Metagenomics enables parallel detection of 176 clinically relevant targets from faecal samples.

Background: Robust identification of pathogens is essential for managing patients with symptomatic infection, yet conventional diagnostic methods focus on a subset of the most prevalent pathogens and genes. Metagenomic next-generation sequencing (mNGS) is a powerful technology that can comprehensively and simultaneously assess a broader range of pathogens and genes in a sample. This study evaluates the clinical (22 targets), analytical (19 targets), and Methods: Diagnostic performance was evaluated relative to conventional pathology testing using 510 clinical faecal samples from patients presenting with gastrointestinal symptoms. Contrived samples were used to assess analytical performance and establish the assay's limit of detection by adding cells to a faecal matrix. Results: Clinical specificity was ≥96% (≥99% for all but Adenovirus F), and median pathogen sensitivity was 91%. VF and AMR gene detection was less sensitive (median 58.7%). The assay was highly reproducible in biological triplicates (27,656/27,808 calls concordant; 99.5%). Importantly, broad mNGS coverage increased diagnostic yield, with 256/510 (50.2%) samples containing one or more additional targets not reported by standard care, and 181/510 (35.5%) containing AMR genes, including carbapenemases. Conclusions: The faecal mNGS assay performed competitively with existing diagnostic techniques while substantially expanding actionable detection in a single assay. These results support stool mNGS as a high-yield second-line or syndromic test for gastrointestinal infection, enabling improved recognition of rare pathogens, co-infections, and resistance determinants.