MATERIALS AND METHODS:
Children under 16 years of age admitted to the Department of Pediatrics at Christian Medical College (CMC), Vellore, with respiratory symptoms or asymptomatic children admitted for management of non-COVID illness in whom rRT-PCR for SARS CoV-2 was requested, were considered for recruitment. Children testing positive for SARS CoV-2 RNA between November 2020-May 2021 were approached 4-6 weeks after PCR positivity and for collection of a blood sample for SARS CoV-2 antibody detection. Informed consent from the child’s parent/guardian and assent was obtained from children aged 8 years and above, prior to recruitment and sample collection. Children above 16 years and those refusing consent were excluded. The study was approved by the Institutional Review Board of CMC, Vellore. Demographic data, clinical presentation and severity were assessed at the time of PCR request. Clinical severity was stratified as asymptomatic, mild-to-moderate or severe (including MIS-C) as per standard definitions6,17 and confirmed by two pediatricians. Outcome of episode was obtained from chart review and electronic medical records.
Detection of SARS CoV-2 in respiratory samples: Confirmation of SARS CoV-2 infection by real-time RT-PCR, was performed as part of routine clinical care as per hospital policy for management of COVID or non-COVID illness. Briefly, respiratory samples (nasopharyngeal, nasal/throat swabs or endotracheal aspirates) were transported in cold chain to the laboratory and processed for testing with appropriate biosafety protocols. Viral nucleic acid was extracted on an automated platform (QiaCube HT, Qiagen GmBh) and tested with a commercial SARS CoV-2 real-time RT-PCR assay (Altona RealStar® SARS-CoV-2 RT-PCR) detecting envelope (E) and spike (S) genes of SARS CoV-2 with a heterologous internal control (IC). Samples were considered positive when both E and S genes amplified with a Ct value (<40). The lower of the Ct values between the two genes was extracted for analysis in this study.
Antibody detection: Blood samples (4-6 ml) collected in Vacutainer® tubes with clot activator (Becton Dickinson Inc., USA), were transported to the laboratory, and centrifuged at 2000 rpm for 10 minutes. Serum was aliquoted and stored at -30°C until testing in batches with appropriate positive and negative controls. SARS CoV-2 specific antibodies were detected on a Cobas e411 analyser based on electrochemiluminescence (ECLIA) for detection of anti-spike (Elecsys® anti-SARS-CoV-2 S, Anti-S) and anti-nucleoprotein antibodies (Elecsys® anti-SARS-CoV-2, Anti-N). Sample testing and reporting were as per manufacturer’s instructions. Sample readouts ≥ 0.8 U/ml and ≥ 1.0 cut-off index (COI) were considered positive for anti-S and anti-N, respectively. The range of detection of the autoanalyzer was 0.01-250. Values above detection limit (>250) were corrected to 251 to allow analysis.
Analytical approach: Baseline characteristics of the cohort are presented as mean (SD) or median (IQR) and compared with t-test or Mann-whitney test as appropriate. Ct values of respiratory samples were categorized into quartiles (Q1-Q4) and relationships with antibody levels (anti-N and anti-S) and clinical severity were compared. Categorical variables were presented as frequency and percentages. Chi-square test was used to compare proportions. One-way analysis of variance (ANOVA) or Kruskal-Wallis test were used for comparison between groups. All analyses were performed using STATA IC/16.0 (StataCorp LLC, College Station, Texas, USA). Graphs were generated using GraphPad Prism 9.