Abstract Background There has been growing demand for fully automated multiplex testing of respiratory viruses in molecular diagnostics following the COVID-19 pandemic. ExiStation™ FA 96 RV developed by BIONEER is a multiplex real-time RT-PCR assay. It is designed for the simultaneous detection and differentiation of SARS-CoV-2, Influenza A, Influenza B, and RSV RNA in human nasopharyngeal swab specimens from individuals showing signs and symptoms of respiratory viral infection. ExiStation™ FA 96 automates the entire process from RNA extraction to real-time RT-PCR for up to 96 samples at once within 100 minutes. Moreover, the system is equipped to decap sample tubes, eliminating the need for handling sample transport media. In this study, the clinical performance of the ExiStation™ FA 96 RV assay was evaluated using nasopharyngeal swab samples. Methods Nasopharyngeal swab samples, totaling 859 samples, are collected according to the procedures by the clinical institution in France and preserved in Virus transport media. Once anonymized and randomly allocated, the swab transport media tube, without capping procedure, are directly placed onto the ExiStation™ FA 96. Subsequently, both extraction and real-time RT-PCR are automatically processed. Clinical sensitivity, specificity, and agreement are analyzed by comparing results with confirmatory reagents SARS-CoV-2 RT-qPCR Reagent Kit, PKAmp™ Respiratory SARS-CoV-2 RT-PCR Panel Kit, and Bosphore SARS-CoV-2/Flu/RSV Panel Kit, as well as comparator reagent Allplex™ Respiratory Panel 1 and Allplex™ SARS-CoV-2 Assay. Results The comparison between confirmatory reagents and the ExiStation™ FA 96 RV resulted in a clinical sensitivity of at least 96.4% (164/164 for SARS-CoV-2, 64/65 for Influenza A, 74/75 for Influenza B, and 53/55 for RSV) and a clinical specificity of 100% (500/500 for all targets). When compared with the comparator reagent, the overall agreement and Cohen's kappa value were both determined to be above 99.1% and 0.989, respectively (664/664 for SARS-CoV-2, 564/565 for Influenza A, 573/575 for Influenza B, and 550/555 for RSV). Additionally, any discrepant samples identified in comparison with the comparator reagent were verified through sequencing analysis. Upon sequencing analysis on a total of 8 samples and aligning the confirmed results with the analysis content, no alterations observed in the analysis of clinical sensitivity and specificity. Conclusions The evaluation of the ExiStation™ FA 96 RV assay showed outstanding clinical performance in detecting four respiratory viruses, SARS-CoV-2, Influenza A, Influenza B, and RSV, with high sensitivity and specificity. This study supports that the ExiStation™ FA 96 RV assay is a reliable tool for simultaneous molecular diagnostics of various respiratory infections in clinical laboratories.
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