Evaluation of the high-throughput RespiV PlexPlus multiplex assay compared to BioFire Respiratory Panel 2.1 plus

During the 2023 Australian winter respiratory virus season, 2,000 clinical samples were prospectively tested over 47 days (July-August 2023) using the BioFire Respiratory Panel 2.1 plus (RP2.1+) assay, which detects 19 viral and 4 bacterial targets. These samples were retrospectively tested with the RespiV PlexPlus (RespiV) assay, which detects 14 viral targets. RespiV demonstrated high overall percentage agreement with RP2.1+ (99.2%), with a negative percent agreement exceeding 99% for the majority of shared targets. Positive percent agreement was more variable across targets, with more positive discordant results for high-prevalence pathogens. Positive discordant results had higher quantification cycle values (median 37.2) than concordant positives (median 27.8), consistent with near-limit-of-detection inter-assay discordance. Discordant results for influenza A virus, influenza B virus, respiratory syncytial virus, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were retested with Xpert Xpress Flu/RSV and Xpress SARS-CoV-2 to generate a consensus result. RP2.1+ and RespiV showed 99.6% overall agreement with the consensus result for these targets. RespiV detected 14 additional influenza A-positive samples, all confirmed by amplicon sequencing. RespiV also identified 19 enterovirus-positive samples, 17 of which were RP2.1+ rhinovirus/enterovirus positive. Conversely, RP2.1+ detected pathogens not included in the RespiV panel, such as 79 non-SARS-CoV-2 coronaviruses and 1 Chlamydophila pneumoniae, with 62 samples negative across all RespiV targets. In summary, both assays show a high degree of concordance for all shared targets. RP2.1+ offers rapid test turnaround and broader pathogen coverage, making it valuable for urgent diagnostics, while RespiV provides higher throughput and is well-suited for centralized, high-volume testing environments.IMPORTANCERapid detection and differentiation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other treatable respiratory pathogens are critical for guiding therapy and infection control. Sample-to-answer multiplex panels, such as the BioFire Respiratory Panel 2.1 plus, allow simultaneous detection of multiple pathogens. These assays are fast, user-friendly, and support random-access testing for small batches, but they are costly and limited in throughput by instrument availability. Conversely, conventional multiplex assays like RespiV PlexPlus are slower and more labor-intensive for small batches but optimized for large batch processing on standard molecular diagnostic platforms. They offer higher throughput with lower consumable costs, making them suitable for centralized laboratories. Recent innovations in both sample-to-answer and conventional multiplexing technologies have expanded detectable targets, with comparable technical performance between formats. Ultimately, the optimal choice depends on healthcare system resources and testing demands.