Evaluation of diagnostic performance of comprehensive respiratory virus panel: comparison of next-generation-sequencing to real-time polymerase chain reaction for detection of respiratory viruses

Respiratory viral infections remain a global health concern, particularly among children, the elderly, and immunocompromised individuals. Although real-time polymerase chain reaction (RT-PCR) is the diagnostic gold standard, its limitations in strain-level typing and mutation tracking highlight the need for complementary approaches such as next-generation sequencing (NGS). We compared a hybridization-based NGS respiratory virus panel (RVP) in comparison with RT-PCR using 81 nasopharyngeal swab (NPS) specimens. The performance metrics included concordance rates, cycle threshold (Ct)-based stratification, co-infection detection, and strain-level classification. Among the 81 NPS specimens, RT-PCR identified respiratory viruses in 56 cases, including eight co-infections. Excluding co-infections, RVP showed 74.5% positive percent agreement, 92.3% negative percent agreement, and 80.8% overall accuracy. The detection and positive concordance rates declined with higher Ct values, and the sequencing depth also decreased. In co-infections, RVP failed to detect low-titer viruses. Strain-level classification was achieved in 65.5% of the positive samples, by subtyping rhinovirus A and C, respiratory syncytial virus A and B, and influenza A (H1N1 and H3N2). NGS panel tests complement RT-PCR by enabling viral detection and strain typing, thereby offering added value to genomic surveillance and outbreak investigations.