Evaluation of the deeplex Myc-TB-targeted next-generation sequencing assay for mycobacterial identification and drug resistance detection

Accurate differentiation of Mycobacterium tuberculosis complex (MTBC) and nontuberculous mycobacteria (NTM), together with early detection of drug resistance, is essential for guiding therapy and infection control. Targeted next-generation sequencing assays, such as the Deeplex Myc-TB (GenoScreen, Lille, France), allow simultaneous identification of species and detection of drug-resistance mutations. We evaluated Deeplex Myc-TB on 304 primary, culture-positive mycobacterial growth indicator tube (MGIT) broths collected between May 2024 and March 2025. The assay generated determinate results for 273 isolates, comprising 29 MTBC and 244 NTM, with 100% agreement for MTBC identification, and 98.8% for NTM. MTBC identification was performed using GeneXpert MTB/RIF, whereas phenotypic drug susceptibility testing (pDST) was performed using either the Sensititre MYCOTB broth microdilution method, or the MGIT-based panel. Among the 29 unique MTBC isolates, 18 (62.1%) had no detectable drug-resistance mutations (DRMs). Phenotypic drug susceptibility testing confirmed susceptibility to all tested drugs for these 18 isolates, resulting in 100% concordance with Deeplex-predicted susceptibility, and no evidence of heteroresistance. The remaining 11 isolates (37.9%) carried at least one DRM, including seven with canonical resistance-conferring mutations. For isolates with canonical DRMs and available CLSI breakpoints, genotypic calls and pDST were concordant, and one isolate met the criteria for multidrug-resistant tuberculosis. All 244 NTM isolates had prior matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) identification; Deeplex confirmed these identifications and provided subspecies-level resolution within the Mycobacterium avium complex and Mycobacterium abscessus. These findings demonstrate the strong diagnostic potential of Deeplex Myc-TB for high-resolution MTBC resistance profiling and precise NTM species and subspecies identification. Integrating targeted sequencing into routine workflows can improve clinical decision-making, support early treatment optimization, and strengthen molecular surveillance of mycobacterial diseases.