SARS-CoV-2 Serosurveys: How antigen, isotype and threshold choices affect the outcome

AbstractBackgroundEvaluating the performance of SARS-CoV-2 serological assays and clearly articulating the utility of selected antigen, isotypes and thresholds is crucial to understanding the prevalence of infection within selected communities.MethodsThis cross-sectional study, implemented in 2020, screened PCR-confirmed COVID-19 patients (n=86), banked pre-pandemic and negative donors (n=96), health care workers and family members (n=552), and university employees (n=327) for anti-SARS-CoV-2 receptor-binding domain (RBD), trimeric spike protein (S), and nucleocapsid protein (N) IgG and IgA antibodies with a laboratory developed Enzyme-Linked Immunosorbent Assay (ELISA) and tested how antigen, isotype and threshold choices affected the seroprevalence. The following threshold methods were evaluated: (i) mean + 3 standard deviations of the negative controls; (ii) 100% specificity for each antigen/isotype combination; and (iii) the maximal Youden index.ResultsWe found vastly different seroprevalence estimates depending on selected antigens, isotypes and the applied threshold method, ranging from 0.0% to 85.4%. Subsequently, we maximized specificity and reported a seroprevalence, based on more than one antigen, ranging from 9.3% to 25.9%.ConclusionsThis study revealed the importance of evaluating serosurvey tools for antigen, isotype, and threshold-specific sensitivity and specificity, in order to interpret qualitative serosurvey outcomes reliably and consistently across studies.