Clinical evaluation of a commercial culture-free targeted next-generation sequencing test for diagnosis of drug-resistant tuberculosis.

UNLABELLED: Rapid molecular diagnostics have significantly improved access to tuberculosis (TB) drug resistance detection and reduced turnaround times. However, these tools remain limited in their capacity to generate comprehensive resistance profiles, particularly for newer and repurposed anti-TB drugs. Targeted next-generation sequencing (tNGS) offers a promising alternative, enabling a broader resolution ofgenomes and faster reporting compared to phenotypic drug susceptibility testing (DST). We conducted a prospective, cross-sectional, multicenter evaluation of the DeepChek 13-Plex KB Drug Susceptibility Testing (ABL Diagnostics S.A., France) tNGS assay for direct detection of TB drug resistance from clinical sputum samples. The study was performed at three reference laboratories in India, South Africa, and Georgia between April 2021 and June 2022. Adults (≥18 years) with confirmed pulmonary TB were enrolled. Sensitivity and specificity were assessed for key anti-TB drugs against a composite reference standard combining phenotypic DST and whole-genome sequencing. Of 832 participants enrolled, 694 (83.4%) were included in the final analysis. Sequencing was successful in 75.6% of samples. Failure rates were higher in samples with low or very low Xpert MTB/RIF categories. The ABL tNGS workflow showed ≥95% sensitivity for rifampicin, isoniazid, and levofloxacin; 92%-93% for pyrazinamide and moxifloxacin; 88% for ethambutol; and 72%-82% for bedaquiline and clofazimine. Specificity was ≥95% for all drugs. The ABL tNGS workflow enables comprehensive resistance profiling, including for new and repurposed TB drugs. However, higher bacillary loads are required to provide a valid test compared to current class-based tNGS assays recommended by the WHO, thus requiring further improvements.

IMPORTANCE: Drug-resistant tuberculosis (TB) threatens progress in global TB control, yet current molecular tests detect resistance to only a few drugs. Targeted next-generation sequencing (tNGS) can read many resistance-related genes at once, offering faster and broader results than conventional culture-based testing. We evaluated a commercial tNGS workflow (DeepChek 13-Plex KB, ABL Diagnostics) for direct detection of drug resistance in sputum samples from adults with pulmonary TB in India, South Africa, and Georgia. Among 832 participants, sequencing produced valid results for most samples with moderate or high bacterial loads. The assay accurately identified resistance to key drugs-including rifampicin, isoniazid, fluoroquinolones, and newer medicines such as bedaquiline and clofazimine-while maintaining high specificity. These findings show that tNGS can deliver comprehensive resistance profiles, supporting tailored treatment for people with drug-resistant TB. Further refinement in sample preparation may expand its use to specimens with lower bacterial counts.

CLINICAL TRIALS: This study is registered with ClinicalTrials.gov as NCT04239326.