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BackgroundChildren infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) usually present minimal symptoms or are asymptomatic. Nevertheless, a subset of children 2-6 weeks after the initial SARS-CoV-2 infection develops a postinfectious SARS-CoV-2-related multisystem inflammatory syndrome in (MIS-C). Recently, transient expansion of TRBV11-2 T cell clonotypes in MIS-C was associated with signatures of inflammation and T cell activation, however, the underlying pathophysiology of MIS-C is not fully understood [1].ObjectivesThe purpose of our project is to characterize the complexity of cell populations and capture cellular heterogeneity to uncover the regulatory networks and interactions that are disrupted during MIS-C flare with simultaneous profiling of gene expression and open chromatin regions from the same nuclei.MethodsSamples of peripheral blood mononuclear cells from patients with MIS-C diagnosed at the University Children's Hospital, University Medical Center Ljubljana, were collected during the initial presentation before any treatment and at 6-12 months in remission. The primary aim is to identify which regulatory networks are driving inflammation in MIS-C flare, for which we are performing single cell Multiome ATAC + Gene Expression Sequencing. To enable simultaneous profiling of epigenomic landscape and gene expression from the same nuclei, we are using Chromium Next GEM Single Cell Multiome ATAC + Gene Expression kit from 10X Genomics.ResultsWe included 32 patients with MIS-C from whom we collected paired blood samples during the initial presentation before treatment and at 6-12 months in remission. In single cell multiomic experiment we included 10 patients with paired samples, with the most viable cell count prior cryopreservation. All samples that are included into multiomic single cell analysis have 75% - 99% viability prior cryopreservation. In the protocol the key is to remove remaining granulocytes causing high mitochondrial RNA burden and extensively optimize the dilution factor of lysis buffer and the length of cell lysis step in order to get intact nuclei with no significant blebbing. Afterward, the single cell ATAC libraries as well as single-cell gene expression libraries are constructed and sequenced. Data are undergoing pairwise analysis to compare the cell population heterogeneity, expression profile and open chromatin landscape in the time of the initial presentation of MIS-C and in the remission, with Cell ranger software as well as with R package scREG [2], and custom scripting. In the second step we will inspect if the resulting altered transcriptomic signature from single-cell experiment is present on larger cohort. In that regard, we will perform bulk transcriptomic profiling on all paired collected samples during the initial presentation of MIS-C before treatment and at 6-12 months in remission.ConclusionThe results of this project are expected to enlighten the underlying pathophysiology of MIS-C flare and thus support clinical decision on more targeted treatment. The identified disrupted networks during MIS-C flare could lead the way to establish an early diagnosis and improve long-term outcome, including prevention of myocardial and neuropsychological impairment. Moreover, a better understanding of the disrupted regulatory networks that are driving inflammation in MIS-C, could lead to new insights into diseases with similar clinical presentations as is Kawasaki Disease.References[1]Sacco, K., Castagnoli, R., Vakkilainen, S. et al. Immunopathological signatures in multisystem inflammatory syndrome in children and pediatric COVID-19. Nat Med 28, 1050–1062 (2022).[2]Duren, Z., Chang, F., Naqing, F. et al. Regulatory analysis of single cell multiome gene expression and chromatin accessibility data with scREG. Genome Biol 23, 114 (2022).AcknowledgementsThis research was supported by Slovenian research agency grant J3-3061 and Interreg ITA-SLO project Cattedra.Disclosure of InterestsNone Declared.
ABSTRACT
Multisystem inflammatory syndrome in children (MIS-C) is a rare, but serious and potentially life-threatening hyperinflammatory complication of infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It typically occurs 2-6weeks after primary infection, which is often mild or asymptomatic. The main symptoms are fever, gastrointestinal symptoms, variable rash, conjunctivitis, mucous membrane, and heart involvement. The differential diagnosis is very broad and includes many other causes of fever such as Kawasaki disease, acute COVID-19, bacterial sepsis, staphylococcal and streptococcal toxic shock syndrome, and gastrointestinal infections. Treatment of MIS-C requires a multidisciplinary approach and early immunomodulatory treatment to stop the hyperinflammatory response. Patients receive high doses of intravenous immune globulins and systemic glucocorticoids. Biologic drugs directed against IL-1 or IL-6 are required in patients with refractory or more severe diseases. With immediate treatment, the prognosis is generally good and cardiac function usually returns to normal. © 2023 Elsevier Inc. All rights reserved.
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Background: In contrast to adults, children are less likely to develop serious disease upon infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) but are at increased risk for infammatory and autoimmune diseases linked to the virus (1). The reported incidence of multisystem infammatory syndrome in children (MIS-C) varied from 0.2 to 11.4/100,000 persons under 21 years (2,3). 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 infam-matory and autoimmune diseases linked to SARS-CoV-2 infection and coronavirus (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 and adolescents (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 587,053 children and adolescents. Only patients who had 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 the number of patients 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 and adolescents were diagnosed with infammatory and autoimmune diseases linked to SARS-CoV-2 (Figure 1). 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 921 children and adolescents after SARS-CoV-2 infection and one in 5870 of all children and adolescents. Cumulative incidence of MIS-C since the start of the pandemic was 17/100,000 children and adolescents. Until December 31, 2021, 92,139 children and adolescents (15.7 %) received at least one dose of COVID-19 vaccine. Three patients presented with infammatory/autoimmune disease after COVID-19 vaccination, including 2 patients with MIS-C and one patient with myositis. 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 and adolescents were hospitalised with severe COVID-19. Seven patients from our cohort were vaccinated against COVID-19 median 8 months after MIS-C and further 6 patients had a SARS-CoV-2 reinfection 3-14 months after MIS-C. None of them experienced SAE or recurrence of MIS-C. COVID-19=coronavirus disease, FVG=Friuli Venezia Giulia region in Italy, MIS-C=multisystem infammatory syndrome in children, SARS-CoV-2=severe acute respiratory syndrome coronavirus 2 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, however long-term data are lacking. 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.
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Background: Despite the low rate of neurological defcits following the SARS-COV-2 infection in the pediatric population, children and adolescents who develop multisystem infammatory syndrome (MIS-C) after being infected with SARS-COV-2 are at a higher risk for neurological abnormalities and brain injury, increasing the risk of adverse cognitive and psychiatric outcome. Objectives: Given the increased risk of central nervous system impairment we chose to conduct a prospective study looking at the cognitive and psychosocial outcome of patients with MIS-C. Methods: Our study included 27 of the 29 patients between 2 to 18 years of age (M = 11.1, SD = 4.4) who were treated for MIS-C from the onset of the SARS-COV-2 pandemic until the beginning of May 2021 at the only tertiary care pedi-atric immunology center in Slovenia. We assessed these patients 6 months after diagnosis using the age-appropriate Wechsler intelligence scales and a battery of neuropsychological test measuring attention, executive function, memory and fne motor skills. We also asked parents to report on patients' psychosocial outcome using the Achenbach Child Behavior Checklist. Results: By using Bayesian statistics to take into account parental education and any potential pre-morbid learning difficulties we found no evidence of impairment on measures of intelligence. However, the posterior distribution of scores on neuropsychological measures indicated that a signifcant proportion of patients scored 1SD bellow expected levels on measures of attention (31%), executive function (28%) and visual memory (35%). Increased symptoms of depression, anxiety and attention difficulties were also reported by parents, although their extent did not rise to a clinically signifcant level. Conclusion: The fndings from our cohort suggest that the cognitive and psychosocial outcome of patients with MIS-C is generally favorable, although up to 35% may experience specifc neuropsychological defcits more than 6 months after diagnosis. The most commonly impaired cognitive domains seem to be attention, executive function and visual memory.
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Introduction: To date, there are few data on SARS-CoV-2 infection in children with rheumatic diseases (RD), including children treated with immunosuppressive therapy. Objectives: To investigate the clinical presentation of SARS-CoV-2 infection in children with RD, whether it affects the disease activity, and if therapy affects the serological response to SARS-CoV-2 in the long term. Methods: This is a partially retrospective study with prospective follow-up of disease activity and serology after SARS-CoV-2 infection. We collected children with RD, treated at UCH Ljubljana, Slovenia, who were exposed to SARS-CoV-2. If a member of the same household had a confirmed SARS-CoV-2 infection, we performed a serology test even if the patient had no symptoms. We collected demographical data, clinical presentation of infection, diagnosis of RD, therapy and disease activity at the time of infection and location of the contact with SARS-CoV-2. Serology for SARS-CoV-2 was tested several times during the follow-up. Results: From July 2020 to May 2021, we identified 36 children with RD, who had been exposed to SARS-CoV-2 (31 female;median age 14.8 years, range 2.5-23 years). The majority (28/36;77%) had juvenile idiopathic arthritis. At the time of SARS-CoV-2 infection 14/36 (39%) children were taking TNFα inhibitors (TNFαi), 4 in combination with methotrexate (MTX), 5/36 MTX only, 2/36 cyclosporine, 2/36 mycophenolate mofetil, 2/36 low dose methylprednisolone, 1/36 azathioprine, 1/36 baricitinib, 1/ 36 anakinra, 1/36 abatacept, 1/36 tocilizumab, 1/36 NSAID, and 5/36 children were in remission without therapy. Seven children (7/36;19%) had a moderate disease course of COVID -19 (fever, musculoskeletal pain, headache, fatigue). Eight children (8/ 36;22%) had no signs at all, but serology was performed in 7 and was positive. A total of 10/36 children (27%) lost the sense of smell and taste. Among them, 2 had no other signs. No child needed hospitalization. In the group of children with moderate COVID -19 infection (4/7 female;median age 15 years, range 11-21 years), 3 were treated with TNFαi, 2 with MTX, 1 with baricitinib, and 1 with NSAIDs. One child had recurrence of uveitis 5 months after infection. So far, no other relapse had been noticed. Contact was known in 27/36 (75%) children and 24/36 (88%) got the virus at home. Only in 9/36 children (25%) the smear was taken and was positive. Serology was determined in 11 children in the first 3 months after infection and in 6 children later than 3 months after infection. In 4 children, serology test had already been performed twice. Only one child treated with adalimumab at the time of the acute infection lost protection at 5 months, while children treated with baricitinib, anakinra and methylprednisolone still had protective antibodies 5-6 months after acute infection. Conclusion: From these preliminary results it appears that children with RD, regardless of the therapy they are taking, are not at serious risk for SARS-CoV-2 infection or for disease relapse. It is possible that the therapy they are taking affects the serological response in the long term, but this needs to be clarified in the future.
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Background: Multisystem inflammatory syndrome in children (MIS-C) is a rare complication of SARS-CoV-2 infection in the pediatric population, caused by extensive activation of immune system. The understanding of the distorted immune response is still in the early stages. Objectives: To analyze comprehensively immune profile in MIS-C patients including detailed serologic response to SARS-CoV-2 in comparison with control groups. Methods: Blood samples of consecutive MIS-C patients were collected at admission. Flow cytometric analysis of all lymphocyte populations including T and B cell differentiation was performed. Immunophenotyping was performed by six-color panels for the detection of lymphocyte subpopulations. Anti-SARSCoV-2 specific antibodies were measured in the patients serum. The IgA and IgG antibodies against S protein, the IgG S1 and S2 specific antibodies, antibodies against nucleoprotein and neutralising antibodies were measured. Patients were assessed for a wide range of auto-antibodies, namely ANA, anti-ENA (Jo-1, PL-7, PL-12, SRP, Mi-2, Ku, Pm/Scl 100, Scl-70), myositis specific antibodies (EJ, MDA-5, TIH-Y, Ro52, SAE-1, SAE-2, NXP-2), anti-dsDNA, anti-phopholipid antibodies (aCl IgA, IgG, IgM, antiβ2GPI IgG, IgM) and ANCA. Control groups to compare specific antibody response consisted of 14 healthy children and 19 healthy adults, who had SARS-CoV-2 infection in the last 2 months. Results: Samples of 20 patients were included (14/20 boys, median age 12.4 years). Patients had higher percentage of double negative T cells and low numbers of of cytokine producing T cells Th1, Th2 and Th17. . Numbers of immune competent and CD21+ transitional B cells were also lowered. All patients had positive antibodies against SARS-CoV-2 including neutralising antibodies. Nine (9/19;47 %) patients had high titer (≥1:160) of neutralising antibodies. Results were compared with 2 control groups;14 healthy children (7/14 boys;median age 8 years,) and 19 healthy adults, who all experienced SARS-CoV-2 infection in the last two months. Patients with MIS-C had significantly higher levels of anti-S IgA (p<0.0001), patients with MIS-C and healthy children had significantly higher titers of anti-S1 (p=0.001) and significantly lower titers of anti-S2 (p=0.016) in comparison to adults (Figure 1). No differences were found in the titers of neutralising antibodies and anti-N antibodies. All patients were ANA negative, 19/20 patients were anti-ENA negative, whereas 1 patient had anti-Ro antibodies in low titre. Three patients had aCL IgG in medium titre and 2 patients anti-beta2GPI IgG in low titre. Patients were negative for all other autoantibodies. Conclusion: The immune response in MIS-C patients is specific with most prominent differences in elevated percentage of double negative T cells and low numbers of Th1, Th2, Th17 and CD21+ transitional B cells. MIS-C patients have distinct serologic response with high anti-S IgA, high anti-S1 and low anti-S2 titres.
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Background: Multisystem inflammatory syndrome in children (MIS-C) was recognized during the 2020 pandemic of SARS-CoV-2. Because of the relative rarity current knowledge is limited, especially in the European Caucasian population. Objectives: To report the epidemiology, clinical and laboratory characteristics of patients with MIS-C in a nationwide cohort study in Slovenia. Methods: This is a nationwide prospective cohort study of all consecutive patients with MIS-C, admitted from the beginning of epidemics to 31st December 2020 to University Medical Centre Ljubljana, Slovenia, the only tertiary care pediatric rheumatology center in the country. The inclusion criteria were meeting the CDC criteria for MIS-C. Infection with SARS-CoV-2 was confirmed in all patients by positive antibodies for SARS-CoV-2. Data were collected from the patients' medical records. Data on the COVID-19 epidemics in Slovenia were collected from National Institute of Public Health. Population data were provided by the Statistical Office of the Republic of Slovenia. Results: Twenty-three patients with MIS-C were diagnosed nationwide in Slovenia, all of them in the second wave of epidemics from 14th September to 31st December 2020. All patients were Caucasian and the estimated prevalence of MIS-C was 5.8/100 000 persons younger than 19 years of age. Detailed analyses were available in 20 patients of which 14 were boys (70 %), median age was 12.4 years (4 months to 17.7 years). Two patients (10 %) were treated in the intensive care unit and none of the patients died. Troponin was elevated in 15/20 (75 %) patients during the disease course, and 7/15 (47 %) of these had normal troponin level at admission. The serum level of troponin closely followed the serum level of CRP. Six out of 20 (30 %) patients had elevated pancreatic enzymes in the second week of the disease after treatment was already given,and one patient developed asymptomatic acute pancreatitis with serum lipase level reaching the maximum of 25μkat/L. All patients had elevated levels of D-dimer with no signs of thrombosis. Five patients (5/20;25 %) had pleural effusions and five patients (5/20, 25 %) had ascites. Half of the patients (10/20;50 %) had hepatosplenomegaly and eight (8/20;40 %) had mesenterial lymphadenopathy. Three patients (3/20;15 %) had radiologic signs of cholecystitis. Two patients had thickened lung parenchyma. All patients received IVIG and systemic glucocorticosteroids. Because of resistant or organ threatening disease 4 patients (4/20, 20%) received high dose methylprednisolone pulse therapy. Biologic therapy with anakinra was started in 2 patients. Nineteen patients (19/20, 95%) received acetylsalicylic acid and prophylactic anticoagulation was prescribed in 15/20 (75%) of patients. The mean follow up was 50 days (14 -122). At the last follow-up visit all patients had normal laboratory parameters of inflammation, troponin, pro-BNP, d-dimer values and normal heart function. Conclusion: A very high incidence of MIS-C, estimated 5.8/100 000 persons under the age of 19 with a predominantly cardiac involvement but very good outcome was noted in European Caucasian population in a nationwide cohort study in Slovenia. Attention to newly described pancreatic involvement should be raised.