Evaluation of a flow cytometry-based surrogate assay (FlowSA) for the detection of SARS-CoV-2 in clinical samples

Abstract The current diagnostic methods for SARS-CoV-2 rely on quantitative RT-PCR. However, the presence of viral RNA in samples does not necessarily reflect the presence of an infectious virus. Therefore, the reliable detection of infectious SARS-CoV-2 in clinical samples is necessary to limit viral transmission. Here, we developed a flow cytometry-based surrogate assay (FlowSA), wherein the presence of infectious SARS-CoV-2 was detected using virus nucleocapsid-specific antibodies. We showed that FlowSA allows the detection of a wide range of viral titers of multiple SARS-CoV-2 variants. Furthermore, the assay was successfully used to detect infectious SARS-CoV-2 in nasopharyngeal swabs from SARS-CoV-2 positive individuals, including those with high Ct values. Notably, FlowSA identified the presence of infectious SARS-CoV-2 in biological specimens that scored negative for cytopathic effect (CPE) in cell culture and would otherwise be considered negative. We propose that FlowSA can be adopted as an alternative to conventional CPE methods for viral diagnostics.