A multiplex assay based on capillary electrophoresis to detect Mycobacterium tuberculosis complex: development and clinical validation.

This study presents a novel multiplex assay based on capillary electrophoresis (CE) for the simultaneous detection of three Mycobacterium tuberculosis complex (MTBC) genes: IS6110, rpoB, and HSP65. Unlike conventional molecular diagnostic methods that target only a single gene, which may lead to misdiagnosis or missed diagnosis, this CE-based multiplex approach provides comprehensive detection to reduce diagnostic errors. Specificity testing with 76 microorganisms representing common respiratory pathogens confirmed 100% analytical specificity with no cross-reactivity, while sensitivity analysis demonstrated detection limits ranging from 10 to 20 copies/mL for all three target genes. In a prospective clinical validation study of 1067 patients suspected of pulmonary tuberculosis, the multiplex assay showed 77.4% sensitivity (CI 74.9%-79.9%), 99.6% specificity (CI 99.2%-100%), 96.0% positive predictive value (CI 94.8%-97.2%), and 97.1% negative predictive value (CI 96.1%-98.1%). Notably, the study identified 6 MTBC strains (4.8% of TB patients) with IS6110 deletions through whole-genome sequencing, which would result in false-negative results for any commercial PCR kits targeting IS6110. This integrated multiplex approach enhances diagnostic accuracy by simultaneously targeting multiple genes; then it offers the potential to reduce misdiagnosis and missed diagnosis of tuberculosis. In summary, the multiplex assay provides a more comprehensive alternative to current single-target molecular methods for MTBC detection. KEY POINTS: • The multiplex assay provides one-run results for IS6110, rpoB, and HSP65. • The multiplex assay is a more comprehensive method to detect MTBC. • This approach can reduce misdiagnosis and missed diagnosis of TB.