SARS-CoV-2 Serosurveys: How Antigen, Isotype and Threshold Choices Affect the Outcome

Abstract

Background. Evaluating the performance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serological assays and clearly articulating the utility of selected antigens, isotypes, and thresholds is crucial to understanding the prevalence of infection within selected communities. Methods. This cross-sectional study, implemented in 2020, screened PCR–confirmed coronavirus disease 2019 patients (n = 86), banked prepandemic and negative samples (n = 96), healthcare workers and family members (n = 552), and university employees (n = 327) for anti–SARS-CoV-2 receptor-binding domain, trimeric spike protein, and nucleocapsid protein immunoglobulin (Ig)G and IgA antibodies with a laboratory-developed enzyme-linked immunosorbent assay and tested how antigen, isotype and threshold choices affected the seroprevalence outcomes. 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. Results. We found vastly different seroprevalence estimates depending on selected antigens and 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%. Conclusions. This study revealed the importance of evaluating serosurvey tools for antigen-, isotype-, and threshold-specific sensitivity and specificity, to interpret qualitative serosurvey outcomes reliably and consistently across studies.