Molecular and Serological Tests for SARS-CoV-2 Detection in Indeterminate Serology: Can We Skip the Second Sample?

Indeterminate serological results for SARS-CoV-2 antibodies create diagnostic uncertainty, requiring repeat testing after 14-21 days to establish seroconversion. This study evaluated whether direct viral detection methods could provide immediate diagnostic information in serum samples with indeterminate antibody results. We analyzed 163 serum samples from clinically healthy individuals collected during March-December 2020 in Bulgaria. Samples were categorized by screening ELISA (IgA/M/G) as positive (n = 69), negative (n = 47), or indeterminate (n = 47). All samples underwent quantitative IgG ELISA, rapid antibody tests, rapid antigen detection (viral nucleoprotein), and RT-nested PCR. Among samples with indeterminate antibody results, 27.7% (13/47; 95% CI: 15.6-42.6%) tested positive by rapid antigen detection and 12.8% (6/47; 95% CI: 4.8-25.7%) by RT-PCR. All PCR-positive samples were also antigen-positive (Cohen's κ = 0.69). Viral detection rates showed a gradient: antibody-positive samples 30.4% (antigen) and 16.4% (PCR), indeterminate samples 27.7% and 12.8%, antibody-negative samples 10.6% and 4.3%, respectively. The algorithm we proposed and the diagnostic methods used enable the application of certain approaches to differentiate infected from uninfected clinically healthy people, in case of intermediate antibody results. Direct viral detection identified evidence of potential SARS-CoV-2 infection in more than one-quarter of sera with indeterminate antibody results. These findings suggest immediate viral detection testing may complement standard serological approaches, though clinical validation through longitudinal studies is essential before routine implementation.