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

Background: Lower 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). Methods: Analytical 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. Results: OT 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. Conclusions: OT 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.