Analytical performance of a multi-target open real-time PCR assay for simultaneous detection of tuberculosis, non-tuberculous mycobacteria, and drug resistance in a high-burden setting

Timely diagnosis of tuberculosis (TB) and drug resistance is crucial for effective disease control. Multiplex open molecular platforms capable of simultaneously detecting Mycobacterium tuberculosis complex (MTBc), non-tuberculous mycobacteria (NTM), and resistance to first-line anti-TB drugs could significantly streamline diagnostic pathways. We evaluated two multiplex real-time PCR assays (MTBc/NTM R-Gene and MTB-RIF/INH R-Gene) using 300 well-characterized samples (150 MTBc-positive and 150 MTBc-negative). Composite reference standards, including culture, MPT64 antigen testing, and line probe assays and phenotypic drug susceptibility testing. The MTBc detection demonstrated 100% sensitivity and specificity (150/150). NTM detection showed 70.0% sensitivity (35/50) and 100% specificity, indicating limitations in NTM species coverage. Rifampicin resistance was detected with 96.0% sensitivity (48/50) and 100% specificity. Isoniazid resistance detection achieved 100% sensitivity and specificity (50/50). High agreement (κ = 0.76–1.00) with established reference standards was observed. This analytical validation confirms that multiplex open real-time PCR assays accurately and simultaneously detect MTBc, NTM, and rifampicin and isoniazid resistance using culture isolates. Clinical validation is warranted. While these platforms offer potential advantages in flexibility and expanded resistance profiling, further studies on clinical diagnostic accuracy, cost-effectiveness, and operational feasibility are essential to determine their practical utility and programmatic impact in high-burden settings.

IMPORTANCE

Despite major advances in molecular diagnostics, critical gaps persist in tuberculosis (TB) detection, particularly the limited identification of isoniazid resistance and inadequate differentiation from non-tuberculous mycobacteria. In this study, a multiplex open real-time PCR assay demonstrated high analytical accuracy for simultaneous detection of Mycobacterium tuberculosis complex, rifampicin and isoniazid resistance, with strong agreement to reference standards. By integrating multi-target detection within a single workflow, this approach addresses key limitations of current diagnostic strategies that rely on rifampicin as a surrogate for multidrug resistance. The open-platform design further offers flexibility, scalability, and reduced dependence on proprietary systems, which are critical considerations in high-burden settings. These findings highlight the potential of next-generation molecular diagnostics to improve early resistance detection, refine treatment decisions, and strengthen TB control efforts globally.