieved target weight reduction. Over half of the participants, 49 (52.7%) with a low level of concern in the EAT-26 score, achieved target weight reduction. indicates that a greater proportion of participants with calorie loss above 500kilocalories, 16(55.2%), could achieve target weight reduction. Among those who were active, a lower proportion, 13 (43.4%), achieved target weight reduction, while among those who were inactive/ sedentarily active, a higher proportion, 53 (53.0%), achieved target weight reduction. Over half of the participants who did not consume alcohol, 59 (52.2%), reached the target weight reduction. A higher proportion of participants with elevated TC 28 (52.8%) and low HDL-C 10 (52.6%) at baseline achieved target weight reduction. Over half of those with WC reduction, 51 (55.4%) achieved the target weight reduction. In , the statistically significant independent determinants of weight reduction were knowledge of Total Cholesterol [TC] (p=0.01) and Low-Density Lipoprotein Cholesterol [LDL-C] (p=0.03). Knowledge of HDL-C (p=0.06) and EAT-26 score (p=0.08) were also independent determinants of weight reduction which showed a tendency of significance. The proportion of variance explained by the model tested was R 2 =0.3928, with the adjusted R 2 equal to 0.2106. The statistical model derived using the coefficients was: Weight reduction = 0.0028 (LDL-C) -0.029(TC) -0.053 (EAT-26) +0.041(HDL-C) The one-way repeated measures ANOVA (RANOVA) test carried out between the participants' baseline weight and the weight at the end of the study also showed that there was a significant effect of counselling using the PILs with the lifestyle modification mnemonic ABC, leading to mean weight reduction from 97.2±16.4kg to 95.6±14.2kg in the study participants [F-statistic (2,128), p<0.05].
This study showed that 50 per cent of the participants were able to meet the recommended weight reduction target, with the majority seen in the participants less than 40 years, and this was corroborated by the report by Atlee et al., in 2017. There was a female preponderance observed among those who were able to achieve target weight reduction similar to a study by Andreyeva et al., 42 with most weight reduction in unmarried participants which was contradictory to an exploratory study with a cross-sectional study design, in which more married participants reduced in weight. Those participants who had greater than or equal secondary school education in this study reached the target for weight reduction. This observation was not supported by the report by Barbering et al., in 2018, in which it was found that education was not related to trying to reduce weight. Majority of those with blue-collar jobs achieved target weight reduction, which differed from a report in which weight reduction was higher in public sector workers. Those who had an income less than 18,000 nairas ($ 51.4-the minimum standard wage as of the period of this study) were able to achieve target weight reduction from an article that individuals with less income could not access weight control methods as well as others. Most of the women who achieved target weight were above 45 years, and the majority of the participants who were able to achieve target weight reduction had a positive family history of obesity in mostly siblings, which was corroborated by a study in which there was a greater weight loss in those with a positive history. A few participants reached the target weight and were informed by health workers, corroborating a report. In this study, those who achieved the target weight reduction had low support. This contradicts the study in which social support groups give improved weight outcomes. Half of the respondents with minimal or mild depression were able to achieve target weight reduction, which was supported by authors who suggested an advantage in treating depression, especially in those who were single. More respondents with normal self-esteem reached the target weight reduction, which agreed with the study in which high levels of confidence and self-esteem affected weight loss. There was a lower level of concern observed in participants with target weight reduction from the EAT-26, supported by reports that documented that one of the barriers to weight reduction was abnormal eating, such as binge eating. Many participants who lost above 500 kilocalories could have more target weight reduction than those who lost less than 500 kilocalories. The mean change in calorie intake was -257.4 kilocalories. Therefore, a reduction in calorie intake was achieved and was supported by a document. Interestingly, in this study, both calorie reducers and calorie gainers were found to achieve weight reduction, which was corroborated by authors who opined that with weight reduction came a reduction in Leptin level and invariably a positive energy balance. Thus, food intake could exceed energy expenditure in those trying to reduce weight. Most of the participants with obesity (88.1%) were documented to ingest a high-calorie diet (p>0.05) in the hospital-based study in South Western Nigeria. Calorie loss, on its own, may not lead to weight reduction and should be combined with exercise as it has been documented that the body will compensate once there is calorie reduction and cause physical activity to decline. Though some participants who reduced in target weight at the end of the study were observed to be more physically active, even in those who were inactive or sedentary, a larger proportion reduced in target weight. A report supported this finding of weight reduction with increased activity.43 In contrast, these bidirectional findings between physical activity, sedentary activity, and obesity outcomes were corroborated by documentation of an inverse association between sedentary activity and abdominal obesity in females. More respondents who did not consume alcohol and participants who did not take cigarettes were able to reach the target weight reduction, which was not in sync with previous reports. , WC helps in predicting health outcomes in both men and women , and more respondents were able to achieve target weight reduction with WC reduction. More of the participants with dyslipidemia, which aligned with a report which documented that dyslipidemia in participants with obesity was 40.7% were able to reduce in target weight. Following multiple linear regression analyses, the statistically significant predictors were TC (p=0.01) and LDL-C (p=0.03). HDL-C (p=0.06) and EAT-26 score (p=0.08) were independent determinants of weight reduction which showed a potential for significance. Counselling with PILs incorporating the lifestyle mnemonic ABC demonstrated a significant effect on the mean weight reduction from the first visit to the last visit.
Counselling with Patient Information Leaflets on the ABC mnemonic for lifestyle modification is effective in weight reduction. The public health implication of this study is that weight reduction could be achieved by enhancing eating attitudes and disclosing the fasting lipid profile of the participants. A statistical model based on the Fasting Lipid Profile for predicting weight reduction among patients with obesity was successfully developed and could be applied in managing obesity among patients. Future studies may include the measurement of Leptin levels in participants.
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Preliminary antifibrotic and vasoconstrictor effects of adrenaline in Schlemm’s canal and suprachoroidal minimally invasive glaucoma surgery in primary open-angle glaucoma | 30fc3ed4-54da-4ba8-8317-5c53c92a4ab1 | 11868348 | Surgical Procedures, Operative[mh] | Glaucoma is the leading cause of irreversible blindness and currently affects 70 million people worldwide . Nowadays, intraocular pressure (IOP) is no longer the only modifiable risk factor in the treatment of glaucoma. Numerous other risk factors can be treated, such as diabetes mellitus, arterial hypertension, and dyslipidemia, all of which have an unfavourable impact on primary open-angle glaucoma (POAG) neurodegeneration . Conventional glaucoma filtration surgery, such as trabeculectomy and glaucoma drainage implant surgery, has been considered the gold standard in glaucoma surgical treatment . Despite exhibiting efficacy at IOP reduction, these incisional surgeries are associated with potential postoperative blinding complications, such as hypotony, long-term risk of endophthalmitis and suprachoroidal haemorrhage . In order to provide a less invasive and safer method for reducing IOP, newer surgical techniques, called minimally invasive glaucoma surgery (MIGS), have been developed and have found their place in the glaucoma treatment paradigm in the last two decades. MIGS refers to a group of IOP-lowering surgical interventions that enhance aqueous humour outflow through Schlemm's canal, the suprachoroidal space or the subconjunctival space . Due to the small device geometry, which reduces trauma to ocular tissues and shortens operation time, MIGS offers enhanced safety profile, predictability, and minimal conjunctival manipulation . MIGS can also be combined with phacoemulsification surgery in patients with both cataract and glaucoma. There are multiple approaches to reduce IOP using MIGS devices. iStent® (Glaukos, Avedro) was the first approved ab interno MIGS implant for use in open-angle glaucoma . It reduces IOP by increasing aqueous outflow from trabecular meshwork and Schlemm’s canal stents . MINIject® (iSTAR Medical SA, Wavre, Belgium) is a new implant developed to target the suprachoroidal space . Its unique flexible design conforms to the shape of the eye and the micropores promote aqueous outflow through the device . Although the evidence supports a good safety profile of using MIGS devices in patients, fibrosis around the implants in the trabecular meshwork (TM) is the main cause of surgical failure in MIGS . The significant postoperative fibrotic tissue formation due to cell proliferation and adhesion limits the success of newly created outflow routes, and makes several new micro-incision devices fail to receive market approval for clinical use . Currently, mitomycin-C (MMC) and 5-fluorouracil (5-FU) are commonly used as non-specific antifibrotic drugs after trabeculectomy. MMC inhibits fibroblast proliferation and 5-FU interferes with cell growth . However, they also carry the risk of severe tissue damage, corneal decompensation and infections , and are therefore too toxic to be used inside the eye in MIGS. Therefore, novel and non-toxic antifibrotic therapies are needed to enhance the long-term effectiveness of MIGS devices. Adrenaline, also known as epinephrine, is endogenously produced by the adrenal glands and has been shown to exhibit distinct antifibrotic effects in glaucoma surgery . We recently investigated the antifibrotic effect of adrenaline by RNA-Sequencing technology and evaluated its impact on fibroblast contractility both in vitro and in three subconjunctival glaucoma surgeries (trabeculectomy, PreserFlo Microshunt, Baerveldt 350 tube surgery). Our results showed that adrenaline substantially decreased conjunctival fibroblast contractility without significant cytotoxicity even at high concentrations of 0.05%, and demonstrated that adrenaline may confer antifibrotic attributes in a concentration-dependent manner by affecting the expression of key cell cycle genes . Given the promising effects in subconjunctival glaucoma surgery, the aim of this study was to investigate the potential benefits of adrenaline on Schlemm's canal and suprachoroidal MIGS devices. By probing the potential antifibrotic effects of adrenaline in these specific locations, this research endeavours to shed light on new strategies to increase the long-term success rates of Schlemm’s canal and suprachoroidal MIGS devices.
Cell culture Collagen contraction assay Light microscopy observation Cell viability assay Real-time quantitative PCRIntracameral injection of adrenaline during MIGS implantation Clinical examination Statistical analyses SV40-immortalized (NTM5) human TM cells were used in this study, and have been characterised and shown to be representative for TM cell studies . Technical replicates were also used in all assays. Human TM cells were cultured in an incubator at 37 °C with 5% CO 2 and 95% humidity. The media for cell culture consisted of Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, ThermoScientific, UK), 10% fetal calf serum (Gibco, ThermoScientific, UK), 100 units/mL penicillin and 0.1 mg/mL streptomycin (Sigma Aldrich, Gillingham, UK). All experiments were conducted in accordance with the Declaration of Helsinki and approved by the West of Scotland Research Ethics Committee (REC 19/WS/0146).
A suspension containing 2 × 10 5 TM cells was centrifuged at 1500 rpm for 5 min. The supernatant was discarded, and the cell pellet was resuspended in 100 µL of fetal calf serum. The collagen gel solution was prepared by 1 mL of Type 1 collagen (Fist Link, Wolverhampton, UK) and 160 µL of concentrated media, which consisted of 1.4 mL of DMEM 10 × (Sigma Aldrich, Gillingham, UK), 140 µL of L-glutamine (ThermoScientific, Loughborough, UK), and 360 µL of 7.5% sodium bicarbonate (Sigma Aldrich, Gillingham, UK). The pH was adjusted to 7.0 by sodium hydroxide before the cells were mixed with the collagen solution. 150 µL of the cell-gel mixture was placed in each MatTek dish and allowed to set for 10 min at 37 °C. The gels were treated with 1.5 mL of different concentrations of adrenaline (0%, 0.0005%, 0.01%) after carefully releasing the gels. The cells in the gels were observed using an Olympus CKX41 inverted microscope, and the gel photos were taken daily over 7 days and analysed using the ImageJ software. The percentage of matrix contractionContraction\;=[] 100$$ C o n t r a c t i o n = A r e a o f g e l a t D a y 0 - A r e a o f g e l a t D a y n A r e a o f g e l a t D a y 0 × 100
Human TM cells were seeded at a density of 1 × 10 5 cells per well in 6-well plates. After 1-day treatment, cells were imaged using an Olympus CKX41 inverted microscope with Olympus CellSens Standard 1.13 software.
Human TM cells were seeded at a density of 6.25 × 10 3 cells per well in a 96-well plate and treated with different concentrations of adrenaline (0%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%) for 1 day. The drug solutions in the 96-well plate were then replaced by 100 µL of fresh media, followed by the addition of 20 µL of the Cell Titer 96 Aqueous one solution (Promega, Southampton, UK). The plate was incubated for 2 h at 37 °C and the absorbance was measured at 490 nm using a PHERAstar FS instrument (BMG Labtech, Aylesbury, UK). The cell viability was calculated as a percentage of the value of untreated (0%) cells.5%, 0.01%) for 1 day. The total RNA was extracted using a RNeasy mini kit (Qiagen, Crawley, UK) and the cDNA was synthesised using a high-capacity cDNA reverse transcription kit (ThermoScientific, Loughborough, UK). RT-qPCR was performed using the QuantiFast SYBR Green PCR kit (Qiagen, Crawley, UK) on a QuantStudio 7 Real-time PCR system. The reaction settings for 40 cycles were as follows: Holding stage: 50 °C for 2 min and 95 °C for 5 min; PCR stage: 95 °C for 5 min and 60 °C for 30 s. The sequences for the forward and reverse primers are presented in Table . The relative gene expressRelative\;Gene\;Expression={2}^{-( {Ct}_{target}- {Ct}_{GAPDH})}$$ R e l a t i v e G e n e E x p r e s s i o n = 2 - ( Δ Ct target - Δ Ct GAPDH )
We tested the effects of adrenaline 0.05% intraoperatively in five patients with POAG undergoing a MIGS device combined with phacoemulsification. Two MIGS devices were included in this study: iStent inject® (Glaukos, Avedro) as a Schlemm’s canal MIGS and MINIject® (iSTAR Medical SA, Wavre, Belgium) as a suprachoroidal MIGS. Informed consent was obtained from all patients. Phacoemulsification surgery was performed as per standard practice. Miochol was injected intracamerally at the end of phacoemulsification to constrict the pupil in preparation for MIGS implantation. All patients then received 0.1 mL of intracameral injection of adrenaline 0.05% before the anterior chamber was filled with viscoelastic and the MIGS device was inserted.
Clinical data, including best-corrected visual acuity (BCVA), IOP, central corneal thickness (CCT), cup-to-disc ratio (C/D), previous glaucoma surgery, previous glaucoma laser, lens status and anti-glaucoma medications were recorded preoperatively and postoperatively in all patients. Blood pressure, heart rate, and blood oxygen saturation were also measured preoperatively, intraoperatively, and postoperatively. Pupil diameter was measured using a measuring scale before and after surgery. To record the positioning of the MIGS, the intraoperative insertion of the implant was captured by video recording during surgery. Postoperatively, gonioscopy was performed in clinic and photographs of the implant in the angle were taken by slit lamp camera (ARC slit lamp imaging camera, Carleton optical, Chesham, Buckinghamshire, UK). We also performed angle scans with an anterior segment OCT (ANTERION® Heidelberg Engineering, Germany) to visualise the implant transversally.
GraphPad Prism was used for graph generation and statistical analysis. All graphs display mean and standard error of the mean (SEM). To determine statistical significance, a one-way ANOVA was performed and P values were calculated. P values, which were statistically significant, were expressed as follows: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Adrenaline significantly decreased matrix contraction in human TM cells Adrenaline did not affect cell viability and cell morphology in human TM cells Adrenaline significantly decreased the expression of key cell cycle genes in human TM cells Intracameral adrenaline injection did not cause any ocular adverse effects during and after MIGS implantation Intracameral adrenaline injection did not significantly affect blood pressure, heart rate, oxygen saturation and pupil size during MIGS implantation All patients had stable blood pressures, heart rates, and oxygen saturations preoperatively, during surgery and postoperatively. Before surgery, the MINIject patients (patients 1, 2, 3) showed a blood pressure of 142/85, 124/84 and 176/80 mmHg, respectively. Their preoperative heart rate was 73, 90 and 72 beats per minute, and their oxygen saturation was 98%, 96% and 99%, respectively. The iStent inject patients (patients 4 and 5) had a preoperative blood pressure of 185/105 and 179/94 mmHg. Their heart rate was 93 and 77 beats per minute, and their oxygen saturation was 96% and 99%, respectively. During surgery, the MINIject patients (patients 1, 2, 3) showed a blood pressure of 143/74, 154/71 and 176/76 mmHg, respectively. Intraoperatively, their heart rate was 60, 93 and 70 beats per minute, and their oxygen saturation was 100%, 97% and 97%, respectively. For the patients who received iStent inject (patients 4 and 5), the blood pressure was 185/105 and 179/94 mmHg during surgery. Intraoperatively, their heart rate was 84 and 59 beats per minute, and their oxygen saturation was 96% and 99%, respectively. After surgery, the MINIject patients (patients 1, 2, 3) showed a blood pressure of 140/73, 154/88 and 176/76 mmHg, respectively. Postoperatively, their heart rate was 60, 93 and 66 beats per minute for the same patients, respectively. Their oxygen saturation after surgery was 98%, 97% and 96%, respectively. For patients who received iStent inject (patients 4 and 5), the blood pressure was 185/100 and 179/94 mmHg after surgery. Postoperatively, their heart rate was 95 and 89 beats per minute, and their oxygen saturation was 96% and 95%, respectively. All patients received intracameral miochol as standard preoperative drug before MIGS implantation. There was no significant change in pupil diameter observed intraoperatively and postoperatively after intracameral adrenaline 0.05% injection, remaining between 6 and 7 mm in diameter in all patients.
A three-dimensional cell-populated collagen contraction assay was carried out to assess the contractility of TM cells after adrenaline treatment, as it has been shown to be a good in vitro model and functional assay to study tissue contraction in the eye . Increasing concentrations of adrenaline decreased the cell proliferation and cell density of TM cells in gels at both day 4 and day 7 (Fig. a). Figure b shows representative gel areas at day 4 and day 7. A dose-dependent decrease in collagen contraction was observed with increasing concentrations of adrenaline throughout the 7 days (Fig. c). At day 4, the TM cell-populated collagen gels treated with 0%, 0.0005%, and 0.01% adrenaline contracted 50.6%, 32.2%, and 5.7%, respectively, while at day 7, they contracted 87.8%, 80.6%, and 7.9%, respectively. The differences in collagen contraction were statistically significant for both 0.0005% adrenaline ( P < 0.05 on day 2, P < 0.0001 on days 3–5, P < 0.001 on days 6–7) and 0.01% adrenaline ( P < 0.05 on day 1, P < 0.01 on day 2, P < 0.0001 on days 3–7), when compared to no drug control 0%.
The potential toxicity of adrenaline on TM cells was assessed by light microscopy observation and a cell viability assay. After 1-day treatment, the cell viability of adrenaline 0%, 0.0001%, 0.0005%, 0.001%, 0.005%, and 0.01% was 100.0%, 108.0%, 99.3%, 97.9%, 92.0%, and 100.7%, respectively. The TM cells on the 6-well plates did not display noticeable changes in cell morphology (Fig. a). All adrenaline concentrations (0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%) did not exhibit a significant decrease in cell viability compared to no drug control 0% ( P > 0.05) (Fig. b).
We tested the effects of adrenaline on the expression of key cell cycle genes in human TM cells to investigate whether adrenaline would have similar effects as previously shown in human Tenon’s fibroblasts . The expression of key cell cycle and fibrosis genes was measured by real-time qPCR after adrenaline treatment (Fig. ). In the G2 phase, ASPM ( P < 0.05), CDCA3 ( P < 0.01), CDCA8 ( P < 0.01), DBF4 ( P < 0.001), and MKI67 ( P < 0.05. The expression of DBF4 gene also showed a significant decrease with 0.0005% adrenaline ( P < 0.01). In the M phase, PLK1 ( P < 0.01), CCNB1 ( P < 0.001), CDC20 ( P < 0.01), and PRR11 ( P < 0.0001) genes were significantly downregulated after 0.01% adrenaline treatment when compared to no drug control 0%, but no significant differences were observed in the 0.0005% adrenaline group. In addition, MRTFB showed a significantly reduced gene expression with 0.01% adrenaline ( P < 0.05), and ACTA2 showed a decreased gene expression with both 0.01% and 0.0005% adrenaline ( P < 0.001).
We next validated our results by injecting adrenaline 0.05% intracamerally in patients with POAG receiving a Schlemm’s canal MIGS (iStent inject) or suprachoroidal MIGS (MINIject), combined with phacoemulsification surgery. Demographically, we gathered data from different age and ethnic groups, which were evenly distributed between both MIGS groups (Table ). All patients in the study were using multiple topical antiglaucoma medications, but none were on oral acetazolamide tablets. Adrenaline exhibited vasoconstrictive effects and significantly reduced intraoperative bleeding in both iStent inject and MINIject patients (Fig. a). None of the patients experienced any intraoperative or postoperative adverse effects due to the intracameral adrenaline 0.05% injection. The MINIject patients (patients 1, 2, 3) had preoperative BCVA (logMAR) of 0.0, 0.6 and 0.1, respectively. For these patients, preoperative IOP was 17, 14 and 14 mmHg, respectively, on at least two anti-glaucoma drops. CCT was found to be 513, 484 and 593 µm at baseline for these patients, and their C/D ratio was 0.8, 0.7 and 0.7, respectively (Table ). One week postoperatively, BCVA was 0.2, 0.6 and 0.0 for patients 1, 2 and 3, respectively. Postoperative IOP was 9, 15 and 16 mmHg on no antiglaucoma medications. The implants were well positioned on gonioscopy (Fig. b) and the surrounding trabecular meshwork tissues were healthy (Fig. c). The iStent inject patients (patients 4 and 5) had preoperative BCVA (logMAR) of 0.5 and 0.2, respectively. For these patients, preoperative IOP was 16 and 27 mmHg, respectively, on at least two anti-glaucoma drops. Their CCT was 546 and 583 µm at baseline, and their C/D ratio was 0.9 and 0.8, respectively (Table ). One week postoperatively, BCVA was 0.5 and 0.2, and IOP was 16 and 14 mmHg, respectively, on no antiglaucoma medications. The implants were correctly positioned (Fig. b) and the surrounding trabecular meshwork tissues were also healthy (Fig. c).
Fibrosis is the most important cause of failure after MIGS. Although the antimetabolites MMC and 5-FU are commonly used to modulate wound healing after subconjunctival glaucoma filtration surgery, they are too toxic to be used intraocularly in MIGS due to the severe sight-threatening side effects, such as hypotonous maculopathy , corneal melting and perforation , and scleral calcification . Therefore, there is a large unmet clinical need to develop an alternative and non-toxic antifibrotic drug that can be used in MIGS. Adrenaline is a safe, cost-effective, and widely available drug in ophthalmic surgery, and has been shown to have a beneficial antifibrotic effect in human Tenon’s fibroblasts . Severe fibrosis of the TM tissue can lead to sustained extracellular matrix accumulation and distortion of the TM framework, resulting in increased resistance to aqueous humour outflow and elevated IOP . It also has an adverse impact on the implantation and is the primary cause of MIGS failure . Our in vitro results indicate that adrenaline also exhibits concentration-dependent antifibrotic effects in human TM cells. Adrenaline 0.0005% and 0.01% significantly decreased the contractility of TM cells by 7.2% and 79.8%, respectively, after a 7-day contraction assay. Meanwhile, no apparent cytotoxicity was observed in TM cells after 1-day treatment with different concentrations of adrenaline (0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%). These findings highlight the potential application of adrenaline in MIGS for its antifibrotic and vasoconstrictor effects, thereby enhancing the surgical success rates of MIGS. Our previous RNA-Sequencing results provide compelling evidence that high concentrations of adrenaline have an impact on cell cycle genes in human Tenon’s fibroblasts . We further examined the expression level of key cell cycle genes and fibrosis-related genes in TM cells after adrenaline treatment. In the G2 phase, ASPM , CDCA3 , CDCA8 , DBF4 , and MKI67 genes were significantly downregulated by adrenaline 0.01%. ASPM is involved in the formation of the mitotic spindle . CDCA3 is a crucial regulator of cell cycle progression from the G2 phase to mitosis . CDCA8 is also a component of the chromosomal passenger complex and is important in mitosis . DBF4 plays a vital role in DNA replication , while MKI67 is closely related to cell proliferation . In the M phase, adrenaline 0.01% significantly decreased the gene expression of PLK1 , CCNB1 , CDC20 , PRR11 . PLK1 is a significant target of the DNA damage checkpoint, allowing cell-cycle arrests at multiple points in the G2 phase and mitosis . CCNB1 is essential for the proper control of the G2/M transition phase. CDC20 also plays a vital role in both nuclear movement prior to anaphase and chromosome separation . PRR11 plays a pivotal role in the accurate regulation from the late S phase to mitosis . The downregulation of these genes indicates that adrenaline inhibits cell cycle progression and suppresses the proliferation of TM cells, causing them to enter a growth arrest phase without undergoing cell death. Moreover, adrenaline 0.01% significantly reduced the expression of MRTFB and ACTA2 genes, while ACTA2 also exhibited decreased level after treatment with adrenaline 0.0005%. MRTFB is one of the major regulators of cytoskeletal gene expressions and is essential for TGF-β-induced fibrosis . ACTA2 is an important downstream gene of the MRTFB/SRF pathway and encodes α-SMA, which plays an important role in myofibroblast differentiation . In this study, the lower expression of MRTFB and ACTA2 genes after adrenaline treatment was consistent with the decreased matrix contraction, indicating a decrease in fibrosis development in TM cells. To validate the feasibility of adrenaline application in MIGS, we further examined the safety and efficacy of adrenaline in POAG patients undergoing Schlemm’s canal MIGS (iStent inject) or suprachoroidal MIGS (MINIject), when combined with phacoemulsification surgery. Different concentrations of adrenaline have been applied in the clinical setting. Adrenaline can be injected into the anterior chamber or added in the infusion solution during cataract surgery , in order to achieve adequate and sustained pupillary dilation. When used in combination with atropine, adrenaline is also effective in the management of intraoperative floppy-iris syndrome due to a powerful synergistic effect on iris dilation. Adrenaline 0.1% exhibits a good safety profile with no impact on blood pressure and heart rate , and adrenaline 0.02% does not increase the risk of postoperative macular oedema . Based on these previous studies, we selected an adrenaline concentration of 0.05% for intracameral injection (0.1 mL) in patients just prior to MIGS implantation in this study. None of the patients experienced any intraoperative or postoperative adverse reactions. Patients receiving these two MIGS devices all exhibited stable blood pressures, heart rates, and oxygen saturations during and after surgery, demonstrating a good safety profile of intracameral adrenaline 0.05%. Patient 2 showed a 30 mmHg increase in blood pressure from preoperative stage to intraoperative stage, which was related to the patient being very anxious. Although adrenaline may induce adverse cardiovascular effects, its systemic absorption is limited , likely due to the presence of the blood-retinal barrier. Nonetheless, it remains contraindicated in patients with hypertension, heart disease, arrhythmias, and narrow angle glaucoma, and the use of adrenaline in these patients requires thorough patient assessment and careful patient selection . The study had a few limitations. While transformed TM cells were used in this study, they may differ from primary TM cells in specific responses. Thus, it would be valuable to include primary TM cells in future in vitro studies. Although the data were carefully collected and analysed, this study was a proof-of-concept study and the sample size of patients was relatively small. With a known genetic variation in the alpha1B-adrenergic receptor , ethnic differences in adrenaline sensitivity also require further consideration. Additionally, the findings presented are specific to POAG patients and two MIGS devices (iStent inject and MINIject), which may not be directly transferable to other MIGS devices. Including individuals of different ethnicities and patients with other types of glaucoma, as well as incorporating a broader range of MIGS devices available on the market, could provide a more comprehensive evaluation for the use of adrenaline in MIGS in the future. Furthermore, this is a short-term study and the early incidence of stent failure is lower than the later failure rate. Currently, it is unknown whether adrenaline affects the cell cycle of other eye cells, such as potentially blocking any stem cell repopulation effects. Further research is needed to determine if this effect could lead to adverse consequences. It also remains unclear whether adrenaline is associated with a better reduction in IOP over time, which requires longer prospective studies. This study is focused on the early fibrotic responses, and moving beyond short-term effectiveness will be crucial to examine longer-term impacts. Given that endothelin-1 (ET-1) is a vasoconstrictor produced by endothelial cells, and that adrenaline can enhance its secretion, measuring ET-1 levels in the aqueous humour might help further understand the mechanism of adrenaline’s vasoconstrictive effects and its impact on aqueous humour outflow in the future. In conclusion, this study showed that adrenaline reduced the contractility of TM cells in a dose-dependent manner, and suppressed the expression of key cell cycle and fibrosis genes with no significant cytotoxicity. The intracameral injection of adrenaline 0.05% in patients undergoing MIGS implantation also demonstrated a good safety profile during and after surgery. Unless contraindicated, we therefore recommend intracameral injections of adrenaline 0.05% as a cheap and safe drug to be used just before MIGS insertion, as it decreases the risk of bleeding from the trabecular meshwork and also exhibits antifibrotic effects by arresting the cell cycle, thereby increasing the postoperative success rates in MIGS.
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Comparison of the effect of SMS-based education and face-to-face teaching on self-care behavior of type 2 diabetic patients in Khorramabad city | a75d045a-e4a7-43a2-9f9b-b0111b7cae96 | 11529012 | Patient Education as Topic[mh] | Diabetes is a chronic and progressive endocrine disease that causes many complications and is one of the most common diseases in the world and Iran . This disease is characterized by three abnormalities: pathological discomfort, impaired insulin secretion, environmental insulin resistance, and excessive hepatic glucose production . Diabetes is the main cause of disabilities such as limb amputation, advanced kidney diseases, and blindness in the active age of life, as well as mortality , and is usually associated with symptoms such as weight loss, excessive drinking, and polyuria . According to the report of the International Diabetes Federation in 2019, diabetes is increasing dramatically and about 463 million adults among the age range with of 20 to 79 have experienced living with this disorder . One of the types of diabetes is type 2 diabetes, which includes impaired glucose homeostasis, impaired insulin secretion and function, and insulin resistance . This type of diabetes, which includes 90 to 95% of all types of diabetes, usually occurs in adulthood. The occurrence of type 2 diabetes has greater than before in the past three decades, with nearly 422 million persons worldwide living with diabetes, the majority of them live in low- and middle-income countries, and about 1.5 million deaths per year are directly related to diabetes . More than 3 million people with diabetes live in Iran . By 2030, Iran will be one of the most prevalent regions in the world in terms of diabetes . Paying attention to the chronic and costly nature of this disease for the general health of the society and the creation of a large financial burden, it is essential to pay serious attention to this problem and its consequences . As shown by research findings, if blood sugar is controlled, it is possible to prevent the long-term complications of this disease, such as neuropathy, nephropathy, and retinopathy or at least they can be delayed . According to WHO, the basis of diabetes treatment is education? One of the primary objectives of diabetes education is to change the patient’s behaviour and improve self-care. Self-care in humans is a developed regulatory mechanism that relies on individuals’ capacity to undertake caregiving tasks independently , and includes the behaviours of following a proper diet, taking medicine on time, self-monitoring of blood sugar, performing regular physical activity, and physical care of the feet . Self-efficacy is also known as a strategy for improving self-care in diabetic patients, which causes self-confidence and adherence to care recommendations in order to control blood sugar in these patients . Studies conducted concerning the self-care behaviours in patients with type 2 diabetic indicate that educational interventions directly and indirectly have been effective in improving the self-care behaviours of these patients . On the other hand, nowadays, new methods of teaching patients, especially diabetic patients, have been developed and proposed, and in this regard, mobile-based educational interventions, as a relatively novel method, It can redirect the emphasis of care and treatment from clinical settings to the patient’s everyday life, where behaviour and attitude changes occur . The use of mobile phones is effective in providing health services and managing chronic diseases . Therefore, considering the importance of self-care in diabetes and the determining role of educational interventions in this regard, using face-to-face and non-face-to-face self-care training, the present study was carried out aiming to compare the impact of SMS-based teaching and face-to-face teaching on self-care behaviours of patients with type 2 diabetic in Khorramabad city.
The present study was a semi-experimental study with pre-test and post-test intervention. The statistical population included all patients with type 2 diabetes referred to the diabetes clinic of Khorram Abad city, Lorestan province who 135 of the eligible patients were selected by simple random sampling method and then by simple random allocation into three SMS-based training groups, the face-to-face training group and the control group were divided. In this research, the sample size was determined from the following relationship, taking into account 10% possible dropout and in order to increase confidence in the results, 45 people in each group and a total of 135 people. [12pt]{minimal}
$$n = } + {Z_{ / }}} )}^ }}}{{{{( )}^ }}}$$ The research inclusion criteria included informed consent for participation in the study, having a minimum level of literacy and understanding the content sent via SMS, having a fixed personal mobile number to receive educational SMS, having the free time for face-to-face teaching and group discussion, not suffering from serious complications of diabetes, and not having special diets due to special diseases or physical problems that limit physical activity. Exclusion criteria included suffering from serious complications of diabetes during the study, suffering from diseases that limit physical activity or nutrition, and unwillingness of the patient to receive educational messages, not participating in more than one training session, and not completing questionnaires completely. For data collection, the Tubert self-care behaviour questionnaire, and self-efficacy questionnaire and demographic information form were used in the current study. Demographic information included age, height, weight, gender, education, employment status, marital status, and type of treatment. Tubert’s self-care behaviour questionnaire, which includes 15 items in the areas of diet compliance, physical activity, foot care, and smoking, was translated and standardized by Ahmadzadeh . Self-efficacy questionnaire is one of the constructs of the standard health belief model questionnaire and its validity and reliability have been confirmed . Cronbach’s alpha for self-care and self-efficacy scale is 0.78 and 0.76 respectively. The research started after submitting the letter of introduction from Iran University of Medical Sciences (IR.IUMS.REC.1397.501). Four days before the start of the intervention, the blood sugar samples of the participants in all groups were taken. In the first session, the investigator explained the aim of the sessions to the participants and informed written consent was obtained. Then, the personal information form and questionnaires were completed by the two case groups and the control group. The training program in the test group, which received self-care training sessions in person, included four 40-minute sessions, so that every week, one training session with lecture, discussion and discussion methods was held in a suitable and comfortable room at Khorramabad Diabetes Clinic. All 45 people of the face-to-face training group were present in all the classes. During the training sessions, the disease of diabetes, its complications, and ways to prevent its progress were explained, and then sports activities, the way of controlling blood sugar, proper diet, the way of using prescribed medicines, including the hours of oral medicines and how to Insulin injections, and self-care trainings were explained to patients. In the second case group, which received self-care training in the form of text messages, the training program was presented in the form of short educational messages via mobile phone, during four weeks and on average, at least three messages per day. For example, one of the short messages about physical fitness and blood sugar monitoring was (for diabetics, walking is the best exercise, or it is better for diabetics to measure their fasting blood sugar after waking up). In order to improve self-efficacy, encouraging messages about adherence to self-care were used. During the intervention, the control group did not receive any educational intervention. However, in order to comply with the ethical principles, an educational CD containing self-care training was given to the control group after the completion of the research. Finally, after two months, the questionnaires were again distributed among the participants and were completed, and the blood sugar levels of the three groups were measured again. The data of the three groups before and after the intervention were entered into SPSS software version 24 and were analysed using Chi-square, Mann-Whitney U and Wilcoxon tests at a significance level of 0.05.
The average age of participants was 51.01 ± 0.967, and the average height and weight were 164.57 ± 1.031 and 75.69 ± 1.101, respectively. At the baseline, the face-to-face teaching, SMS-based teaching, and control groups were matched in terms of demographic variables, including age, height, weight, gender, education, employment status, marital status, and type of treatment, and no statistically significant difference were seen between the groups (Table ). Table indicates that at the baseline, average score of self-efficacy and self-care, and blood sugar were not significant between the face-to-face teaching, SMS-based and control groups. But at the end of the intervention, the average score of self-efficacy and self-care in the face-to-face and SMS-based group was significantly higher than the control group ( p -value < 0.001) and the mean blood sugar score in the face-to-face and SMS-based group is lower compared to the control group, which is a statistical difference it shows significance. The average scores of self-care and self-efficacy after the educational intervention in the SMS-based teaching group were higher than the face-to-face case group, but it does not show a significant difference.
The finding of the current study indicated a significant increase in the average self-care score in the two test groups after the intervention, and the SMS-based and face-to-face educational interventions caused a decrease in the average blood sugar score in the two test groups, while in the control group, the average blood sugar score compared to It does not show a statistically significant difference before the intervention. This means that both training methods (face-to-face and mobile SMS) have been effective in improving self-care and reducing blood sugar levels. Although the impact of SMS-based training was greater than face-to-face training in the two test groups, there was no statistically significant difference. The results of this study are consistent with the findings of the study by Khoshnudifar et al. . entitled “Investigation of the effect of combined training in comparison with face-to-face training on the self-care behaviours of patients with type 2 diabetes”, and the study by Haghighinejad et al. , on the comparison of SMS-based training and group training in diabetes self-management. The studies of Abaza et al. and Agbede et al. on the effect of SMS-based intervention on self-care of diabetic patients and the studies by Yari et al. and Rahnavard et al. on the SMS-based education and self-care-based education diabetic patients show that SMS-based education improves self-care in diabetic patients. Regardless of the method of implementation and the type of educational intervention, it seems that self-care of the disease is important for all groups of diabetic patients. However, the effect of intervention and type of training cannot be ignored. Choosing the appropriate educational method can help control the disease, reduce its complications, and increase people’s ability to take care of themselves and make correct decisions about the disease. Finally, it can increase the quality of life of patients with diabetes . Technologies such as mobile phones and text messages, which are currently part of people’s daily lives, have the potential to influence self-care and improve people’s health . The mobile phone short message service provides the possibility of immediate and direct sending of a short message to people at any time and place. These messages are asynchronous, which means that they can be seen and read at any time that is convenient for the recipient of the message . Consequently, it has attracted the attention of the researchers to develop studies on the use of this service for improving the quality of care in diabetic patients . The other finding of this study is the increase in the average score of self-efficacy in two groups of SMS-based and face-to-face teaching after the educational intervention, which indicates a statistically significant difference. Besides, Katz and Nordwall and Peymani et al. , found that interventions based on mobile phone messages improve the self-efficacy of diabetic patients. Research indicates that individuals with high self-efficacy are more inclined to engage in challenging behaviours, possess a more favourable interpretation of health-related behaviours, and exhibit greater control over their behaviours . It seems that the availability of mobile phones and sending educational text messages to remind them will promote healthy behaviours, because in this way, the lack of financial resources, the lack of physical space, and the difficulty of accessing different groups of society are eliminated. Consequently, the SMS system, with the ability to send information at the desired time, easy to use and not having problems related to face-to-face training, including training space and financial resources, can be used instead of face-to-face training in order to improve the self-care behaviour of diabetic patients. Limitation One of the limitations of the self-report study was that participants were assured of confidentiality. The small size of the sample and lack of measuring the long-term stability of the results were other limitations of this research.
The educational interventions based on face-to-face teaching and teaching through mobile phone short messages in the field of self-care was not significant. Therefore, it is suggested to use the SMS system in educating patients regarding self-care, disease management, and control of its complications, regardless of the costs, time and place limitations of face-to-face training and the emergence of new diseases such as Covid-19 requiring keeping a distance.
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Curriculum Klinische Akut- und Notfallmedizin – Schwerpunkt Innere Medizin | cfd7f158-ffb2-4977-ab94-b25f5e2b2e0d | 11098871 | Internal Medicine[mh] | Der Begriff „Notfallmedizin“ wird in Deutschland bis heute häufig mit der Tätigkeit im Rettungsdienst assoziiert. Jedoch ist die Notfallmedizin in Deutschland heutzutage weit mehr und umfasst neben der prähospitalen Notfallmedizin – dem Rettungsdienst oder besser der Rettungsmedizin – die sektoren- und gebietsübergreifende klinische Akut- und Notfallmedizin in Notfallzentren, Notaufnahmen und Notfallkliniken. Die Bedeutung der klinischen Akut- und Notfallmedizin im Gesundheitssystem zeigt sich bei der wichtigen Weiterentwicklung der Strukturen in Anlehnung an den Beschluss des gemeinsamen Bundesausschusses (G-BA) zu einem gestuften System von Notfallstrukturen . Der in der Notaufnahme tätige Arzt muss ein breites Spektrum an fächerübergreifendem theoretischem Wissen (Methoden- bzw. kognitive Kompetenzen) und praktischen Fähigkeiten (Handlungskompetenzen) beherrschen. In einigen europäischen Ländern existiert ein „Facharzt für Unfall- und Notfallmedizin“, in anderen ein „Facharzt für Notfallmedizin“. Die europäischen notfallmedizinischen Gesellschaften haben dafür ein Curriculum erstellt, in dem stichwortartig das breite Spektrum der Notfallmedizin symptomen- und befundorientiert dargestellt ist; eine deutsche Curriculumversion hat die Deutsche Gesellschaft Interdisziplinäre Notfall- und Akutmedizin e. V. (DGINA) ausgearbeitet . In Deutschland basiert dagegen die Weiterbildung zur Tätigkeit in der Notfallmedizin auf einer Facharztqualifikation – z. B. Innere Medizin – ergänzt durch die Zusatz-Weiterbildung (Z-WB) „Klinische Akut- und Notfallmedizin“ (erwerbbar nach der Facharztweiterbildung) und die am prähospitalen Rettungsdienst orientierte Z‑WB „Notfallmedizin“ (erwerbbar bereits während der Facharzt-Weiterbildung; siehe 1.3.1. und 1.3.2.). Die erforderlichen Qualifikationen sind in den beiden Z‑WB der Bundesärztekammer (BÄK) stichwortartig aufgeführt. Eine zentrale Säule in der gebietsübergreifenden Versorgung stellt das Gebiet der Inneren Medizin mit seinen Schwerpunkten dar. In großen zentralen Notaufnahmen oder Notfallzentren fällt mindestens die Hälfte aller Patienten entsprechend ihrer Diagnosen in den internistisch-konservativen Bereich . Die Bedeutung der Inneren Medizin in der klinischen Akut- und Notfallmedizin wird aufgrund der demografischen Entwicklung mit einer zunehmenden Zahl betagter Notfallpatienten an Bedeutung weiter zunehmen . Viele Akut- und Notfallpatienten weisen in der Regel mehrere prognoserelevante internistische Begleiterkrankungen auf, die im Rahmen der Behandlung ebenfalls berücksichtigt werden müssen. Die BÄK wird dem Stellenwert der Inneren Medizin in der klinischen Akut- und Notfallmedizin insofern gerecht, als dass die (Muster‑)Weiterbildungsordnung ([M-]WBO) zum Facharzt für Innere Medizin bzw. für Innere Medizin und Schwerpunkt einen gut fundierten, ausführlichen notfall- und intensivmedizinischen Weiterbildungsblock vorsieht, mit einer verpflichtenden 6‑monatigen Tätigkeit unter Anleitung in der klinischen Akut- und Notfallmedizin und einer 6‑monatigen Tätigkeit unter Anleitung in der Intensivmedizin. Allerdings kann der Weiterbildungsblock im Rahmen der internistischen Weiterbildung nicht das gesamte Spektrum der klinischen Akut- und Notfallmedizin abbilden, sodass der Internist, der zukünftig im Bereich der Notfallmedizin tätig sein möchte, im Anschluss an die Facharztqualifikation die bereits genannte Z‑WB „Klinische Akut- und Notfallmedizin“ und ggf. auch schon während seiner Facharztweiterbildung die Z-WB „Notfallmedizin“ zu absolvieren hat (siehe 1.3.1. und 1.3.2). Das vorliegende Curriculum „Klinische Akut- und Notfallmedizin – Schwerpunkt Innere Medizin“ zu Ausbildungsinhalten der Inneren Medizin in der Notaufnahme der Deutschen Gesellschaft für Internistische Intensivmedizinunter Einbeziehung der Deutschen Gesellschaft für Palliativmedizin (DGP) dient einerseits dazu, Empfehlungen zu Ausbildungsinhalten der Inneren Medizin in der Notaufnahme zu geben. Insbesondere dient es aber dazu, die internistischen Inhalte der Zusatz-Weiterbildung „Klinische Akut- und Notfallmedizin“ (siehe 1.3.2.) „mit Leben zu füllen“, die relevanten internistischen akut- und notfallmedizinischen Themen zu benennen, die Lerninhalte zu deklarieren und letztendlich einen umfassenden Überblick über die internistischen Inhalte der Akut- und Notfallmedizin zu geben. Auch die Weiterbildungsinhalte der Z‑WB „Notfallmedizin“ (siehe unter 1.3.1.) werden im Curriculum ausgewiesen und benannt. Dieses Curriculum kann zudem als Leitfaden für den Erwerb der erforderlichen Kenntnisse genutzt werden und es definiert und kategorisiert die notwendigen praktischen Fähigkeiten für eine bestmögliche Versorgung der akut- und notfallmedizinischen Patienten aus internistischer Sicht. Das Curriculum repräsentiert umfassend die Position und das Verständnis der DGIIN, der DGIM samt Schwerpunktgesellschaften sowie des BDI, welche Expertise, Kenntnisse, Fertigkeiten und auch berufsethische Qualitäten künftige Notfallmediziner in der Inneren Medizin besitzen sollen. Die Gliederung des Curriculums in einen allgemeinen und einen schwerpunktspezifischen Teil ermöglicht– bereits in der Notaufnahme – der Behandlung der dem „Problem“ zugrunde liegenden internistischen Erkrankung. Die Autoren erhoffen sich zudem, dass dieses Curriculum auch bei berufspolitischen und standesorganisatorischen Fragestellungen sowie Diskussionen entsprechende Berücksichtigung finden wird. Für die Vorstände der DGIIN, der DGIM und deren Schwerpunktgesellschaften sowie des BDI inkl. DGG und DGP (Palliativmedizin).Prof. Dr. Matthias Kochanek, Präsident der Deutschen Gesellschaft für Internistische Intensivmedizin und Notfallmedizin e. V. (DGIIN) Prof. Dr. Andreas Neubauer, Vorsitzender der Deutschen Gesellschaft für Innere Medizin e. V. (DGIM) Prof. Dr. Wulf Ito, Präsident der Deutschen Gesellschaft für Angiologie – Gesellschaft für Gefäßmedizin e. V. (DGA) Prof. Dr. Günter Stalla, Past-Präsident der Deutschen Gesellschaft für Endokrinologie e. V. (DGE) Prof. Dr. Heiner Wedemeyer, Präsident der Deutschen Gesellschaft für Gastroenterologie, Verdauungs- und Stoffwechselkrankheiten e. V. (DGVS) Prof. Dr. med. univ. Markus Gosch, Präsident der Deutschen Gesellschaft für Geriatrie e. V. (DGG) Prof. Dr. Hermann Einsele, Geschäftsführender Vorsitzender der Deutschen Gesellschaft für Hämatologie und Medizinische Onkologie e.V. (DGHO) Prof. Dr. Bernd Salzberger, Past-Präsident der Deutschen Gesellschaft für Infektiologie e. V. (DGI) Prof. Dr. Holger Thiele, Präsident der Deutschen Gesellschaft für Kardiologie – Herz- und Kreislaufforschung e. V. (DGK) Prof. Dr. Hermann Pavenstädt, Präsident der Deutschen Gesellschaft für Nephrologie e. V. (DGfN) Prof. Dr. Claudia Bausewein, Präsidentin der Deutschen Gesellschaft für Palliativmedizin e. V. (DGP) Prof. Dr. Wolfram Windisch, Präsident der Deutschen Gesellschaft für Pneumologie und Beatmungsmedizin e. V. (DGP) Prof. Dr. Christof Specker, Präsident der Deutschen Gesellschaft für Rheumatologie e. V. (DGRh) Christine Neumann-Grutzeck, Präsidentin des Berufsverbands Deutscher Internistinnen und Internisten e. V. (BDI) 1. Weiterbildung „Klinische Akut- und Notfallmedizin – Schwerpunkt Innere Medizin“ 1.1. Die (Muster‑)Weiterbildungsordnung ([M-]WBO) der Bundesärztekammer 1.2. Klinische Akut- und Notfallmedizin in der (M-)WBO zum Facharzt für Innere Medizin und zum Facharzt für Innere Medizin und Schwerpunkt 1.3. Akut- und notfallmedizinische Zusatz-Weiterbildungen (Z‑WB) der Bundesärztekammer (BÄK) 1.3.1. Zusatz-Weiterbildung (Z‑WB) „Notfallmedizin“ 1.3.2. Zusatz-Weiterbildung (Z‑WB) „Klinische Akut- und Notfallmedizin“ 1.4. Empfehlung zu Weiterbildungsinhalten der Inneren Medizin in der Notaufnahme 2. Adressaten des Curriculums „Klinische Akut- und Notfallmedizin – Schwerpunkt Innere Medizin“ 2.1. Weiterzubildende 2.2. Weiterbilder 2.3. Gremien und Ärztekammern 3. Erforderliche Qualifikationen 3.1. Theoretische Kenntnisse – praktische Fähigkeiten – professionelles Verhalten 3.2. Kompetenzgraduierung – Level I, II und III 4. Durchführung der Z‑WB für Internisten und Schwerpunktinternisten unter Einbeziehung des Curriculums 4.1. Weiterbildungsinhalte und Weiterbildungsdauer 4.2. Aktivitätsnachweise und Einbindung von DGIIN, DGIM und BDI 4.3. Mindestmengen 5. Curriculum: Anforderungen an den Weiterzubildenden 6. Curriculum: Anforderungen an den Weiterbilder und an die Weiterbildungsstätte 7. Curriculum: Dokumentation der Zusatz-Weiterbildung 8. Curriculum: Akkreditierung der Weiterbildungsstätte 9. Zertifizierung des Weiterzubildenden 10. Vorgesehene Aktualisierung des Curriculums 11. Ziele des Curriculums: internistische Weiterbildungsinhalte der Akut- und Notfallmedizin umfassend und aktuell präsentieren! 11.1. Allgemeine Aspekte der Klinischen Akut- und Notfallmedizin – Schwerpunkt Innere Medizin 11.1.1. Allgemeiner Teil – Struktur- und Prozessqualität (Tab. ) 11.1.2. Allgemeiner Teil – Erstdiagnostik, Initialtherapie und Indikationsstellung zur weiterführenden Behandlung (Tab. ) 11.1.3. Allgemeiner Teil – Schockraumversorgung (Tab. ) 11.1.4. Allgemeiner Teil – Diagnostik und Monitoring (Tab. ) 11.1.5. Allgemeiner Teil – generelle Therapieverfahren (Tab. ) 11.1.6. Allgemeiner Teil – Hygienemaßnahmen (Tab. ) 11.1.7. Allgemeiner Teil – Pharmakotherapie (Tab. ) 11.2. Angiologische Aspekte in der Akut- und Notfallmedizin (Tab. ) 11.3. Endokrinologische, diabetologische und metabolische Aspekte in der Akut- und Notfallmedizin (Tab. ) 11.4. Gastroenterologische Aspekte in der Akut- und Notfallmedizin (Tab. ) 11.5. Geriatrische Aspekte in der Akut- und Notfallmedizin (Tab. ) 11.6. Hämatoonkologische Aspekte in der Akut- und Notfallmedizin (Tab. ) 11.7. Infektiologische Aspekte der Akut- und Notfallmedizin (Tab. ) 11.8. Kardiologische Aspekte der Akut- und Notfallmedizin (Tab. ) 11.9. Nephrologische Aspekte der Akut- und Notfallmedizin (Tab. ) 11.10. Palliativmedizinische Aspekte der Akut- und Notfallmedizin (Tab. ) 11.11. Pneumologische Aspekte der Akut- und Notfallmedizin (Tab. ) 11.12. Rheumatologische Aspekte der Akut- und Notfallmedizin (Tab. ) 11.13. Toxikologische Aspekte der Akut- und Notfallmedizin (Tab. ) 1.1. Die (Muster‑)Weiterbildungsordnung ([M-]WBO) der Bundesärztekammer1.2. Klinische Akut- und Notfallmedizin i1.3. Akut- und notfallmedizinische Zusatz-Weiterbildungen (Z-WB) der Bundesärztekammer (BÄK)1.4. Empfehlung zu Weiterbildungsinhalten der Inneren Medizin in der NotaufnahmeDer Begriff „Weiterbildung“ im engeren Sinne ist ein Terminus der (Muster‑)Weiterbildungsordnung ([M-]WBO) mit Prüfungsabschluss, für die die Bundesärztekammer und die Landesärztekammern verantwortlich zeichnen. Die Präambel der (M-)WBO führt dazu Folgendes an : „Ärztliche Weiterbildung beinhaltet das Erlernen spezieller ärztlicher Fähigkeiten und Fertigkeiten nach abgeschlossenem Studium der Humanmedizin und nach Erteilung der Erlaubnis zur Ausübung der ärztlichen Tätigkeit. Im Interesse der Patienten werden die in der Ausbildung geprägten ärztlichen Kompetenzen und Haltungen während der Weiterbildung vertieft. Kennzeichnend für die Weiterbildung ist die vertiefende Anwendung ärztlicher Kenntnisse in der Berufsausübung. Die Weiterbildung erfolgt in strukturierter Form, um in Gebieten die Qualifikation als Facharzt, darauf aufbauend eine Spezialisierung in Schwerpunkten oder in einer Zusatz-Weiterbildung zu erhalten … Die Weiterbildung wird in angemessen vergüteter hauptberuflicher Ausübung der ärztlichen Tätigkeit an zugelassenen Weiterbildungsstätten durchgeführt. Sie erfolgt unter Anleitung befugter Ärzte in praktischer Tätigkeit und theoretischer Unterweisung sowie teilweise durch die erfolgreiche Teilnahme an anerkannten Kursen … Die Weiterbildungsbezeichnung ist der Nachweis für erworbene Kompetenz. Sie dient der Qualitätssicherung der Patientenversorgung und der Bürgerorientierung.“ Die Novellierung der (Muster‑)Weiterbildungsordnung ([M-]WBO) der Bundesärztekammer ist im November 2018 mit der Publikation erfolgreich zum Abschluss gebracht worden. Die (M-)WBO beinhaltet neben der Gebiets‑, Facharzt- und Schwerpunkt-Weiterbildung auch die darauf aufbauenden Zusatz-Weiterbildungen (Z‑WB), die gebietsübergreifend erworben werden können. Derzeit erarbeitet die Bundesärztekammer für alle Weiterbildungsordnungen inkl. der Zusatz-Weiterbildungen den sog. fachlich empfohlenen Weiterbildungsplan (FEWP) zur Konkretisierung der Weiterbildungsinhalte. Die FEWPs werden nach Fertigstellung in die Weiterbildungsordnungen als Ergänzung der kognitiven und Methodenkompetenz- und Handlungskompetenzspalten integriert werden. Die Fachgesellschaften haben die Möglichkeit, bei einer Aktualisierung der (M-)WBO („Novellierung“) durch die BÄK beratend die Weiterbildungsinhalte mit zu definieren: In Vorbereitung einer Novellierung der (M-)WBO durch die BÄK werden die Fachgesellschaften von der BÄK aufgefordert, Vorschläge hinsichtlich der Weiterbildungsinhalte zu unterbreiten. Nach Fertigstellung der Novellierung durch die BÄK legt diese die (M-)WBO-Novellierung dem Ärztetag zur Beschlussfassung vor. Nach Zustimmung des Ärztetags leitet die BÄK die novellierte (M-)WBO als „Muster“-Vorschlag den Landesärztekammern zur Umsetzung zu, wobei diese die Möglichkeit haben, durch Modifikationen die (M-)WBO der BÄK in die definitive Weiterbildungsordnung für ihren jeweiligen Ärztekammerbereich (WBO) umzugestalten. In dieser Phase besteht wiederum für Mitglieder der Fachgesellschaften, die in Gremien der Landesärztekammer mitarbeiten, die Möglichkeit – dieses Mal auf der Ebene der jeweiligen Landesärztekammer – beratend Modifikationen der Weiterbildungsinhalte vorzuschlagen. In der (M-)WBO zum Facharzt für Innere Medizin bzw. zum Facharzt für Innere Medizin und Schwerpunkt ist festgelegt, dass jeder zukünftige Facharzt im Rahmen seiner 60- bzw. 72-monatigen Weiterbildungszeit unter Befugnis im Gebiet Innere Medizin 6 Monate in der Notfallaufnahme und 6 Monate in der Intensivmedizin ableisten muss. Die notfallmedizinische Weiterbildung im Rahmen der Weiterbildung zum Facharzt für Innere Medizin und zum Facharzt für Innere Medizin und Schwerpunkt ist eine gute Basis für die Mitarbeit im Bereich der Akut- und Notfallmedizin zur Behandlung internistischer Patienten und zur Mitbehandlung internistischer Komorbiditäten bei nichtinternistischen Patienten. Notfall- und intensivmedizinische Maßnahmen im Gebiet Innere Medizin: Die gemeinsamen Inhalte der Facharztweiterbildungen im Gebiet Innere Medizin sind für die Notfall- und Intensivmedizin im Folgenden zusammengefasst. Kognitive und Methodenkompetenz (Kenntnisse): Differenzierte Beatmungstechniken. Handlungskompetenz (Erfahrungen und Fertigkeiten): Stufendiagnostik und Therapie bei akut einsetzenden Leitsymptomen, z. B. Dyspnoe, Thoraxschmerz, Bauchschmerz, passagere und persistierende Bewusstseinsstörungen, Fieber, Erbrechen, Durchfall; Diagnostik und Therapie akuter und vital bedrohlicher Erkrankungen und Zustände insbesondere respiratorische Insuffizienz, Schock, kardiale Insuffizienz, akutes Nierenversagen, sonstige Ein- und Mehrorganversagen, Koma und Delir, akute Enzephalopathie, Sepsis und Intoxikationen; Kardiopulmonale Reanimation; intensivmedizinische Behandlung von Patienten mit Funktionsstörungen von mindestens 2 vitalen Organsystemen; Analgosedierung von intensivmedizinischen Patienten; atemunterstützende Maßnahmen bei intubierten und nichtintubierten Patienten einschließlich Beatmungsentwöhnung bei langzeitbeatmeten Patienten; Therapie von Stoffwechselentgleisungen; Notfallsonographie; Notfallbronchoskopie; passagere Schrittmacheranlage; Punktions- und Katheterisierungstechniken (insbesondere zentralvenöse Zugänge und arterielle Gefäßzugänge). Facharzt für Innere Medizin und KardKardiologie noch zusätzlich einen Weiterbildungsblock für kardiovaskuläre Notfall- und Intensivmedizin. Kognitive und Methodenkompetenz (Kenntnisse): Herzunterstützende Verfahren. Handlungskompetenz (Erfahrungen und Fertigkeiten): Behandlung des Herz-Kreislauf-Versagens in der Akutphase; Management der Postreanimationsphase; Akutbehandlung von Patienten mit akuten und bedrohlichen Herz-Kreislauferkrankungen, insbesondere akutes Thoraxschmerzsyndrom, auch in Notaufnahme und Chest Pain Unit, Intermediate Care und internistischer Intensivmedizin; invasives hämodynamisches Monitoring; Organ-unterstützende Verfahren, z. B. nichtinvasive und invasive Beatmung, intraaortale Ballongegenpulsation, perkutane Herz-Lungen-Maschine, extrakorporale Membranoxygenierung, perkutane Herzunterstützungssysteme; Akutbehandlung des Herz-Kreislauf-Schocks, insbesondere des kardiogenen Schocks; Mitbehandlung des Multiorgan-Dysfunktions-Syndroms. Facharzt für Innere Medizin und Infektiologie: Neben den gemeinsamen Inhalten zur Notfall- und Intensivmedizin für alle internistischen WBOs (siehe oben) enthält die WBO für den Facharzt für Innere Medizin und Infektiologie noch zusätzlich einen Weiterbildungsblock für Infektiologische Notfälle. Kognitive und Methodenkompetenz (Kenntnisse): Akute lebensbedrohliche Infektionen und infektiologische Notfälle. Handlungskompetenz (Erfahrungen und Fertigkeiten): Beurteilung des Schweregrads von Infektionen; Erkennung und Behandlung einschließlich Erstversorgung von Infektionen mit hoher Kontagiosität; interdisziplinäre Beratung und Akutbehandlung bei lebensbedrohlichen Infektionen; Erkennung und Therapie der Sepsis und des septischen Schocks, auch in interdisziplinärer Zusammenarbeit. Für die Tätigkeiten in der prähospitalen und klinischen Akut- und Notfallmedizin hat die Bundesärztekammer 2 Zusatz-Weiterbildungen geschaffen, die Z‑WB „Notfallmedizin“ und die Z-WB „Klinische Akut- und Notfallmedizin“. Die Z‑WB „Notfallmedizin“ kann bereits während einer Facharztausbildung absolviert werden und fokussiert mit den 50 Notarzteinsätzen vor allem auf die „onen.“ Dagegen vermittelt die Z‑WB „Klinische Akut- und Notfallmedizin“ einem fertigen Facharzt die zusätzlichen interdisziplinären Kompetenzen zur „Erstdiagnostik und Initialtherapie von Notfall- und Akutpatienten im Krankenhaus sowie zur1.3.1. Zusatz-Weiterbildung (Z-WB) „Notfallmedizin“ 1.3.2. Zusatz-Weiterbildung (Z-WB) „Klinische Akut- und Notfallmedizin“ ] Definition: „Die Zusatz-Weiterbildung Notfallmedizin umfasst die Erkennung drohender oder eingetretener Notfallsituationen und die Behandlung von Notfällen sowie die Wiederherstellung und Aufrechterhaltung akut bedrohter Vitalfunktionen.“ Die Mindestanforderungen gemäß § 11 MWBO für diese Z‑WB sind 24 Monate Weiterbildung in einem Gebiet der unmittelbaren Patientenversorgung im stationären Bereich unter Befugnis an Weiterbildungsstätten, davon 6 Monate in der Intensivmedizin oder Anästhesiologie plus anschließend 50 Notarzteinsätze im öffentlichen Rettungsdienst (Notarzteinsatzfahrzeug oder Rettungshubschrauber) unter Anleitung eines verantwortlichen Notarztes, davon können bis zu 25 Einsätze im Rahmen eines standardisierten Simulationskurses erfolgen. Die Weiterbildungsinhalte der Z‑WB gliedern sich in organisatorische, einsatztaktische Grundlagen; die Untersuchung des Notfallpatienten; Leitsymptome: Handlungskompetenz (Erfahrungen und Fertigkeiten) in der Einleitung einer symptomorientierten Erstbehandlung bei → Bewusstseinsstörungen/neurologischen Defiziten, → akuter Atemnot, → Brustschmerz, → Blutungen, → Schock, → Herzrhythmusstörungen, → akutem Abdomen/Bauchschmerzen, → psychischen Störungen, → Fieber; diagnostische Maßnahmen: Handlungskompetenz (Erfahrungen und Fertigkeiten) → Durchführung und Befunderstellung des Elektrokardiogramms im Notfall, → Applikation und Bewertung des Basismonitorings einschließlich Besonderheiten des kindgerechten Monitorings beim Transport, Messung und Bewertung der Kapnometrie und Kapnographie; therapeutische Maßnahmen: kognitive und Methodenkompetenz (Kenntnisse) u. a. in Grundlagen der transkutanen Schrittmachertherapie; Handlungskompetenz (Erfahrungen und Fertigkeiten) u. a. bei der Durchführung von Defibrillation oder Kardioversion, auch als Simulation. , ] Definition: „Die Zusatz-Weiterbildung Klinische Akut- und Notfallmedizin umfasst in Ergänzung zu einer Facharztkompetenz die Erstdiagnostik und Initialtherapie von Notfall- und Akutpatienten im Krankenhaus sowie die Indikationsstellung und Koordination der weiterführenden fachspezifischen Behandlung in interdisziplinärer Zusammenarbeit.“ . Die Mindestanforderungen gemäß § 11 MWBO für diese Z‑WB sind die Facharztanerkennung in einem Gebiet der unmittelbaren Patientenversorgung plus 6 Monate Intensivmedizin, die auch während der Facharztweiterbildung abgeleistet werden können, plus 80 h-Kurs-Weiterbildung gemäß § 4 Abs. 8 in allgemeiner und spezieller Notfallbehandlung plus 24 Monate klinische Akut- und Notfallmedizin in einer interdisziplinären Notfallaufnahme unter Befugnis an Weiterbildungsstätten . Die Weiterbildungsinhalte der Z‑WB gliedern sich in übergreifende Inhalte ; symptomorientierte Erstdiagnostik und Initialtherapie ; alters- und geschlechtsspezifische Notfälle (Kindes- und Jugendalter, Schwangerschaft, geriatrische Patienten); notfallmedizinische Kernverfahren ; organbezogene und spezifische Notfallsituationen. Die Weiterbildungsinhalte zu organbezogenen und spezifischen Notfallsituationen beinhalten keine Handlungskompetenz (Erfahrungen und Fertigkeiten), wohl aber kognitive und Methodenkompetenz (Kenntnisse) in Form der Differenzialdiagnostik und Therapieoptionen organbezogener Notfälle: → kardiovaskuläre Notfälle, → hämatologische und onkologische Notfälle; → immunologische Notfälle, → Infektionskrankheiten und Sepsis, → endokrine und metabolische Notfälle, → Flüssigkeits- und Elektrolytstörungen; → gastrointestinale und hepatologische Notfälle, → respiratorische Notfälle; → nephrologische und urologische Notfälle, → dermatologische Notfälle; → Notfälle im Hals‑, Nasen‑, Ohren‑, Mund- und Nackenbereich; → gynäkologische Notfälle, → muskuloskelettale Notfälle, → neurologische Notfälle, → neurochirurgische Notfälle, → ophthalmologische Notfälle; → psychiatrische Notfälle und Verhaltensstörungen, → Trauma (stumpf/penetrierend), → akute Notfälle durch Umwelteinflüsse, thermische, hyper- und hypobare Exposition und elektrischen Strom. Das vorliegende Curriculum beschreibt die internistischen Weiterbildungsinhalte der präklinischen und klinischen Akut- und Notfallmedizin, die für die Betreuung internistischer akut- und notfallmedizinischer Patienten zu erwerben sind, aber auch für die Mitbetreuung nichtinternistischer Akut- und Notfallpatienten – Patienten mit internistischen Komorbiditäten und bei internistischen Notfällen – beherrscht werden sollen. Das Curriculum wurde gemeinsam von Mitgliedern der Deutschen Gesellschaft für Internistische Intensiv- und Notfallmedizin (DGIIN), der Deutschen Gesellschaft für Innere Medizin (DGIM) samt deren Schwerpunktgesellschaften sowie des Berufsverbands Deutscher Internistinnen und Internisten (BDI) erstellt. Das Curriculum soll dem Facharzt für Innere Medizin bei der Absolvierung der Zusatz-Weiterbildung „Klinische Akut- und Notfallmedizin“ die Weiterbildungsinhalte aus dem Bereich der Inneren Medizin aufzeigen. Das Curriculum inkludiert auch die internistischen Weiterbildungsinhalte der auf die präklinische Notfallmedizin fokussierten Zusatz-Weiterbildung „Notfallmedizin“. Selbstredend umfasst dieses Curriculum nur die internistischen Weiterbildungsinhalte und nicht das gesamte restliche notfallmedizinische Spektrum. Die aufgeführten Weiterbildungsinhalte berücksichtigen auch die entsprechenden Weiterbildungsinhalte des European Core Curriculum for Emergency Medicine der European Society for Emergency Medicine und der Section for Emergency Medicine der European Union of Medical Specialists (UEMS; sowie internationale Weiterbildungskonzepte ). 2.1. Weiterzubildende2.2. Weiterbilder2.3. Gremien und ÄrztekammernZiel dieses Curriculums ist es, aus Sicht der DGIIN, der DGIM samt deren Schwerpunktgesellschaften, des BDI und unter Einbeziehung der Palliativmedizin aufzuzeigen, welche Kompetenzen auf dem Gebiet der internistischen Akut- und Notfallmedizin heutzutage benötigt werden. Dem in den Z‑WB „Klinische Akut- und Notfallmedizin“ oder „Notfallmedizin“ Weiterzubildenden soll das Curriculum die Möglichkeit geben, seine Zusatzweiterbildungszeit hinsichtlich der internistischen Weiterbildungsinhalte so effizient wie möglich zu strukturieren, sich gut auf die Prüfung vorzubereiten und das Erlernte anschließend im Sinne eines „berufslebenslangen“ Qualifizierens zu bewahren und auszubauen. Wie bereits im Vorwort ausgeführt erlaubt die Gliederung des Curriculums in einen allgemeinen und einen schwerpunktspezifischen Teil es dem Weiterzubildenden, die Notfallmedizin nicht nur als Beseitigung eines akuten „Problems“ zu sehen, sondern als möglichst früher Beginn der Behandlung der dem „Problem“ zugrunde liegenden internistischen Erkrankung bereits in der Notaufnahme. Das Curriculum möchte aber nicht nur die Weiterzubildenden, sondern auch die Weiterbilder in den Z‑WBs „Klinische Akut- und Notfallmedizin“ sowie „Notfallmedizin“ erreichen und aufzeigen, welche internistischen Inhalte und Fertigkeiten nach Ansicht von DGIIN, DGIM und BDI in den Z‑WB vermittelt werden sollen, um alle Aspekte der Inneren Medizin in der Akut- und Notfallmedizin für die Patienten bestmöglich einsetzen zu können. Und schließlich soll das Curriculum den für die Zusatz-Weiterbildungen verantwortlichen Gremien der Ärztekammern das breite Spektrum der internistischen Akut- und Notfallmedizin aufzeigen, das nach Ansicht von DGIIN, DGIM und BDI in die Z‑WBs „Klinische Akut- und Notfallmedizin“ bzw. „Notfallmedizin“ einfließen soll. 3.1. Theoretische Kenntnisse – praktische Fähigkeiten – professionelles Verhalten3.2. Kompetenzgraduierung – Level I, II und IIIDie Qualifikation der Weiterzubildenden wird durch „theoretische Kenntnisse“ („knowledge“; TK), „praktische Fähigkeiten“ („skills“; PF) und beruflich-professionelles Verhalten („behaviours and attitudes“; BV) erworben und aufrechterhalten. Diese Klassifizierung „knowledge“, „skills“ und „behaviours and attitudes“ ist international akzeptiert und auch auf nationaler Ebene – z. B. in Deutschland – im Einsatz. Die Bundesärztekammer verwendet in der (M-)WBO die Begriffe „ Kognitive und Methodenkompetenz – Kenntnisse “ und „ Handlungskompetenz – Erfahrungen und Fertigkeiten“ , die unschwer mit den in diesem Curriculum verwandten Begriffen „theoretische Kenntnisse“ und „praktische Fähigkeiten“ gleichgesetzt werden können. Dagegen ist der für den Berufsalltag wichtige Weiterbildungsinhalt „beruflich-professionelles Verhalten“ in der (M-)WBO nicht explizit abgebildet. Die TK-PF-BV-Einteilung bildet die Basis der Klassifikation dieses Curriculums. Dieses standardisierte Vorgehen erleichtert den Vergleich der Zusatzweiterbildungsinhalte der Akut- und Notfallmedizin mit den Weiterbildungsinhalten der internistischen Intensivmedizin und der internistischen Schwerpunkte, z. B. der Kardiologie : Die „theoretischen Kenntnisse“ (TK, „ knowledge “) definieren sich aus den stichwortartig aufgeführten Themenschwerpunkten bzw. den kognitiven und Methodenkompetenzen der internistischen Weiterbildungsinhalte der Z‑WB „Klinische Akut- und Notfallmedizin“ und der Z‑WB „Notfallmedizin“ (siehe Tab. – ). Die theoretischen Wissensanteile sind das essenzielle Fundament der Kompetenzentwicklung. Die „praktischen Fähigkeiten“ (PF, „ skills “) bzw. Handlungskompetenzen beschreiben die effektive Anwendung von theoretischem Wissen zur Lösung von Problemen, zu klinischen Entscheidungsfindungen und – aufbauend auf Erfahrung und Training – zur Durchführung von Prozeduren. Simulationstraining (siehe Kurse der DGIIN) im Team stellt eine sinnvolle Ergänzung in der kompetenzorientierten Zusatz-Weiterbildung dar und führt zu einer vertrauensvollen interdisziplinären und interprofessionellen Zusammenarbeit. Die „ skills “ sollten sich allerdings nicht allein auf die fachpraktischen, die sog. „ hard skills “, beschränken, sondern persönliche, soziale und methodische Kompetenzen, sog. „s oft skills “, mit einschließen. Das „beruflich-professionelle Verhalten“ (BV, „behaviours and attitudes“) muss der Akut- und Notfallmediziner lernen und beruflich „leben“ im Umgang mit Patienten und Angehörigen, allen beteiligten Berufsgruppen – sowohl interdisziplinär als auch interprofessionell – und anderen Akteuren im Gesundheitswesen. Das sehr aufgefächerte gesamte Methodenspektrum der internistischen Inhalte in der Akut- und Notfallmedizin muss zwar von jedem Weiterzubildenden „gewusst“ werden, nicht jeder Akut- und Notfallmediziner kann aber alle Spezialkenntnisse bzw. Techniken der integrierten internistischen Schwerpunktfächer – wie z. B. die Behandlung eines dekompensierten Vitiums oder die extrakorporale Reanimation (eCPR) – selbständig durchführen. Insofern muss hinsichtlich der „Eindringtiefe“ im Beherrschen praktischer Fähigkeiten zwangsläufig eine Selektion vorgenommen werden zwischen dem selbständigen bzw. nichtselbständigen Beherrschen der breiten Palette notfallmedizinischer Methoden und Techniken und dem Wissen um erweiterte Methoden und Techniken im Speziellen. Letztere können einerseits anhand weiterführender, auf dem Curriculum „Klinische Akut- und Notfallmedizin – Schwerpunkt Innere Medizin“ aufbauender Curricula der internistischen Schwerpunktfächer erworben werden, z. B. dem Curriculum „Kardiovaskuläre Intensiv- und Notfallmedizin“ der Deutschen Gesellschaft für Kardiologie – Herz- und Kreislaufforschung e. V. (DGK; ); andererseits dienen dazu die Empfehlungen fächerübergreifender Konsensuspapiere unter Einbeziehung der Deutschen Interdisziplinären Vereinigung für Intensiv- und Notfallmedizin (DIVI), z. B. die Empfehlungen zur extrakorporalen Reanimation (eCPR; ). Hinsichtlich der Kompetenzgraduierung orientiert sich das vorliegende Curriculum ebenfalls an der von der Europäischen Gesellschaft für Kardiologie (ESC) beschriebenen Graduierung mit den Kompetenzlevels I–III: Kompetenzlevel I für praktische Fähigkeiten („skills“): Erfahrung bei der Auswahl der geeigneten diagnostischen oder therapeutischen Maßnahmen und der Interpretation der erhaltenen Ergebnisse; Erfahrung bei der Suche nach einer geeigneten Behandlung, zu der der Patient überwiesen werden soll; Level I erfordert zwar umfassende theoretische Kenntnisse der Methoden, jedoch keine Beherrschung der Techniken. Kompetenzlevel II für praktische Fähigkeiten („skills“): Level II geht über Level I hinaus: Zusätzlich zur Level-I-Kompetenz soll der Weiterzubildende sich praktische Erfahrungen aneignen und bewahren, aber nur als nichtselbständiger und nicht als eigenverantwortlicher Untersucher (der Weiterzubildende assistiert oder führt eine spezielle Technik oder Prozedur unter Anleitung durch). Kompetenzlevel III für praktische Fähigkeiten („skills“): Level III geht über Level I und Level II hinaus. Der Weiterzubildende soll lernen und die Kompetenz bewahren, eigenständig für ein diagnostisches oder therapeutisches Verfahren die Indikation zu erkennen, die Technik oder die Prozedur durchzuführen, die Daten zu interpretieren und Komplikationen zu beherrschen. In den Tab. – sind die Level-Angaben mit einem * gekennzeichnet. 4.1. Weiterbildungsinhalte und Weiterbildungsdauer4.2. Aktivitätsnachweise und Einbindung von DGIIN, DGIM und BDI4.3. MindestmengenWeiterbildungsinhalte und -dauer für die Z‑WB „Klinische Akut- und Notfallmedizin“ und die Z‑WB „Notfallmedizin“ sind unter 1.3. beschrieben. Die von DGIIN, DGIM und BDI im Einklang mit den Z‑WB empfohlenen internistischen Weiterbildungsinhalte finden sich im Curriculum. Obligat ist die Dokumentation der erworbenen Weiterbildungsinhalte für die Z‑WB „Klinische Akut- und Notfallmedizin“ und die Z‑WB „Notfallmedizin“ entsprechend der WBO der zuständigen Ärztekammer. Der Besuch nationaler und internationaler Fachtagungen/Kongresse mit internistisch-akutmedizinischen und internistisch-notfallmedizinischen Programmteilen ist empfehlenswert. Auf nationaler Ebene bieten sich dabei die Kongresse und Kurse der DGIIN/ÖGIAIN, der DIVI und DGINA an. Die Weiter- und Fortbildungsveranstaltungen und -kurse der DGIIN gehen gezielt auf die im Curriculum genannten Zusatzweiterbildungsinhalte ein. Beim Erlernen von Techniken spielen das persönliche Handanlegen und die praktische Erfahrung eine große Rolle. Zwar ist die Zahl der durchgeführten Untersuchungen keine Garantie dafür, dass die Prozedur vom Weiterzubildenden kompetent beherrscht wird; dennoch vermittelt die Durchführung einer bestimmten Anzahl bei ausgewählten Prozeduren eine gewisse Sicherheit, die in Bezug auf den Patienten gefordert werden muss. Die notfallmedizinischen Weiterbildungsinhalte für das Fachgebiet Innere Medizin enthalten keine entsprechenden Richtzahlen, ebenso wenig wie die Z‑WB „Klinische Akut- und Notfallmedizin“. In der Z‑WB „Notfallmedizin“ werden folgende Richtzahlen genannt: Indikationsstellung und Durchführung von Repositionen bei Frakturen und Luxationen: 5 sowie Sicherung der Atemwege durch endotracheale Intubation einschließlich Videolaryngoskopie: 50. Jeder Facharzt, wie z. B der Facharzt für „Innere Medizin“ bzw. „Innere Medizin und Schwerpunkt“, der die Z‑WB „Klinische Akut- und Notfallmedizin“ anstrebt und anschließend als Akut- und Notfallmediziner arbeiten möchte, soll sich im Rahmen seiner Z‑WB-Zeit die von den Ärztekammern in der WBO geforderte Kognitive und Methodenkompetenz (Kenntnisse) und Handlungskompetenz (Erfahrungen und Fertigkeiten) aneignen. Das vorliegende Curriculum versucht, die von den Ärztekammern vorgeschriebenen internistischen Weiterbildungsinhalte entsprechend den Vorstellungen von DGIIN, DGIM und BDI anhand der geforderten theoretischen Kenntnisse, der praktischen Fähigkeiten und des beruflich professionellen Verhaltens „mit Leben zu füllen“. Hilfreich – sowohl für die Teamarbeit als auch für die Patientensicherheit – sind auch das Lernen mit digitalen Medien und Ausbildungskonzepte, die eine Simulation von Fällen und Szenarien aller Art anbieten . Die zugehörige Qualifikationen sind die Z‑WB-Prüfungen „Klinische Akut- und Notfallmedizin“ und „Notfallmedizin“ durch die jeweilige Landesärztekammer. Der Weiterbilder für die Inhalte des Curriculums „Klinische Akut- und Notfallmedizin – Schwerpunkt Innere Medizin“ arbeitet in Vollzeit- bzw. in überwiegender Tätigkeit in einer/einem interdisziplinären Notaufnahme/Klinik für Akut- und Notfallmedizin/Zentrum für Notfallmedizin, in der/dem sich der fachärztliche Weiterbilder „Innere Medizin“ besonders für die internistischen Akut- und Notfallpatienten verantwortlich zeichnet. Die Weiterbildungsstätte sollte – unterstützt von der Klinikleitung – mit einer adäquaten Prozess- und Strukturqualität ausgestattet sein, um den Weiterzubildenden die in Abschn. 11 aufgeführten Weiterbildungsinhalte zu ermöglichen. Sehr zu begrüßen ist die staatlich finanzierte notfallmedizinische Weiterbildung in manchen Ländern wie Schweden . Anzustreben ist hinsichtlich des Erstellens eines Qualitätskriterienkatalogs für Weiterbilder und Weiterbildungsstätten eine Kooperation der DGIIN mit den Landesärztekammern, wie dies im Fall der Weiterbildung zum Facharzt für Innere Medizin und Kardiologie bereits realisiert ist. Das Curriculum gibt dem in klinischer Akut- und Notfallmedizin Weiterzubildenden die Möglichkeit, sich systematisch die internistischen Weiterbildungsinhalte zu erarbeiten und sie mit den internistischen Inhalten der Z‑WB „Klinische Akut- und Notfallmedizin“ und der Z‑WB „Notfallmedizin“ abzugleichen. Die erlernten und geübten Weiterbildungsinhalte sollen entsprechend der Z‑WB-Version der zuständigen Ärztekammer anhand des (elektronischen) Logbuchs und der Zeugnisse der Weiterbilder dokumentiert werden. DGIIN, DGIM und BDI unterstützen die Weiterzubildenden beratend und anhand des Fortbildungs- und Kursangebots bei der strukturierten Z‑WB entsprechend dem Curriculum. Eine zusätzliche Zertifizierung der in diesem Curriculum vorgeschlagenen Weiterbildungsinhalte ist nicht vorgesehen. Der Weiterbilder wird gebeten, im Weiterbildungszeugnis nicht nur die Erfüllung der in der Z‑WB geforderten Weiterbildungsinhalte zu dokumentieren, sondern auch die in diesem Curriculum vorgelegten Weiterbildungsinhalte. Hinsichtlich der Akkreditierung der Weiterbildungsstätte gilt das unter Abschn. 6 Gesagte. Eine darüber hinaus gehende Akkreditierung durch die DGIIN als Fachgesellschaft ist nicht vorgesehen. Die zuständige Landesärztekammer dokumentiert die erfolgreich abgeleistete Z‑WB „Klinische Akut- und Notfallmedizin“ und die Z‑WB „Notfallmedizin“ mit einer Prüfung. Eine zusätzliche Prüfung der Curriculumweiterbildungsinhalte ist nicht vorgesehen. Dem in der klinischen Akut- und Notfallmedizin Tätigen und mit der Zusatz-Weiterbildung „Klinische Akut- und Notfallmedizin“ Qualifizierten obliegt die Verantwortung für das „berufslebenslange“ Aufrechterhalten dieser Qualifikation durch Teilnahme an Fortbildungsveranstaltungen und Kursen (z. B. der DGIIN, DIVI, DGINA), durch Kongressbesuche (z. B. Jahrestagung der DGIIN/ÖGIAIN, DIVI, DGINA) und mit kontinuierlichem Fachliteraturstudium . Eine darüber hinaus gehende Zusatzqualifizierung internistischer Schwerpunktgesellschaften erweitert das Spektrum im Sinne einer weiterführenden Spezialisierung. Eine Aktualisierung des Curriculums ist spätestens in 5 Jahren (2029) vorgesehen. Ziel des Curriculums ist es, die in der Akut- und Notfallmedizin tätigen Ärztinnen und Ärzte in die Lage zu versetzen, bei ihrer Tätigkeit das gesamte Spektrum der Inneren Medizin in Bezug auf Diagnostik, Monitoring und Therapie kompetent beim Patienten anzuwenden. Dies schließt umfassende Kenntnisse, Erfahrungen und Fertigkeiten in der Gesprächsführung mit Patienten und Angehörigen mit ein. Besondere Kenntnisse und praktische Erfahrungen soll der Akut- und Notfallmediziner auch bezüglich der Anwendung von Arzneimitteln bei Notfallpatienten erwerben, da häufig Arzneimittelmetabolismus und -elimination infolge von Organdysfunktionen des Notfallpatienten alteriert sind. Dies gilt insbesondere auch für alte und geriatrische Patienten in der Notaufnahme/in der Notfallklinik, mit Frailty, Sarkopenie, geriatrischen Syndromen, kognitiver Dysfunktion, Polypharmazie und Polypragmasie . Und schließlich wird die zunehmende Digitalisierung in der Akut- und Notfallmedizin auch die Weiterbildungsinhalte und damit auch zukünftige Auflagen dieses Curriculums entscheidend prägen. 11.1. Allgemeine Aspekte der k11.2. Angi11.4. Gastroenterolog11.5. Geriatrische Aspekte in der Akut- und Notfallmedizin (Tab.11.6. Hämatoonkologische Aspekte in der Akut- und Notfallmedizin (Tab.11.7. Infektiologische Aspekte der Akut- und Notfallmedizin (Tab.11.8. Kardiologische Aspekte der Akut- und Notfallmedizin (Tab.11.9. Nephrologische Aspekte der Akut- und Notfallmedizin (Tab.11.10. Palliativmedizinische Aspekte der Akut- und Notfallmedizin (Tab.11.11. Pneumologische Aspekte der Akut- und Notfallmedizin (Tab.11.12. Rheumatologische Aspekte der Akut- und Notfallmedizin (Tab.11.13. Toxikologische Aspekte der Akut- und Notfallmedizin (Tab. ) Grundlagen und Standards.Das soll gewusst und gekonnt werden. Toxidrome, Antidote, primäre Giftelimination : Der Akut- und Notfallmediziner benötigt insbesondere das Wissen um Toxidrome (sympathomimetisch, anticholinerg, Opiat, Serotonin, cholinerg) und der toxidromspezifischen Therapie. Der Akut- und Notfallmediziner soll auch einen Überblick über die wichtigsten Antidote und deren Dosierung sowie über die Möglichkeiten der primären Giftelimination haben. 11.1.1. Allgemeiner Teil – Struktur- und Prozessqualität (Tab.11.1.3. Allgemeiner Teil – Schockraumversorgung (Tab.11.1.4. Allgemeiner Teil – Diagnostik und Monitoring (Tab.11.1.5. Allgemeiner Teil – generelle Therapieverfahren (Tab.11.1.6. Allgemeiner Teil – Hygienemaßnahmen (Tab.11.1.7. Allgemeiner Teil – Pharmakotherapie (Tab. ) Grundlagen und Standards.Das soll gewusst und gekonnt werden.Das soll gewusst und gekonnt werden.Die Qualität der Zusammenarbeit in dem interprofessionellen und interdisziplinären Team einer Notaufnahme/Notfallklinik/einem Notfallzentrum hat einen erheblichen Einfluss auf die Qualität der dortigen Patientenversorgung und die Patientensicherheit. Verständnis dafür, welche strukturellen Faktoren und Prozesse diese Qualität beeinflussen und welche Ergebnisse als relevant eingestuft werden müssen. Dazu gehören auch medikolegale Aspekte, wie die Einwilligung in medizinische Eingriffe, das Betreuungsrecht und die Anwendung freiheitsentziehender Maßnahmen sowie die allgemeinen Rechtsgrundlagen und Haftungsfragen. Kenntnisse im Qualitätsmanagement, im Risikomanagement und in der Fehlerkultur in der Notaufnahme sowie Kenntnisse der unterschiedlichen Sektoren und Schnittstellen in der klinischen Akut- und Notfallmedizin. Umfassender Überblick und Kenntnis der Patientenersteinschätzung, einem der wichtigen organisatorischen Systeme. ) Grundlagen und Standards.Das soll gewusst und gekonnt werden.Kernelemente der Arbeit des klinischen Akut- und Notfallmediziners ist die differenzialdiagnostische Abklärung von zur Vorstellung führenden Leitsymptomen unter Berücksichtigung einer zeitkritisch einzuleitenden Notfalltherapie. Nach Stellung einer Arbeitsdiagnose ist die folgende Notfalldiagnostik leitlinienkonform durchzuführen. Weiterhin ist die Entscheidung für oder gegen eine stationäre Aufnahme bzw. eine ambulante Weiterbehandlung zu treffen. Im Fall einer stationären Aufnahme soll der Notfallmediziner die Indikationen/Kriterien für die Notwendigkeit einer engmaschigen Überwachung der Patienten auf einer geeigneten Behandlungseinheit (z. B. Beobachtungsstation) kennen bzw. erkennen . ) Grundlagen und Standards.Das soll gewusst und gekonnt werden.Die Anzahl kritisch kranker, nichttraumatologischer Schockraumpatienten, die in Notaufnahmen bzw. Notfallzentren eingewiesen werden, ist bis zu 4‑mal höher als die Anzahl schwerverletzter Patienten . Ein entsprechendes Weißbuch „Nichttraumatologisches Schockraummanagement“ ist verfügbar . Die Wahl einer geeigneten Behandlungseinrichtung, der Einsatz einer adäquaten bildgebender Diagnostik, die Etablierung von Algorithmen und Behandlungspfaden sowie die Koordination und Abstimmung der Übergabe sind unabdingbare Bestandteile der Kenntnisse und Fähigkeiten eines Akut- und Notfallmediziners. Insbesondere die Übergabe von Notfallpatienten bedarf einer gewissen Strukturierung und Vereinheitlichung unter Einbeziehung und Beachtung aller beteiligter Berufsgruppen und Prozesse. ) Grundlagen und Standards.Das soll gewusst und gekonnt werden.Standardrepertoire: Die Akutdiagnostik und das Monitoring gehören zum Standardrepertoire der notfallmedizinischen Betreuung von Notfallpatienten inkl. kritisch kranker Patienten. Im Rahmen der Diagnostik soll die gezielte (Fremd‑)Anamnese sowie die körperliche Untersuchung und damit der „klinische Blick“ stets die Basis bei der Aufnahme eines Notfallpatienten darstellen. Bei der Anamneseerhebung hat die Interaktion mit dem Rettungsdienst inkl. das Verständnis für die im Rettungsdienst eingesetzten Übergabealgorithmen eine besondere Bedeutung. Wesentliche Fähigkeiten des Notfallmediziners sind das rasche Stellen einer Arbeitsdiagnose unter Berücksichtigung zeitkritischer und potenziell lebensbedrohlicher Differenzialdiagnosen, das schnelle Einleiten geeigneter Notfalltherapien sowie die Entscheidung über die Notwendigkeit einer stationären Weiterbehandlung und die Festlegung eines geeigneten Stationstyps . Diagnostik: In der Notfallmedizin findet die bildgebende Diagnostik überwiegend am Krankenbett statt – „ bedside ultrasonography “ (fokussierte Sonographie ), da Transporte ggf. mit einem erhöhten Patientenrisiko oder einer Zeitverzögerung verbunden sind. Alle diagnostischen Verfahren sollten stets im klinischen Kontext sowie im interdisziplinären fachärztlichen Team erfolgen. Monitoring: Für das Monitoring stehen dem Notfallmediziner sowohl nichtinvasive als auch invasive Werkzeuge zur Verfügung. Die Europäische Gesellschaf für Intensivmedizin (ESICM) empfiehlt ein klinisches sowie ein hämodynamisches Monitoring auch zur Identifizierung der Schockursache und zur Überprüfung der therapeutischen Maßnahmen und des Ansprechens auf die Therapie . Das Basismonitoring soll die klinische Untersuchung (z. B. Symptome/Zeichen von Stauung und Hypoperfusion bei akuter Herzinsuffizienz) und nichtinvasive Überwachungsverfahren (z. B. die Messung der peripheren Sauerstoffsättigung) beinhalten. Das erweiterte Monitoring reduziert sich in der Notaufnahme zumeist auf eine kontinuierliche invasive Blutdruckmessung. Ergänzende hämodynamische Messverfahren sollen auf der Intensivstation durchgeführt werden. Das Basismonitoring soll immer 2 unabhängig voneinander agierende Vitalparameter des Patienten einbeziehen und beinhaltet neben der Bestimmung der Atemfrequenz, der Körpertemperatur sowie der peripheren Sauerstoffsättigung die nichtinvasive Blutdruckmessung (ggf. invasiv), die Beurteilung des 12-Ableitungs-Elektrokardiogramms (EKG) und auch Grundkenntnisse in der leitsymptomorientierten Notfallsonographie. Ziel der fokussierten Notfallsonographie inkl. Echokardiographie ist das frühzeitige Erkennen bzw. der Ausschluss wichtiger kritischer Diagnosen. Bei den allermeisten Leitsymptomen kann die bettseitige Notfallsonographie hier eine rasche Diagnose z. B. des Pneumothorax bei Dyspnoe oder der Cholezystitis bei akutem Abdomen ermöglichen. Die strukturierte Ultraschallausbildung in der internistischen Intensiv- und Notfallmedizin (SIN) basiert auf einem 2‑Stufen-Konzept, das von den 3 nationalen Fachgesellschafen DGIIN, DGK und DEGUM vertreten wird . Das Konzept umfasst ein Basislevel (SIN-I) und ein Expertenlevel (SIN-II), die – aufeinander aufbauend mithilfe moderner Lehrmethoden – eine leitsymptomorientierte Sonographie für die Notfall- und Intensivmedizin vermitteln. Es werden sowohl theoretische Kenntnisse als auch praktische Fertigkeiten gelehrt und im Rahmen einer Prüfung kontrolliert. Ziel ist es, die in nationalen und internationalen Leitlinien empfohlenen Vorgaben zum Einsatz der Sonographie in der Notfall- und Intensivmedizin in der klinischen Praxis standardisiert zu etablieren. Anamnese, körperliche Untersuchung und sämtliche bettseitige Untersuchungen, wie EKG, Labordiagnostik (u. a. „ Point-of-care-testing “[POCT]-Verfahren), fokussierte Sono‑/Echokardiographie sowie das Monitoring, bilden die Basis der notfallmedizinischen Betreuung von Notfallpatienten inklusive der kritisch kranken Patienten. Neben diesen Inhalten sollen die Grundlagen sämtlicher radiologischer Untersuchungsverfahren (Röntgen, Computertomographie, Magnetresonanztomographie) von der Überprüfung der Indikation bis hin zur Planung bzw. Durchführung eines Patiententransports (Intra- und Interhospitaltransport) nach den Regeln eines Intensivtransports (inklusive Komplikationsmanagement) in der Zusatz-Weiterbildung vermittelt werden. Der Akut- und Notfallmediziner soll in der Lage sein, selbstständig und eigenverantwortlich eine fokussierte Sonographie inkl. Echokardiographie, abhängig vom jeweiligen Ausbildungslevel (beginnend von „ focused echocardiography in emergency life support “ [FEEL] bis hin zu Grundzügen der transösophagealen Echokardiographie) durchzuführen. Weiterhin sollte er eine flexible Bronchoskopie im Rahmen des Atmungs- und Beatmungsmanagements durchführen können. Häufige Punktionen – insbesondere Pleura- und Aszitespunktion – sollen selbstständig durchgeführt werden. Hinsichtlich der Hirntoddiagnostik zur Feststellung des irreversiblen Hirnfunktionsausfalls soll er über die theoretischen Kenntnisse verfügen, um gemeinsam mit dem interdisziplinären Team die Indikation für eine organerhaltende Therapie initiieren zu können. ) Grundlagen und Standards.Das soll gewusst und gekonnt werden.Die Betreuung eines Notfallpatienten erfordert ein breites Spektrum an allgemeinen Therapieverfahren, das weit über das übliche Spektrum bei der Behandlung einer speziellen internistischen Erkrankung im stabilen Zustand des Patienten hinausgeht. Die Entscheidung zur Therapie hat dabei rasch und gezielt zu erfolgen und erfordert den Erwerb spezieller manueller Fähigkeiten. Kathetertechnik: Die sonographieunterstützte Anlage zentralvenöser (V. jugularis, V. subclavia, V. femoralis) und arterieller (A. femoralis, A. radialis, A. brachialis) Zugänge sowie verschiedene Punktionstechniken und Drainageanlagen (Pleura, Perikard, Aszites, Liquor) sollen während der Z‑WB erlernt und sicher beherrscht werden. Hämodynamische Therapie: Einer differenzierten hämodynamischen Therapie mit verschiedenen Volumenersatzstoffen und vasoaktiven Substanzen kommt bei den verschiedenen Schockformen vor allem in der Akutphase einer kritischen Erkrankung eine zentrale Rolle zu. Zur hämodynamischen Therapie gehört eine profunde klinische Einschätzung ebenso dazu wie ein leitlinienorientiertes hämodynamisches Monitoring (Tab. , Top 4.3) unter Einbeziehung von Zielparametern der Organperfusion . Die sonographische Point-of-care-Evaluation ist ein elementarer Bestandteil (Tab. , Top. 4.4). Eine kardiale Elektrotherapie (Defibrillation, Schrittmacher, Kardioversion) soll der Notfallmediziner sicher durchführen können. Bezüglich der venoarteriellen extrakorporalen Membranoxygenierung im Rahmen der Reanimation (eCPR) sind theoretische und praktische Kenntnisse Bestandteil dieses Curriculums. Notfallmediziner in Notaufnahmen, in denen diese mechanische Herz-Kreislauf-Unterstützungssysteme unter Reanimationsbedingungen oder im kardiogenen Schock eingesetzt werden, sollen sich die für die Mitbetreuung dieser Patienten erforderlichen praktischen Fähigkeiten strukturiert aneignen (extrakorporaler Life Support: ; extrakorporale Reanimation: ). Respiratorische Therapie: O 2 -Therapie und ein modernes Atemwegsmanagement spielen häufig eine zentrale Rolle in der Akutversorgung eines Notfallpatienten. Hier sollen die notfallmäßige Sicherung der Atemwege einschließlich endotrachealer Intubation sowie der Algorithmus bei schwieriger Intubation, inkl. alternativer Atemwege wie z. B. Larynxtubus und Larynxmaske, aber auch die Koniotomie sicher beherrscht werden. Eingehende Kenntnisse in der Atemphysiologie bis hin zum leitlinienorientierten und differenzierten Atmungs- und Beatmungsmanagement sind zentrale Ziele der Z‑WB. Hiermit gehen auch Kenntnisse in der modernen Analgosedierung einher. Organersatzverfahren: Zentraler Bestandteil der Notfallmedizin ist das Erkennen der Notwendigkeit des Einsatzes von Organersatzverfahren. Während es in der klinischen Akut- und Notfallmedizin primär um die Prävention/Abmilderung des Organversagens geht, beschäftigt sich der Intensivmediziner mit dem Ersatz der Organfunktion bis zu deren Rekompensation. Dennoch soll auch der Notfallmediziner die wichtigsten Indikationen zum Nierenersatzverfahren (z. B. Intoxikation, lebensbedrohliche Hyperkaliämie) kennen. (siehe auch Abschn. 11.9, Tab. ). Auch die Anlage verschiedener Zugänge zur Durchführung entsprechender extrakorporaler Verfahren unter Reanimationsbedingungen oder im kardiogenen Schock gehört zum Tätigkeitsfeld. Infektions‑/Sepsistherapie: Eine zentrale Rolle in der klinischen Akut- und Notfallmedizin kommt schweren Infektionskrankheiten bis hin zur Sepsis und zum septischen Schock zu (siehe auch Abschn. 11.7, Tab. ). Hier soll der internistische Notfallmediziner eine entsprechend rasche Diagnostik und Therapieeinleitung gemäß den „sepsis bundles“ und Sepsisleitlinien initiieren und durchführen können. Hier kommt gerade der ersten Stunde nach Sepsisdiagnose eine große Bedeutung zu („1 h bundle“), um die noch immer hohe Letalität in der Sepsis zu reduzieren. Indikation, Auswahl und Dauer einer antiinfektiven Therapie bei primären und sekundären Infektionen sollen auch bei kalkuliertem Ansatz beherrscht werden, ebenso wie Prinzipien der Hämodynamik und Herz-Kreislauf-Therapie im septischen Schock. Therapie mit Blutprodukten: Der Notfallmediziner soll sicher Blutprodukte einsetzen können. Organspendemanagement: Er muss einen potenziellen Organspender erkennen, über die theoretischen Kenntnisse für einen irreversiblen Hirnfunktionsausfall verfügen und gemeinsam mit dem interdisziplinären Team die Indikation zur organerhaltenden Therapie stellen können (siehe auch Tab. , Top 4.13). Notfalltherapie am Lebensende: Die Gesundheitssysteme in Ländern mit hohem Einkommen sind mit einer wachsenden Zahl von älteren Patienten mit zunehmenden Komorbiditäten und der steigenden Nachfrage nach technologisch fortschrittlicher Versorgung konfrontiert. Die Hälfe aller Sterbefälle in Deutschland ereignet sich im Krankenhaus und nicht selten in der Notaufnahme. Die Weichen für die Aufnahme auf eine Beobachtungsstation oder eine Intensivstation werden oft in der Notaufnahme gestellt. Somit stellt sich die Frage nach einer angemessenen Inanspruchnahme einer Akuttherapie am Lebensende. Für eine qualitativ hochwertige Versorgung am Lebensende („ end-of-life care “) sind Kompetenz in der Entscheidungsfindung, kommunikative Fähigkeiten sowie die Zusammenarbeit eines gut funktionierenden interdisziplinären Teams erforderlich . Dabei nehmen die ärztliche Indikation und der Patientenwille eine zentrale Rolle in einem komplexen multiprofessionellen und interdisziplinären Entscheidungsprozess ein. Eine der Kernaufgaben von Ärztinnen und Ärzten der Notaufnahme ist in der Beachtung und der Umsetzung ethischer Grundprinzipien zu sehen . Begleitung, Unterstützung und Führung der Angehörigen schwerstkranker Patienten ist ebenfalls Kernelement ärztlicher Prozesse im medizinischen Alltag. ) Grundlagen und Standards.Das soll gewusst und gekonnt werden.Auch Selbstschutz! In Zeiten zunehmender ambulanter und nosokomialer Infektionen und der steigenden Zahl resistenter Erreger rückt die Bedeutung der Hygiene im Arbeitsalltag eines Mediziners, insbesondere aber eines Notfallmediziners, zunehmend in den Vordergrund. Der hygienische Selbstschutz ist ebenso wichtig wie das unbedingte Vermeiden einer Übertragung potenziell tödlicher Keime. Der Akut- und Notfallmediziner soll sich nicht nur theoretisches Wissen und praktische Fähigkeiten, wie keimarmes oder steriles Arbeiten, aneignen, sondern auch die Fähigkeit besitzen, die Aufmerksamkeit eines jeden Mitarbeiters zu schärfen und seine Patienten und deren Angehörige zu schulen. Das Wissen um resistente Erreger, Isolationsmaßnahmen und grundsätzliche Hygienemaßnahmen kann die Prognose der Patienten entscheidend verbessern. Dabei ist eine gute interdisziplinäre Zusammenarbeit mit den Gesundheitsämtern und Krankenhaushygienikern unerlässlich. Die gestörten Organfunktionen des Notfallpatienten können sowohl Pharmakokinetik als auch Pharmakodynamik erheblich beeinträchtigen. Auch die Pharmakotherapie in der Schwangerschaft und im höheren Alter ist mit zu berücksichtigen. Pharmakokinetik und Pharmakodynamik: Der Akut- und Notfallmediziner soll bei seinen Patienten auf Besonderheiten bei der Pharmakotherapie gefasst sein und eine sichere und wirksame Arzneimittelbehandlung unter Berücksichtigung der Besonderheiten von Pharmakokinetik und Pharmakodynamik durchführen können. Hierzu gehört auch das Verständnis von Verteilungsvolumina, Proteinbindung und Eliminationskinetiken sowie deren Veränderungen besonders bei kritisch kranken Patienten. Weiterhin muss er Standarddosierungen, Applikationsformen, Nebenwirkungsprofile, Interaktionen und Toxizitäten notfallmedizinischer Pharmaka kennen. Antiinfektiva: Besonders wichtig in der Notaufnahme sind profunde Kenntnisse der Antibiotikatherapie zur Infektionsprophylaxe sowie zur Infektions- und insbesondere Sepsistherapie. Eine empirische, oft kalkulierte Primärtherapie soll selbstständig indiziert werden können. Von großer Bedeutung ist die Pharmakokinetik spezifischer Antibiotika, konzentrations- und zeitabhängiger Antibiotika und der Antibiotika mit unterschiedlicher Gewebegängigkeit. Ebenso wichtig sind Kenntnisse zu Dosisanpassungen, Dosierungsintervallen und prolongierten Laufzeiten. Probleme der Antibiotikatherapie bei Multiresistenzen, Antibiotikaprophylaxen vor operativen Eingriffen und der Einfluss der kritischen Erkrankung auf Plasmaspiegel und Gewebegängigkeit sollen von notfallmedizinischer Seite in Zusammenarbeit mit ABS-Programmen adressiert werden könnDas soll gewusst und gekonnt werden.gefäßmedizinischen Kenntnisse. Thromboembolische Erkrankungen: Tiefe Venenthrombose (TVT) und Lungenarterienembolie (LAE): Diagnostik und Therapie der TVT sowie der LAE erfordern rasches und leitlinienorientiertes Handeln unter Berücksichtigung der Klinik und der sonographischen sowie angiologischen oder radiologischen (CT-)Gefäßdiagnostik, ggf. unter Einbeziehung interventioneller bzw. operativer Therapieverfahren. Erforderlich sind ein adäquates Standardmonitoring sowie die kontinuierliche Überwachung von Patienten mit instabilen Kreislaufverhältnissen (z. B. intermediär hohes Risiko). Zudem müssen seltenere, aber akut lebensbedrohliche Krankheitsbilder, wie die obere Einflussstauung bei Verschluss der V. cava superior oder die Phlegmasia coerulea dolens, erkannt und unmittelbar der Therapie zugeführt werden. Akute und kritische Extremitätenischämie: Rasches interdisziplinäres Handeln ist bei der kritischen Extremitätenischämie – insbesondere der akuten Arm- oder Beinischämie – erforderlich unter Einbeziehung der konservativ-medikamentösen, interventionellen bzw. operativen Therapieverfahren. Er soll die Dringlichkeit der Therapie anhand klinischer Parameter einschätzen und eine akute Ischämie, die eine unmittelbare Therapie erfordert, von einer kritischen Extremitätenischämie abgrenzen können. Akute Mesenterialischämie: Die akute mesenteriale Ischämie ist ein in höherem Alter häufiges Krankheitsbild, das Überleben der Patienten hängt entscheidend von der raschen Diagnosestellung und Einleitung gezielter radiologischer (CT-)Diagnostik ab. Die interdisziplinäre Therapieeinleitung ist zu koordinieren. Akut entzündliche Gefäßerkrankungen (Vaskulitiden) (siehe auch Abschnitt 11.12 inkl. Tab. ): Differenzialdiagnostisch sind bei schwerkranken Gefäßpatienten auch Vaskulitiden in Betracht zu ziehen. Die frühzeitige Diagnostik und adäquate Therapie, ggf. durch Einbezug von Kollegen mit einer fundierten angiologischen Fachexpertise, sind oft ausschlaggebend für die Vermeidung oder Begrenzung schwerwiegender Organschäden. ) Grundlagen und Standards.Das soll gewusst und gekonnt werden.Das Weiterbildungsziel ist der Erwerb der für die Betreuung von akut- und notfallmedizinischen Patienten erforderlichen Kenntnisse der Klinik, Diagnostik und Therapie relevanter endokriner, diabetologischer und metabolischer Störungen. Zu erkennen sind Regulationsstörungen integrativer neuroendokriner Signalpfade beim kritisch kranken Patienten, wie Diabetes insipidus, hypophysäres Koma, Insuffizienz adenohypophysärer Achsen, insbesondere der kortikotropen Achse, und das Non-thyroidal-illness-Syndrom; zum anderen Nebennierenerkrankungen wie die Addison-Krise, mineralokortikoide Insuffizienz (auch z. B. bei adrenogenitalem Syndrom) und Phäochromozytom. Wichtig ist auch die leitlinienorientierte Hydrokortisonsubstitution bei absoluter oder relativer Nebenniereninsuffizienz, z. B. bei Patienten mit septischem Schock oder bei diagnostischen oder operativen Eingriffen. Weiterhin von Relevanz sind Schilddrüsenerkrankungen, wie thyreotoxische und Myxödemkrise, der Diabetes mellitus mit hypoglykämischer sowie hyperosmolarer hyperglykämischer Entgleisung und diabetischer Ketoazidose sowie die Besonderheiten bei schwerer Adipositas und nach bariatrischen Operationen. Differenzialdiagnostisch sollte der Akut- und Notfallmediziner auch an seltene, aber gravierende Stoffwechselstörungen denken, wie beispielsweise Gicht, metforminassoziierte Laktatazidose, arzneimittelbedingte Ketoazidose oder auch akute hepatische PorphyriDas soll gewusst und gekonnt werden.gastroenterologischer Störungen . Gastrointestinale Blutungen: Fähigkeit zur Diagnose und nichtinterventionellen Therapie von gastrointestinalen (GI-)Blutungen : Differenzierung zwischen oberer und unterer GI-Blutung und Kenntnis der verschiedenen Ursachen gastrointestinaler Blutungen. Die Unterschiede in der Risikobewertung und im Management von akuten varikösen und nichtvarikösen Blutungen sind dem Akut- und Notfallmediziner vertraut. Er beherrscht die initiale pharmakologische Behandlung bei akuten GI-Blutungen , die Kreislaufstabilisierung und das differenzierte Volumen- sowie das Blutprodukte- und Gerinnungsmanagement. Das Wissen um die Möglichkeiten der endoskopischen Blutungsstillung sowie der interventionellen Radiologie und der chirurgischen Intervention ist ein wichtiger Bestandteil der Tätigkeit. Er kennt die Maßnahmen zur Vorbereitung zur Endoskopie und der periinterventionellen Betreuung bei endoskopischen Eingriffen. Die interdisziplinäre Zusammenarbeit ist eng und umfasst die Schwerpunkte Gastroenterologie, interventionelle Radiologie und Viszeralchirurgie. Akute und chronische Lebererkrankungen: Erforderlich ist die Fähigkeit zur Diagnose und Therapie von akuten und chronischen Lebererkrankungen. Von besonderer Bedeutung sind die korrekte klinische Einordnung erhöhter Leberwerte sowie Kenntnisse über Definition, Diagnostik, Ursachen, spezifische Therapieoptionen und Risikostratifizierung des akuten und akut-auf-chronischen Leberversagens. Weitere wichtige Bestandteile der Tätigkeit sind die Diagnostik und Therapie der Komplikationen der dekompensierten Leberzirrhose, insbesondere von Aszites, spontan-bakterieller Peritonitis, hepatorenalem Syndrom, hepatischer Enzephalopathie, und das nichtinterventionelle Management der varikösen Blutung . Fundierte Kenntnisse der Schockbehandlung und des differenzierten Blutprodukte- und Blutgerinnungsmanagement sind notwendig. Die Abdomensonographie mit den Schwerpunkten Leber und Lebergefäße soll beherrscht werden. Die interdisziplinäre Zusammenarbeit erfolgt insbesondere mit der Gastroenterologie. Erkrankungen der Gallenwege: Erforderlich ist die Fähigkeit zur Diagnose und Therapie von Erkrankungen der Gallenwege. Der Akut- und Notfallmediziner beherrscht die Differenzialdiagnostik des rechtseitigen Oberbauchschmerzes und des prä-/intra-/posthepatischen Ikterus. Die Behandlung der akuten Cholezystitis und Cholangitis als Erkrankungen der Gallenwege steht in der Notaufnahmestation im Vordergrund. Eine enge interdisziplinäre Zusammenarbeit mit der Gastroenterologie und der Viszeralchirurgie ist unabdingbar. Das Wissen um die Indikationsstellung und das richtige Timing von Cholezystektomie und notfallmäßiger endoskopischer retrograder Cholangiographie (ERC) ist notwendig. Die Abdomensonographie mit den Schwerpunkten Leber, Gallenblase, Gallenwege soll beherrscht werden. Pankreatitis: Der Akut- und Notfallmediziner kennt die Prinzipien der Diagnostik und Therapie der akuten Pankreatitis und des akuten Schubs einer chronischen Pankreatitis. Er ist vertraut mit den Diagnosekriterien und Ursachen der akuten Pankreatitis und kennt die Verlaufsformen und Komplikationen der akuten Pankreatitis. Er beherrscht die differenzierte Volumentherapie bei akuter Pankreatitis und kennt Maßnahmen zur Beurteilung des intravasalen Volumens. Er weiß um die Prinzipien der Schmerztherapie bei akuter Pankreatitis. Wichtig ist das Wissen um die Indikationen zur CT-Diagnostik bei akuter Pankreatitis im Hinblick auf diagnostische bzw. prognostische Fragestellungen. Er hat profunde Kenntnisse der Abdomensonographie mit den Schwerpunkten Pankreas, Gallenwege, Gallenblase und beherrscht die Messung und Interpretation des intraabdominellen Drucks. Die interdisziplinäre Zusammenarbeit erfolgt schwerpunktmäßig mit der Gastroenterologie. Erkrankungen des Magen-Darm-Trakts: Der Akut- und Notfallmediziner soll die Fähigkeit zur Diagnose und Therapie von Erkrankungen des Magen-Darm-Trakts besitzen. Im Vordergrund steht die differenzialdiagnostische klinische Beurteilung abdomineller Schmerzen sowie von Erbrechen und Diarrhö. Er hat profunde Kenntnisse von Diagnostik und Therapie des mechanischen und paralytischen Ileus, der gastrointestinalen Ischämie, der Divertikulitis, des akuten Schubs chronisch-entzündlicher Darmerkrankungen und von Hohlorganperforationen und konsekutiver Peritonitis. Eine enge interdisziplinäre Zusammenarbeit mit den Fachbereichen Gastroenterologie, Viszeralchirurgie und Radiologie ist notwendig. Er ist mit den Indikationen und Verfahren der radiologischen Abdominaldiagnostik vertraut und beherrscht die Abdomensonographie. Er kennt die Indikationen zur endoskopischen Bolus- bzw. Fremdkörperextraktion und ist mit der periinterventionellen Betreuung bei endoskopischen Eingriffen vertraut. ) Grundlagen und Standards.Das soll gewusst und gekonnt werden.Der Anteil der über 70-jährigen Notfallpatienten liegt in der Notaufnahme bei 30 % . Insofern ist es für den klinischen Notfallmediziner wichtig, Spezifika dieser Patienten zu kennen . Dazu zählen unter anderem das Verständnis von Grundbegriffen der Geriatrie und Gerontologie, wie Sarkopenie und Frailty , altersspezifische Einschränkungen der Organfunktionen, geriatrische Syndrome, kognitive Dysfunktion, Multimorbidität, Polypharmazie und Polypragmasie sowie Arzneimittelinteraktionen und die häufigsten Verordnungskaskaden . Besonderheiten in Symptomatik und Diagnostik: Bei geriatrischen Patienten zeigen sich akute Erkrankungen meist durch unspezifische Symptome wie funktionelle oder kognitive Veränderung, Stürze oder Delir. Bis zu 20 % der älteren Patienten geben im Notfall unspezifische Beschwerden an und 51–59 % dieser Älteren mit unspezifischen Beschwerden haben ein akut behandlungsbedürftiges Problem . Auch in der Diagnostik sind die Besonderheiten beim geriatrischen Patienten zu beachten. Durch die meist vorbestehende Multimorbidität können Akutsymptome überlagert und Befunde fehlinterpretiert werden. Dies kann auch Auswirkungen auf die Beurteilung der Behandlungsdringlichkeit haben . Realistische Therapieoptionen, Patientenwünsche und Vorsorgeplanung: Wesentlich ist es, die machbaren Therapieoptionen in der Akut- und Notfallmedizin mit den Patientenwünschen in Kooperation mit dem Patienten abzugleichen, ggf. auch mit Angehörigen, betreuenden Ärzten und Pflegeeinrichtungen und auch in Absprache mit Geriatern und Palliativmedizinern. Die vorausschauende Vorsorgeplanung („advance care planing“) stellt einen wichtigen Pfeiler der adäquaten Behandlung dar. ) Grundlagen und Standards.Das soll gewusst und gekonnt werden.Obwohl es für Deutschland keine Zahlen gibt, zeigt eine internationale Studie , dass länderspezifisch 24–42,5 % der Krebsfälle in der Notaufnahme diagnostiziert werden. Zusätzlich stellen sich aber auch viele Patienten mit einer schon bekannten Krebserkrankung in der notfallmedizinischen Versorgung vor, aufgrund von Komplikationen entweder im Rahmen der Krebserkrankung oder therapieassoziiert. Der Akut- und Notfallmediziner soll nicht nur die typischen Komplikationen von Krebserkrankungen und deren Therapie, sondern auch Krebsneuerkrankungen erkennen und entsprechende diagnostische und therapeutische Schritte in Kooperation mit einem Hämatoonkologen bzw. anderen Fachdisziplinen einleiten können. Psychoonkologie: Patienten stellen sich entweder mit Komplikationen der Krebserkrankung, deren Therapie oder mit ersten Symptomen einer Krebserkrankung vor. Die Versorgung dieser Patienten stellt häufig eine besondere psychische Belastung für alle Beteiligten dar. Eine entsprechende empathische Kommunikation im notfallmedizinischen Setting sollte sowohl im Team als auch mit dem Patienten und den Angehörigen durchgeführt werden. Auch palliativmedizinische Aspekte einer Versorgung in der Notfallaufnahme (siehe Tab. zu Abschn. 11.10.) sollten bekannt sein und umgesetzt werden. ) Grundlagen und Standards.Das soll gewusst und gekonnt werden.Infektionserkrankungen gehören nach wie vor zu den häufigsten Ursachen für eine Vorstellung von Patienten in der Notaufnahme. Das Spektrum reicht von Bagatellinfektionen bis hin zu schweren, akut lebensbedrohlichen Infektionen. Gerade bei schweren Infektionen, wie z. B. der Sepsis, ist die zeitnahe Diagnose und der Beginn einer adäquaten Therapie essenziell. Die Komplexität der Erkrankungen steigt dabei nicht zuletzt aufgrund des Alters und zunehmender Komorbiditäten der versorgten Patienten weiter an. Zusätzlich ist die Notaufnahme für viele Patienten mit der Frage einer Prophylaxe nach möglicher Exposition gegenüber einem infektiösen Erreger die erste Anlaufstelle. Es ist wichtig, dass der Akut- und Notfallmediziner Erfahrung in der klinischen Präsentation, schnellen, aber rationalen Diagnostik und adäquaten Therapie der häufigsten Infektionserkrankungen besitzt. Neben dem Erkennen des klinischen Bilds spielt nicht nur das Behandeln einer akuten Infektion eine wichtige Rolle, sondern auch das Bahnen eines weiteren stationären Aufenthalts durch die initiale Diagnostik und Therapie. Neben dem Behandeln von Infektionen muss der Akut- und Notfallmediziner auch Kenntnisse in der Indikationsstellung und Durchführung der wichtigsten Postexpositionsprophylaxen besitzen. Es wird außerdem vorausgesetzt, dass der Akut- und Notfallmediziner Kenntnisse über die wichtigsten antiinfektiven Therapien hat. Er soll in der Lage sein, letztere an lokale Gegebenheiten sowie im Rahmen von „Antibiotic Stewardship“ in Zusammenarbeit mit Kollegen anderer Fachrichtungen anzupassen. ) Grundlagen und Standards.Das soll gewusst und gekonnt werden.Die kardiovaskuläre Notfall‑, Akut- und Intensivmedizin macht aufgrund der Häufigkeit kardiovaskulärer Erkrankungen einen beträchtlichen Teilbereich dieser Disziplinen aus. Bei einem relevanten Anteil der Patienten ist eine kompetente Versorgung zeitkritisch, um die bestmögliche Diagnostik und Therapie zu gewährleisten. Bei den kardialen Erkrankungen ist von besonderer Bedeutung, dass nicht nur einige eigenständige Krankheitsbilder von zentraler Relevanz sind, sondern auch kardiovaskuläre Erkrankungen als Begleitumstände anderer Krankheitsbilder hochprävalent sind. Erforderlich sind grundlegende Kenntnisse der kardiovaskulären Anatomie, Physiologie und Pkardialen Erkrankungen als eigenständige Krankheitsbilder und auch deren Relevanz als Komorbiditäten, der Verfahren zur Diagnosestellung (inkl. bildgebender Verfahren) und Kenntnisse der Therapie akuter Funktionsstörungen. Das Erkennen eines kardialen Problems (myokardial, strukturell, rhythmologisch) muss gewährleistet sein mit dem Ziel, häufige zeitkritische Therapiemaßnahmen und nachgeschaltete kardiologische Spezialdiagnostik und/oder -therapie einzuleiten. Insbesondere sollen notwendige medikamentöse Therapiestrategien selbstständig angewendet und Sofortmaßnamen, wie kardiopulmonale Reanimation, Kardioversion und Defibrillation, eigenständig durchgeführt werden können. Kenntnisse der (Notfall‑/Basis‑)Echokardiographie sollen vorhanden sein, ebenso wie grundlegende Kenntnisse der Sonographie und der Befundung konventioneller Röntgenaufnahmen des Thorax sowie der thorakalen Computertomographie und kardialen Magnetresonanztomographie. Hypertensive Dringlichkeit und hypertensiver Notfall : Bei einer Prävalenz der arteriellen Hypertonie von 30–50 % in Deutschland ist es nicht verwunderlich, dass jeder 10. Hochdruckpatient im Lauf seines Lebens eine hypertensive Notfallsituation erleidet. Bei etwa 5 von 1000 Fällen ist eine hypertensive Notfallsituation der Aufnahmegrund in der Notaufnahme. Blutdruckwerte > 180/110 mm Hg mit Organschädigung (akutes Lungenödem: 30 %; Schlaganfall: 20 %; Myokardinfarkt: 20 %; akute Nierenschädigung: 5 %) werden als hypertensiver Notfall klassifiziert und bedürfen einer sofortigen Behandlung in der Notaufnahme. Die Letalität eines nicht adäquat behandelten hypertensiven Notfalls wird auf etwa 10 % innerhalb der nächsten 5 Jahre geschätzt. Blutdruckwerte > 180/110 mm Hg ohne Endorganschädigung werden als hypertensive Dringlichkeit bezeichnet; hierbei wird eine zeitnahe ambulante Abklärung empfohlen. Kardiologische(s) Notfalldiagnostik, -behandlung und -monitoring: Für die zeitgerechte Diagnostik, Überwachung und Therapie von Akut- und Notfallpatienten ist eine Reihe von Fähigkeiten von besonderer Relevanz. Dazu gehören die Notfallechokardiographie/-sonographie, das hämodynamische (Basis‑)Monitoring und das Management der akuten Herzinsuffizienz (inklusive des akuten Rechtsherzversagens und infolge eines dekompensierten Vitiums), das Erkennen und Versorgen des akuten Koronarsyndroms (ACS) inkl. des Patienten mit Myokardinfarkt (NSTEMI und ST-Hebungs-Infarkt [STEMI]), das Management des (infarktbedingten) kardiogenen Schocks , das Erkennen und die Behandlungsinitiierung eines Perikardergusses bzw. einer Perikardtamponade, Kenntnisse zu mechanischen Kreislaufunterstützungsverfahren sowie Notfallrhythmusdiagnostik und akutes Arhythmiemanagement. Darüber hinaus sind die Fähigkeiten zur Reanimation/Advanced Life Support (ALS) essenziell, ebenso wie diejenigen zu den Leitsymptomen der Themenkomplexe „Dyspnoe und Brustschmerz“, „Hypertensive Notfälle“, „Hypotonie und Synkope“ und „Herzerkrankung bei speziellen Patientengruppen“ (HIV, Schwangerschaft, Tumorpatienten, Patienten mit pulmonalarteriellem Hochdruck, Transplantationspatienten, akutes Rechtsherzversagen). Die Pharmakotherapie in der kardiovaskulären Notfallmedizin soll beherrscht werden, ebenso wie das Basismanagement bei notfälligen Devicepatienten mit Herzschrittmacher oder Defibrillator . Spezifische diagnostische und therapeutische Fähigkeiten: Der Akut- und Notfallmediziner soll über Kenntnisse verfügen, die es ihm ermöglichen, eigenständig oder in Kooperation folgende diagnostische und therapeutische Maßnahmen in Bezug auf oben genannte Krankheitsbilder anzuwenden. Die diagnostischen Fähigkeiten beinhalten: die EKG-Beurteilung ; die transthorakale Echokardiographie sowie Grundzüge der transösophagealen Echokardiographie ; den fokussierten Gefäßultraschall ; Indikation zur Linksherzkatheteruntersuchung; Indikation zur Rechtsherzkatheteruntersuchung; Basiskenntnisse in der Herzschrittmacher- und Deviceabfrage; Indikation zur Perikardpunktion und Myokardbiopsie; kardiale Biomarker; Basiskenntnisse in der Indikationsstellung und Diagnostik mittels Kardio-CT und Kardio-MRT. Die therapeutischen Fähigkeiten beinhalten: die Absprache mit einem Facharzt für Innere Medizin und Kardiologie zur Indikationsstellung zur akuten perkutanen Koronarintervention (PCI); die leitliniengerechte Initiierung einer Inotropika‑/Vasopressorentherapie; die passagere perkutane Schrittmachertherapie, Kardioversion, Defibrillation und Überstimulation; die Postreanimationsphase einschließlich des zielgerichteten Temperaturmanagements; die Indikationsstellung mechanischer Herz-Kreislauf-Unterstützungssysteme nach Anlegen in einem Herzkatheterlabor oder im Schockraum; die systemische Thrombolyse sowie Grundprinzipien der Thrombektomie und Thrombusfragmentierung inklusive der Indikationsstellung in Absprache mit dem jeweiligen Fachkollegen; ein adäquates medikamentöses Therapiemanagement des hypertensiven Notfalls. ) Grundlagen und Standards.Das soll gewusst und gekonnt werden.Grundlagenkenntnisse der Nierenphysiologie, Nierenfunktion und Urindiagnostik sind erforderlich, ebenso das Wissen um die für die Akutmedizin relevanten akuten und chronischen Nierenerkrankungen. Die akute Nierenfunktionseinschränkung („acute kidney injury“, AKI) ist das häufigste sepsisassoziierte Organversagen in der Notfall- und Intensivmedizin. Dafür werden Kenntnisse und Handlungskompetenz benötigt: zur spezifischen Pathophysiologie des AKI sowie des Organ-Crosstalks; zur Diagnostik, Differenzialdiagnostik und Therapie kardiorenaler, hepatorenaler sowie pulmorenaler Syndrome als Manifestation von Systemerkrankungen; vertieft zur Indikation und Durchführung einer medikamentösen Akuttherapie des AKI sowie der Indikationen zur Einleitung einer Nierenersatztherapie; zur Beherrschung der standardisierten Nierensonographie, unter anderem zum Ausschluss prä- und postrenaler Ursachen des AKI; zum Verständnis für Langzeitfolgen eines AKI einschließlich der Notwendigkeit einer nephrologischen Nachsorge. Chronische Nierenerkrankung : Einteilung der chronischen Nierenerkrankung („chronic kidney disease“, CKD) in Schweregrade und der sich daraus ergebenden medizinischen Konsequenzen; Kenntnisse der sich daraus ergebenden Labor- sowie Urindiagnostik; Verständnis, dass Patienten mit chronischer Dialysetherapie rasch an Zentren mit Dialysemöglichkeit und Nephrologie weiterzuvermitteln sind. Thrombotische Mikroangiopathie (TMA) : Diagnostik und Therapie der verschiedenen Formen einer mit einem AKI einhergehenden TMA, insbesondere einer thrombotisch-thrombozytopenischen Purpura (TTP) und eines hämolytisch-urämischen Syndroms (HUS) anhand der klinischen Trias „Thrombopenie, Hämolyse und Fragmentozyten“; Verständnis, dass Patienten mit einem TMA-Verdacht sofort in nephrologische oder hämatologische Behandlung weiter verwiesen werden müssen. Elektrolytstörungen : Physiologie und Pathophysiologie der Volumen- und Osmoregulation sowie der Homeostase des Kalium‑, Kalzium‑, Phosphat- und Magnesiumhaushalts; Elektrolytstörungen und die damit verbundenen akut bedrohlichen Krankheitsbilder der Notfallmedizin; Akuttherapie vital bedrohlicher Elektrolytstörungen einschließlich der Indikationen zur Durchführung einer Akutdialyse . Störungen des Säure-Basen-Haushalts : Bedeutung der Aufrechterhaltung eines konstanten pH-Werts für Zellphysiologie und Elektrolythaushalt sowie der Kompensationsmechanismen zur Konstanthaltung des pH-Werts; zielführende Diagnostik einer Störung des Säure-Basen-Haushalts inkl. der Durchführung einer arteriellen oder venösen Blutgasanalyse und deren Interpretation, auch mit Bestimmung der Anionenlücke. ) Grundlagen und Standards Das soll gewusst und gekonnt werden.]. Die demographische Entwicklung und die Weiterentwicklung medizinischer Behandlungsmöglichkeiten führen dazu, dass insbesondere die Innere Medizin mit einer zunehmenden Zahl an multimorbiden Patienten mit komplexen Erkrankungssituationen und palliativmedizinischem Behandlungsbedarf konfrontiert ist. Neben den onkologisch/hämatologisch Erkrankten betrifft dies auch die große Gruppe der Patienten mit progredientem Organversagen (COPD, Herzinsuffizienz, Niereninsuffizienz) sowie hochaltrige, gebrechliche und demente Patienten. In Bezug auf eine angemessene Therapiezielfindung stellen die sehr unterschiedlichen Krankheitsverläufe die Behandler – insbesondere in Akut- und Notfallsituationen – häufig vor große Herausforderungen (Abb. ). Notfälle bei Patienten in palliativer Situation lassen sich in folgende Konsultationsanlässe clustern : Probleme ohne Zusammenhang zur Grunderkrankung; neu aufgetretene Probleme im Zusammenhang mit der Grunderkrankung; therapieassoziierte Probleme; Exazerbation bekannter Symptome; „total pain“; Überforderung des sozialen Umfelds. Es gilt in diesen Situationen – basierend auf der mutmaßlichen Prognose – neben dem üblichen notfallmedizinischen Therapieziel „Hilfe zum Leben“, auch weitere mögliche Ziele zu eruieren, wie „Hilfe im Sterben“ oder „Hilfe zur rechten Zeit zu sterben, bis dahin aber bestmöglich zu leben“ . Um den in „Grundlagen und Standards“ genannten Anforderungen an eine palliativmedizinisch orientierte Notfallbehandlung gerecht zu werden, sollen die in Tab. aufgeführten Weiterbildungsziele (WZ; siehe auch ) berücksichtigt werden. ) Grundlagen und Standards.Das soll gewusst und gekonnt werden.Erforderlich sind grundlegende Kenntnisse der Lungenanatomie, -physiologie und -pathophysiologie, Kenntnisse der für die klinische Akut- und Notfallmedizin relevanten akuten und chronischen Lungenerkrankungen, der Verfahren zur Diagnosestellung (inkl. Materialentnahme) und Kenntnisse der Therapie akuter Funktionsstörungen des Organs. Das Erkennen eines A(temwegs)- und B(eatmungs)-Problems soll gewährleistet sein und die Diagnose einer akuten Lungenerkrankung inkl. der Lungenarterienembolie soll selbständig gestellt werden. Notwendige Therapieverfahren, wie Maskenbeatmung, nichtinvasive Beatmung, Notfallintubation und invasive Beatmung, sowie grundlegende Beatmungseinstellungen sollen selbstständig durchgeführt werden können. Grundlegende Kenntnisse der Lungen- und Pleurasonographie sollen vorhanden sein, ebenso wie grundlegende Kenntnisse der Echokardiographie und der Befundung konventioneller Röntgenaufnahmen des Thorax. Er soll selbstständig einen Pneumothorax/Hämatothorax diagnostizieren, Kenntnisse der klinischen und radiologischen Zeichen einer Spannungskomponente bei Pneumothorax haben und stadienadaptiert therapieren können (inkl. Anlage einer Thoraxdrainage). Die Indikationsstellung für die diagnostische und therapeutische Bronchoskopie (z. B. bei der Aspiration von Fremdkörpern, aber auch z. B. bei Polytrauma) soll beherrscht werden; ggf. kann die Durchführung in Kooperation mit einem Pneumologen erfolgen. Bei respiratorischem Versagen jedweder Art und Genese sind grundlegende Kenntnisse der Pathophysiologie und der Behandlung zur Sicherstellung der Oxygenierung und Dekarboxylierung erforderlich. Bei obstruktiven Ventilationsstörungen sind Kenntnisse zu Indikationen und Kontraindikationen einer antiobstruktiven Therapie erforderlich, ebenso Kenntnisse und praktische Erfahrungen mit verschiedenen Möglichkeiten der Beatmung (nichtinvasiv, invasiv). Die Fähigkeit zur Einleitung einer nichtinvasiven Beatmung und Kenntnisse grundsätzlicher Beatmungseinstellungen zur Oxygenierung und Dekarboxylierung, insbesondere zur Therapie der Hyperkapnie, sollen vorhanden sein. Kenntnisse des typischen Verlaufs, der Akut- und Langzeitprognose und akuter und mittel-/langfristiger therapeutischer Optionen bei obstruktiven Ventilationsstörungen sind erforderlich. Bei restriktiven Ventilationsstörungen sind Kenntnisse erforderlich zu Indikationen und Kontraindikationen einer Beatmungstherapie, bezüglich. Bei Lungenblutungen sollen im Kontext der Grunddiagnose gekannt und beherrscht werden: Ätiologie und Pathogenese, Ersteinschätzung, therapeutische Optionen (in interdisziplinärer Zusammenarbeit mit interventionellen Radiologen und Thoraxchirurgen) sowie deren stadienadaptierte Therapie zur akuten Sicherung des Gasaustauschs. Interstitielle Lungenerkrankungen , Vaskulitiden, Lungenblutungen und pulmonal-arterielle Hypertonie sollen in enger Zusammenarbeit mit dem Pneumologen und/oder Rheumatologen behandelt werden können. Er soll Lungenarterienembolien erkennen und behandeln können und Kenntnisse der Pathophysiologie und der Risikostratifizierung haben. Ebenso sind eingehende Kenntnisse der Therapieoptionen bis hin zu interventionellen Therapieverfahren und der Indikation zur venoarteriellen ECMO erforderlich. Entzündliche und infektiöse Lungenerkrankungen (Bronchitis, Bronchiolitis, Pneumonien mit und ohne Erguss/Empyem und schwere Pneumonien mit septischem Verlauf) sind häufige akutmedizinische Krankheitsbilder. Hier soll die Fähigkeit zur risikoadaptierten (Differenzial‑)Diagnosestellung und oft rasch notwendigen Therapieeinleitung, v. a. einer effektiven kalkulierten antiinfektiven Therapie, vorhanden sein. Wichtig sind auch Kenntnisse häufiger und akuter Komplikationen von malignen Erkrankungen der Lunge und des Thorax sowie von Lungenmetastasen und deren Prognose. ) Grundlagen und Standards.Das soll gewusst und gekonnt werden.Entzündlich rheumatische Erkrankungen sind durch autoimmunologische bzw. autoinflammatorische Prozesse des Immunsystems gekennzeichnet, die mit einer entkoppelten immunologischen Entzündungsreaktion und folgender Zerstörung des körpereigenen Gewebes einhergehen. Zusätzlich werden auch nichtentzündliche Gelenkerkrankungen wie die Arthrose der Rheumatologie zugeordnet. Entscheidend für die bestmögliche Betreuung von Patienten mit rheumatologischen Erkrankungen in der Notaufnahme ist eine fundierte Diagnostik . Zu den akutmedizinisch relevanten rheumatologischen Erkrankungen zählen entzündliche Gelenkerkrankungen, Kollagenosen, das Antiphospholipidantikörpersyndrom (APS), die Polymyalgia rheumatica, die Riesenzellarteriitis (inkl. Arteriitis temporalis), die Kleingefäßvaskulitiden (ANCA-assoziierte Vaskulitiden) und autoinflammatorische Syndrome inkl. kristallinduzierter Arthritiden. Die antiinflammatorische, immunsuppressive und immunmodulatorische Therapie rheumatologischer Erkrankungen birgt für die Patienten die Gefahr infektiöser Komplikationen. Dies erfordert Kenntnisse über die Besonderheiten der Diagnostik und Therapie bei Infektionen unter immunsuppressiver Therapie. Relevant sind diejenigen Kenntnisse in klinischer Toxikologie, die für eine selbstständige Diagnostik und (Primär‑)Behandlung der häufigsten und gefährlichsten Intoxikationen nötig sind. Bei der zielführenden Diagnostik und Therapie der zahlreichen und komplexen Medikamentenintoxikationen empfiehlt sich die Kooperation mit den Giftnotrufzentralen. |
Subsets and Splits