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

Abstract Background Timely diagnosis of tuberculosis and drug resistance remains a cornerstone of 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-tuberculosis drugs could streamline diagnostic pathways. Methods We conducted a laboratory-based evaluation of two multiplex real-time PCR assays (MTBc/NTM R-Gene and MTB-RIF/INH R-Gene) using 300 well-characterized samples, including 150 MTBc-positive culture isolates (including rifampicin-resistant, isoniazid-resistant, and drug-susceptible strains) and 150 MTBc-negative samples (50 NTM isolates and 100 mycobacteria-negative specimens). Composite reference standards included culture, MPT64 antigen testing, and line probe assay corroborated by phenotypic drug susceptibility testing for resistance profiling, with NTM speciation performed using a dedicated line probe assay. DNA extraction was performed using the QIAamp DNA Mini Kit (QIAGEN, Germany), followed by amplification on a real-time PCR platform according to manufacturer instructions. The diagnostic performance was assessed against composite reference standards. Results The analytical performance for detecting MTBc demonstrated 100% sensitivity and specificity (150/150). NTM detection showed 70.0% sensitivity (35/50) and a specificity of 100%, highlighting limitations in coverage of NTM species. Rifampicin resistance was detected with a sensitivity of 96.0% (48/50) and specificity of 100%, whereas isoniazid resistance detection was 100% sensitive and specific (50/50). Agreement with established reference standards was high ({kappa}=0.76-1.00) within this analytical context. Interpretation This analytical validation demonstrates that multiplex open real-time PCR assays can accurately and simultaneously detect MTBc, NTM, and rifampicin and isoniazid resistance using culture isolates. While these platforms offer potential advantages in flexibility and expanded resistance profiling, additional studies on clinical diagnostic accuracy, cost-effectiveness analyses, and operational feasibility are required to determine their practical utility and programmatic impact in high-burden settings