Clinical evaluation of a fully automated, laboratory- developed multiplex RT-PCR assay integrating dual- target SARS-CoV-2 and influenza A/B detection on a high- throughput platform

Introduction. Laboratories worldwide are facing high demand for molecular testing during the severe acute respiratory syndrome coronavirus 2 (SARSCoV-2) pandemic, which might be further aggravated by the upcoming influenza season in the northern hemisphere. Gap Statement. Given that the symptoms of influenza are largely indistinguishable from those of coronavirus disease 2019 (COVID-19), both SARS- CoV-2 and the influenza viruses require concurrent testing by RT- PCR in patients presenting with symptoms of respiratory tract infection. Aim. We adapted and evaluated a laboratorydeveloped multiplex RT- PCR assay for simultaneous detection of SARS- CoV-2 (dual target), influenza A and influenza B (SC2/InflA/InflB- UCT) on a fully automated highthroughput system (cobas6800). Methodology. Analytical performance was assessed by serial dilution of quantified reference material and cell culture stocks in transport medium, including pretreatment for chemical inactivation. For clinical evaluation, residual portions of 164 predetermined patient samples containing SARS- CoV-2 (n=52), influenza A (n=43) or influenza B (n=19), as well as a set of negative samples, were subjected to the novel multiplex assay. Results. The assay demonstrated comparable analytical performance to currently available commercial tests, with limits of detection of 94.9 cp ml(-1) for SARSCoV-2, 14.6 cp ml(-1) for influenza A and 422.3 cp ml(-1) for influenza B. Clinical evaluation showed excellent agreement with the comparator assays (sensitivity of 98.1, 97.7 and 100 % for Sars- CoV-2 and influenza A and B, respectively). Conclusion. The SC2/InflA/InflB- UCT allows for efficient highthroughput testing for all three pathogens and thus provides streamlined diagnostics while conserving resources during the influenza season.

Automated summary

This study developed a laboratory-developed multiplex RT-PCR assay for simultaneous detection of SARSCoV-2, influenza A, and influenza B, demonstrating comparable analytical performance to current commercial tests and excellent agreement in clinical evaluation. The SC2/InflA/InflB-UCT allows for efficient high-throughput testing for all three pathogens, providing streamlined diagnostics and resource conservation during the influenza season.