Evaluation of novel PCR-CTPP for simultaneous detection of Mycobacterium tuberculosis complex and identification of RpoB H526D point mutation

Abstract

Background: Tuberculosis (TB) is a chronic and highly contagious disease caused of the acid-fast bacilli, Mycobacterium tuberculosis complex (MTC). Currently, it has been reported that rifampicin-mono resistant M. tuberculosis containing histidine to aspartate replacement at residue 526 (H526D) of the beta-subunit RNA polymerase enzyme (RpoB) increased the cell wall permeability and approximately eight times more susceptible to vancomycin. Objectives: To develop and evaluate the Polymerase chain reaction with confronting two-pair primers (PCR-CTPP) for simultaneous detection of MTC and identification of RpoB H526D mutation. Materials and methods: PCR-CTPP was implemented for TB diagnosis. Reaction and profile were optimized and applied for detection in a total of 308 clinical samples. Sensitivity and specificity of PCR-CTPP was calculated in comparison to the acid-fast bacilli (AFB) staining and standard culture method. In addition, microscopic observation drug susceptibility (MODS) assay was modified for vancomycin susceptibility determination in 15 clinical isolates. Results: Limit of detection of PCR-CTPP was approximately 2×103 bacilli and no cross-detection to other mycobacteria. PCR-CTPP was evaluated in 308 clinical samples. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of PCR-CTPP versus standard culture method were 90.95%, 79.31%, 91.78% and 77.53%, respectively. In addition, comparison of developed PCR-CTPP versus AFB staining were also represented. MODS was performed in fifteen samples, two multidrug-resistant (MDR) strains containing RpoB H526D were susceptible against vancomycin. Conclusion: The established PCR-CTPP is highly sensitive, specific for investigation of MTC and identification of RpoB H526D mutation. This method could be useful for TB diagnosis together with precision medicine application in vancomycin susceptibility determination in TB patients in the future.