Analytical performance verification of the Deeplex Myc-TB assay using well-characterized Mycobacterium tuberculosis proficiency testing isolates

Rapid and accurate detection of drug-resistant Mycobacterium tuberculosis is critical for effective patient management and tuberculosis (TB) control. Targeted next-generation sequencing (tNGS) assays such as Deeplex® Myc-TB offer rapid detection of resistance-associated mutations with much more comprehensive coverage than the conventional routine molecular assays. This study evaluated the performance of the Deeplex assay in detecting resistance to first- and second-line anti-TB drugs using well-characterized proficiency testing (PT) samples. A total of 41 isolates received as part of the World Health Organization (WHO) global Tuberculosis (TB) Drug-Susceptibility Testing (DST) Proficiency Testing (PT) Program, organized and administered by the TB Supranational Reference Laboratory (SRL) in Antwerp, Belgium, were included. Phenotypic drug susceptibility testing (LJ and MGIT) and whole-genome sequencing (WGS) served as reference (judicial) standards. Deeplex tNGS was used to analyze remnant DNA, and the results were analyzed using the Deeplex web app and TBProfiler using a composite interpretation rule. Concordance, sensitivity, and specificity were assessed across multiple drug classes. The Deeplex Myc-TB demonstrated 100% specificity across all drug targets. Sensitivity was 100% for rifampicin (17/17), pyrazinamide (7/7), fluoroquinolones (13/13), and linezolid (6/6), and 94.1% for isoniazid (16/17). The false-susceptible INH result was due to a katG c.45_46insA mutation outside the assay target region. For repurposed/new drugs, sensitivity was 80.0% (4/5) for clofazimine and 44.4% (4/9) for bedaquiline due to five phenotypically resistant strains lacking detected resistance variants. Overall concordance ranged from 87.5% (bedaquiline) to 100% (rifampicin, pyrazinamide, fluoroquinolones, linezolid). Limitations were observed for bedaquiline and clofazimine, where mutations in mmpR5 (Rv0678) were largely detected, but atpE -mediated resistance was not identified due to limited assay coverage. Notably, the assay consistently detected several recurrent mutations for the different drugs. The Deeplex assay is a highly specific tool for detection of major TB drug resistance-associated mutations, demonstrating strong concordance with phenotypic and WGS-based methods. However, gaps in target coverage, particularly for certain rare or non-targeted mutations, may result in missed resistance. These findings highlight the importance of understanding local resistance mutation patterns when implementing tNGS-based resistance detection.