Evaluation of a Flow Cytometry-Based Surrogate Assay (flowsa) for the Detection of SARS-CoV-2 in Clinical Samples

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. ### Competing Interest Statement The authors have declared no competing interest. ### Clinical Protocols ### Funding Statement This project received funding from the Netherlands Organization for Health Research and Development (ZonMw) [1], grant 10430012010023. The funders had and will not have a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The study design protocol of this cohort including ethics information and documentation has been described elsewhere (13). Briefly, this study has been approved by the Medical Ethical Review Committee of the UMCG (METc 2020/158) and follows international standards for the ethical conduct of research involving human subjects. All procedures employed in the clinical and related laboratory. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present work are contained in the manuscript