ONETest PathoGenome: A Multi-Cohort Evaluation of an Optimized NGS Assay for Detection of Lower Respiratory Pathogens in Bronchoalveolar Lavage

Massoumi Alamouti, S.; Nguyen, H. D.; Daneshpajouh, H.; Moshgabadi, N.; Kwok, B. S.; Houck, H. J.; Stazyk, G.; Patrick, T.; Kartikeya, C.; Starostik, P.; Qadir, M. A.; Rand, K. H.

doi:10.64898/2026.03.26.714510

bioRxiv · 10.64898/2026.03.26.714510

ONETest PathoGenome: A Multi-Cohort Evaluation of an Optimized NGS Assay for Detection of Lower Respiratory Pathogens in Bronchoalveolar Lavage

Massoumi Alamouti, S.·Nguyen, H. D.·Daneshpajouh, H.·Moshgabadi, N.·Kwok, B. S.·Houck, H. J.·Stazyk, G.·Patrick, T.·Kartikeya, C.·Starostik, P.·Qadir, M. A.·Rand, K. H.

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Abstract

BackgroundLower respiratory tract infections (LRTIs) remain diagnostically challenging when culture and molecular assays are negative or delayed. We evaluated ONETest Pathogenome (OT), an automated hybrid-capture metagenomic assay with core-genome enrichment probes, for direct pathogen detection in bronchoalveolar lavage (BAL). MethodsAnalytical performance (LoD, precision, continuity) was assessed using whole-cell spike-ins into culture-negative BAL fluid. Technical performance was assessed in 119 specimens profiled by OT and whole-metagenome shotgun sequencing (WmGS, cohort 1). Clinical accuracy was evaluated in 360 specimens (cohort 2) benchmarked against routine bacterial and acid-fast bacillus (AFB) culture. Laboratory-developed test (LDT) validation included 43 specimens (cohort 3) benchmarked to bacterial and AFB culture. ResultsOT uses 6.2 million probes covering core genomes across 50 microbial families (>250 respiratory pathogens). In BAL specimens, OT increased normalized on-target microbial abundance 26-fold versus that of WmGS while preserving within-sample microbial diversity. In cohort 2, OT achieved species-level sensitivity of 80% and specificity of 99% across culture-confirmed isolates and detected [≥]1 culture-confirmed organism in 100/115 culture-positive specimens (87%), while applying species-specific background baselines to mitigate overcalling. Additive yield was 21% (76/360), with 7.5% (27/360) of specimens having [≥]1 additional finding supported by orthogonal testing. In LDT validation, OT identified [≥]1 culture-confirmed organism in 34/40 culture-positive specimens (85%) with one OT-positive/culture-negative specimen. ConclusionsOT is an assay with a turnaround time <24 h complementary to culture that improves pathogen detection and expands microbiologic findings through additional detections and co-detections, including slow-growing organisms that may require prolonged incubation by conventional methods.

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Massoumi Alamouti, S., Nguyen, H. D., Daneshpajouh, H., Moshgabadi, N., Kwok, B. S., Houck, H. J., Stazyk, G., Patrick, T., Kartikeya, C., Starostik, P., Qadir, M. A., Rand, K. H.. 2026-03-30. ONETest PathoGenome: A Multi-Cohort Evaluation of an Optimized NGS Assay for Detection of Lower Respiratory Pathogens in Bronchoalveolar Lavage. https://doi.org/10.64898/2026.03.26.714510

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