High-throughput multiplex detection of respiratory pathogens: multicenter evaluation of the NxTAG Respiratory Pathogen Panel v2 assay

Respiratory tract infections remain a major cause of global morbidity, highlighting the need for rapid and accurate diagnostic testing. This multicenter study evaluated the clinical and analytical performance of the updated NxTAG Respiratory Pathogen Panel v2 (RPPv2), a high-throughput multiplex molecular assay designed to detect 21 viral and bacterial respiratory pathogens. A total of 2,145 nasopharyngeal swab specimens, including both prospectively enrolled and archived specimens, were analyzed across five U.S. clinical sites. The NxTAG RPPv2 was assessed against FDA-cleared molecular assays and bi-directional sequencing, with contrived specimens used to supplement low-prevalence targets. Valid results were obtained for 99.7% of specimens after a single retest, with minimal performance differences between fresh and frozen specimens. Among viral targets, 15 of 18 (one viral target did not have clinical positive specimens for analysis) achieved ≥95% positive percent agreement (PPA) across the prospective and pre-selected arms. For bacterial targets, Mycoplasma pneumoniae and Chlamydia pneumoniae showed PPA values of 92.3% and 100%, respectively. Lower PPA values were observed for parainfluenza 4 (90.3%). Negative percent agreement (NPA) was consistently high across targets, with all viral and bacterial analytes achieving ≥99.2%. Reproducibility across sites and operators was 99.9%. Overall, the NxTAG RPPv2 demonstrated high accuracy, reproducibility, and operational efficiency, supporting its clinical utility for rapid and reliable diagnosis of respiratory tract infections.