Evaluation of Sensitivity of Molecular Methods for Detection of Rifampin-Resistant Strains Amongst Drug-resistant Mycobacterium tuberculosis Isolates

Background: Resistance to rifampin in multidrug-resistant M. tuberculosis (MDR-TB) is one of the major threats to the global public health system. Objectives: The aim of the present study was to explore the molecular characterization of resistance against rifampin amongst multidrug-resistant (MDR) and extensively drug-resistant (XDR) isolates obtained from patients. Methods: In this study, we used XDR (n = 6), MDR (n = 9) and rifampin sensitive (n = 39) isolates whose drug susceptibility patterns were identified by proportion method. A simple single-step multiplex-allele specific-polymerase chain reaction (MAS-PCR) assay was optimized to detect the three most frequent mutations in rifampin resistance-determining region (RRDR) fragment of rpoB gene. Additionally, single-strand conformational polymorphism (SSCP)-PCR and Sequencing were utilized. Results: Out of nine MDR isolates, five were detected by MAS-PCR as rifampin resistant (sensitivity; 55.5%). In comparison with the proportion method, the sensitivity of SSCP for XDR, MDR and rifampin sensitive isolates were 83.3%, 77.7% and 97.4%, respectively. The DNA sequencing revealed that three of the 6 XDR isolates had several silent mutations, one isolate had one silent mutation, one strain had no mutations and only one isolate had mutations at codon 531. Conclusions: In sum, although the molecular methods are rapid, they are not able to identify resistance against rifampin efficiently.