From Triplex to Quadruplex: Enhancing CDC’s Respiratory qPCR Assay with RSV Detection on Panther Fusion® Open Access™

The overlapping circulation of influenza (Flu), SARS-CoV-2 (SC2), and respiratory syncytial virus (RSV) continues to challenge clinical laboratories, particularly in settings with limited automation and fragmented healthcare coverage. This study expanded the CDC Flu-SC2 assay by incorporating a laboratory-developed test for RSV A/B detection into a fully automated quadruplex RT-qPCR (LDRA) on the Panther Fusion® Open Access™ system. The design, based on more than 8,000 RSV genomic sequences targeting the conserved M gene, achieved optimal amplification efficiencies (97–105%) and full multiplex compatibility. Analytical assessment established limits of detection between 9.6 and 37.8 copies per reaction, absence of cross-reactivity with 30 respiratory pathogens, and inclusivity for 33 viral variants. Commutability and diagnostic performance among the LDRA, CE-marked Allplex™ SARS-CoV-2/FluA/FluB/RSV, and IVD-marked Panther Fusion® SARS-CoV-2/Flu A/B/RSV Assays were evaluated using 405 nasopharyngeal UTM-conserved swabs. The LDRA demonstrated excellent concordance (overall agreement≥98%, k > 0.95), strong diagnostic accuracy, and reliable detection of mixed infections. This quadruplex provides a fully automated, rapid, and accurate solution for the simultaneous detection of influenza A, influenza B, SARS-CoV-2, and RSV viruses, enhancing molecular diagnostic capacity and supporting equitable, timely clinical decision-making in middle-income healthcare systems, such as that of the Dominican Republic.