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.