Refined fully automated RT-qPCR assay for simultaneous detection of SARS-CoV-2, Influenza A/B, and RSV with target optimization for improved variant resilience

Background: Continuous viral evolution is likely to cause a loss of performance or failure of existing diagnostic assays. Here, we delineate the adaptation and validation of an operational laboratory developed (LDT) multiplex RT-qPCR for simultaneous detection of SARS-CoV-2, influenza A/B (FluA/B) and respiratory syncytial virus (RSV) on a high-throughput, fully automated platform. Furthermore, we explore the integration of an alternative SARS-CoV-2 target to enhance assay robustness Methods: An adaption of the operational assay (PMID:38820916) was performed and a novel SARS-CoV-2 target (macrodomain, Mac1) was implemented. Analytical performance of the adapted assay was evaluated using digital-PCR based standards or international reference material and clinical performance was assessed on clinical samples with a CE-IVD comparator assay. Results: Analytical sensitivity (lower limit of detection (LoD)) was 70.9 IU/ml for SARS-CoV-2, and 112-474, 919 and 1,720 cp/ml for influenza A, influenza B and RSV, respectively, with linear ranges of 26.3-36.4 ct (SARS-CoV-2), 26.8-37.7 ct (FluA), 28.5-37.9 (FluB) and 25.6-38.2 (RSV). Clinical performance evaluation confirmed improved performance (e.g. FluA/B detection -1.6/-4.81 ct) and comparable performance to the CE-IVD assay (excellent correlation of the SARS-CoV-2 assays, more effective detection of influenza B). Overall, positive/negative agreement was 94%/96% (SARS-CoV-2), and 100%/100% (FluA/B, RSV). Conclusion: The adapted LDT assay as a focused syndromic assay provides reliable detection of major respiratory viruses on a high-throughput platform. The strategic targeting of conserved genomic regions ensures diagnostic resilience, while automated integration facilitates scalable laboratory operations. This approach facilitates robust pathogen surveillance and expedites clinical decision-making during periods of co-circulation and epidemic surge.