Objectives Accurate diagnosis of existing and emerging respiratory pathogens is important. We evaluated the capability of unbiased metagenomic next generation sequencing (mNGS) to identify pathogenic RNA viruses from two cohorts of nasopharyngeal (NP) swabs previously tested by commercial multiplex respiratory diagnostics. Methods NP swabs (N = 100) in viral transport media (VTM) were assessed using mNGS for this study. Cohort 1 (N = 52) consisted of symptomatic individuals who tested negative for SARS-CoV-2, influenza A/B, and RSV by the Xpert Xpress CoV-2/Flu/RSV Plus multiplex respiratory virus panel and were tested by mNGS for undetected pathogens. Cohort 2 (N = 48) included symptomatic individuals who were positive (N = 26) or negative (N = 22) by the ePlex RP2 multiplex respiratory pathogen panel. Samples were positive for influenza A (N = 8), rhinovirus/enterovirus (N = 5), RSV (N = 4), adenovirus (N = 3), parainfluenza (N = 2), seasonal coronaviruses (N = 2), and human metapneumovirus (N = 1), as well as a rhinovirus/enterovirus/human metapneumovirus co-infected sample (N = 1). mNGS results were compared with ePlex RP2 findings, and symptomatic negative samples were evaluated for additional pathogen detection. Results Cohort 1 contained 8% (4/52) viral and 19% (10/52) bacterial reads. In cohort 2, positive concordance between ePlex RP2 and mNGS was 31% (8/26). mNGS did not identify any viral reads in ePlex RP2-negative samples. However, it detected other microbial reads, such as Acanthamoeba castellanii, in 21% (10/48) of samples. Conclusion In this study, targeted multiplex amplification methods demonstrated better overall sensitivity in NPs of symptomatic respiratory individuals than mNGS. Other mNGS approaches may produce different results. This study suggests that mNGS may offer adjunctive information, including the detection of rare pathogens, which may be helpful in some clinical contexts.
Hardick et al. (2026) studied this question.