A novel multiplex assay for species-level identification of non-tuberculous mycobacteria in culture-positive respiratory specimens based on capillary electrophoresis: development and clinical validation

Nontuberculous mycobacterial pulmonary disease (NTM-PD) is rising globally, driven predominantly by Mycobacterium avium complex (MAC), yet treatment is highly species-dependent. We developed a Multiplex assay targeting four clinically important non-tuberculous mycobacteria (NTM) species– M. avium , M. intracellulare , M. abscessus , and M. fortuitum –and compared it with metagenomic next-generation sequencing (mNGS), using colony-based multigene Sanger sequencing as the reference method. Among 101 specimens, 63 (62.4%) yielded polymicrobial results, of which mixed-species detection of M. avium – M. intracellulare accounted for 46 (73.0%). The Multiplex assay and mNGS showed no statistically significant difference in overall accuracy in this cohort (82.2% vs. 81.2%; raw agreement 83.2%, P = 1.000). The Multiplex assay achieved 100.0% sensitivity for all four species, with specificity of 87.8% and 87.5% for M. avium and M. intracellulare , respectively, and >98% for M. abscessus and M. fortuitum ; mNGS showed the converse pattern, with lower MAC sensitivity (88.5%, 92.2%) but higher MAC specificity (93.9%, 95.8%). Requiring only a single PCR reaction without sequencing or bioinformatic analysis, the Multiplex assay showed no statistically significant difference in diagnostic accuracy from mNGS in this culture-positive cohort, without requiring library preparation or sequencing, representing a promising candidate for further evaluation as a routine NTM species-identification tool.