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

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• Evidence summary: RNA pathogen detection workflows: Summary The single-assay results are a UCSF respiratory RNA metagenomic assay at 93.6% (103 of 110) sensitivity against original clinical respiratory panel testing, as reported by its developers, and the developers' own prospective validation of an Oxford nanopore workflow on 344 nasopharyngeal swabs (preprint). With thresholds fixed on a separate derivation set, the nanopore workflow detected 51% (133 of 260) of PCR-positive targets in the samples passing quality control, from 85% for RSV to 19% for rhinovirus/enterovirus, and 99.8% of PCR-negative targets were not detected; criteria chosen after the validation raised sensitivity to 58%, and the authors attributed all nine false positives to barcode crossover. Its swabs were selected mostly as PCR-positive at Ct 30 or below, with about one negative per eight positives, and reads were matched only to the 23 panel pathogens, so it does not test detection outside the panel. Three comparisons of several workflows add proxy evidence. On 88 nasal washings scored against 1144 respiratory virus PCR results (Carbo et al. 2022), all five classifiers detected 22 or 23 of the 24 PCR-positive results at species level at a read-count cut-off chosen on the same data, but 31% to 58% of their positive calls were PCR-negative. When 13 European clinical virology laboratories each ran their own pipeline on the same 13 clinical datasets (de Vries et al. 2021, preprint), 10 to 13 samples were correctly called positive, and the three pipelines that found every sample reported 6, 2 and 0 additional PCR-negative viruses. In the CAMI II blood metagenome challenge (Meyer et al. 2022), 4 of 10 submissions listed the causal virus. In a developer benchmark of five virus profilers on one shared database (Tisza et al. 2026, preprint), the model-estimated probability of reporting the correct family for reads from seven divergent viruses at 10 read pairs was 0.974 for EsViritu and 0.9998 for Metabuli but 0.045 for Kraken2, 0.012 for Centrifuger and 0.0009 for Sylph at the authors' headline settings, and Metabuli reported a median of 18 false species in seven pathogen-free libraries against 0 for EsViritu. None of this is clinical validation: the cohorts are small or single-site, reference databases and reporting rules differ, and the benchmark data are mostly synthetic. (amp-20261007-rna-pathogens-source: result amp-20261007-rna-pathogens-result; rna-pathogen-20261009-source-carbo2022-supp: Supplementary Table 1; rna-pathogen-20261009-source-devries2021-supp: Supplementary Tables 2 and 4; rna-pathogen-20261009-source-meyer2022-supp: Supplementary Table 39; articles: Methods and Results; rna-pathogen-20261011-sanderson2026-source: Results 'Validation Set: Diagnostic performance', 'Performance by pathogen'; rna-pathogen-20261011-tisza2026-source-supp: Supplemental Tables S2 and S6)
• false_positive_cause: rna-pathogen-20261011-sanderson2026-protocol-validation: False positive cause All nine false-positive detections in the validation set were potentially attributable to barcode crossover from another sample on the same run with many reads for the same pathogen. (Results 'Validation Set: Diagnostic performance' paragraph 5)
• Relevance of target-level detection against routine multiplex PCR, validation set (Sanderson et al. 2026) to "Select an RNA pathogen-detection workflow for diagnostic testing": Assessed by Target-level detection of respiratory pathogens against routine multiplex PCR, validation set (Sanderson et al. 2026) (rna-pathogen-20261011-sanderson2026-source: Results 'Validation Set: Diagnostic performance')

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