Detection and characterization of mutations in Rifampicin resistant Mycobacterium tuberculosis clinical isolates by DNA sequencing.

ABSTRACT

Background: Multiple Drug Resistant Tuberculosis (MDR-TB) is increasing because of widespread application and results in selection of mutants resistant to other components of short course chemotherapy. Resistance to Rifampicin can be considered as a surrogate marker for MDR-TB and the target gene for detection of rifampicin resistance is the rpo gene. Aims: To detect and characterize mutations in the rpo B region of Rifampicin resistant isolates of Mycobacterium tuberculosis by automated DNA sequencing. Methods: Absolute concentration method was used to determine the MIC of Rifampicin for 44 M. tuberculosis isolates (21 respiratory, 3 ocular, 3 cerebrospinal fluid and 17 biopsies). Automated DNA sequencing was performed in the ABI 310 Genetic Analyser. Results: Five isolates (2 sputa and one each from bronchoalveolar lavage, lymph node and endometrial biopsies) were rifampicin resistant with MIC greater than 128 mg/ml. Three of the five isolates showed mutations. Two of the isolates had the common missense mutation at codon 531(Ser®Leu), the other isolate showed three insertions and two of them did not show any mutation in the sequenced rpo B region. Conclusions: DNA sequencing technique is a rapid, conclusive and more advantageous technique than the conventional susceptibility testing for detection of rifampicin resistance in terms of the risk involved and time consumption.