Clinical validation of a novel metagenomic nanopore sequencing method for detecting viral respiratory pathogens: diagnostic accuracy study

BackgroundClinical metagenomics (CMg) offers high-throughput respiratory pathogen detection with a wider range than targeted, probe-dependent diagnostics. Sequencing cost and the challenges of high host biomass in non-invasive samples are barriers to the use of CMg in high-throughput respiratory pathogen detection. MethodsWe optimised a long-read sequencing workflow to detect RNA viruses in nasopharyngeal swabs, employing pathogen enrichment and ONT sequencing. As a pre-requisite for agnostic pathogen detection, we first derived quality control (QC) criteria and diagnostic thresholds against a gold-standard comprising 23 pathogen targets detected by routine multiplex PCR. We validated this workflow using 344 prospectively collected upper respiratory tract samples submitted for routine testing. FindingsUsing pre-defined QC and positivity criteria, the workflows sensitivity versus PCR was 51% (95%CI: 45%-57%) (133/260 positive targets detected) (ranging from 19%-85% across pathogens with >20 gold-standard detections), and specificity 99.8% (95%CI: 99.6%-99.9%) (3836/3845 negative targets not detected). Sensitivity improved to 58% using post-hoc optimised thresholds, 61% only considering RNA pathogens, 70% excluding rhinovirus/enterovirus and 83% excluding samples with qPCR Ct values [&ge;]35. Read crossover from multiplex sequencing contributed most (7/9) false-positives: only 2 plausible additional pathogens were identified (rhinovirus and coronavirus OC43). 41 respiratory syncytial virus (RSV), 13 influenza A and 10 rhinovirus/enterovirus were successfully sub-typed by sequencing. Multiplexed nanopore sequencing costs were {pound}112/sample. InterpretationAlthough CMg has substantial diagnostic potential, this validation study demonstrates the technical limitations of current metagenomic sequencing methods applied to viral detection in upper respiratory tract samples with high host and low pathogen biomass. Its greater sensitivity at higher viral loads demonstrates the importance of identifying the most appropriate use cases to maximise its utility and value. FundingNational Institute for Health and Care Research Oxford Biomedical Research Centre. Research in contextO_ST_ABSEvidence before this studyC_ST_ABSClinical metagenomics (CMg) provides the potential for rapid respiratory pathogen detection with a wider range than targeted, probe-dependent diagnostics. Searching PubMed for studies published between Jan 1 2018 and December 1 2025 using the terms respiratory tract infection AND metagenomics AND diagnosis, with no language restrictions yielded 734 items. We identified 15 clinical studies assessing the diagnostic performance of a metagenomic workflow for respiratory samples. Only 3 of these included upper respiratory tract samples (nasopharyngeal swabs); the remaining studies exclusively investigated invasive samples from the lower respiratory tract (bronchioalveolar lavage fluid). One of the three relevant studies assessed detection of viral pathogens in 83 specimens from critical care patients as part of pan-microbial nanopore sequencing metagenomic assay; only 2 samples included were nasopharyngeal samples. The second validated a high-depth short-read CMg workflow in 191 samples (85% nasopharyngeal), reporting 93.6% sensitivity and 98.8% specificity across eight respiratory pathogens detected by respiratory PCR. This workflow required substantial equipment and sequencing costs. A third study validated a nanopore sequencing CMg workflow, with lower laboratory footprint and sequencing costs than the second study. In 359 nasopharyngeal samples, they reported 61% sensitivity and 100% specificity against four respiratory viral targets detected by PCR. The costs of CMg mean that routine deployment as a high-throughput test with broad use will require demonstrating good performance across a range of common respiratory pathogens (such as can be assayed with commercially available extended multiplex PCR testing) and in upper respiratory tract samples. Added value of this studyThis prospective study is the first to evaluate nanopore sequencing for detecting a broad range of common respiratory viruses in nasopharyngeal samples. We validated a CMg workflow employing a modernised SISPA step for viral amplification, in 344 prospectively collected non-invasive samples, from which 19 different respiratory pathogens were detected by routine PCR testing. Using pre-defined bioinformatic thresholds overall sensitivity was 51% compared with PCR. This could be improved to 83% by limiting analysis to RNA viruses with Ct <35 and using post-hoc exploratory bioinformatic criteria. Sequencing costs were 45% lower than short-read sequencing. Implications of all the available evidenceOur results demonstrate rigorous bioinformatic thresholds are essential in multiplexed CMg sequencing to reduce false-positive detections, a particular danger with imperfect barcode de-multiplexing. However these thresholds impair true-positive detection in samples with a high ratio of host-to-pathogen biomass. Further research should focus on identifying in which samples and clinical settings CMg can offer greater value in patient care.