Molecular detection of multi-drug resistance genes in Mycobacterium tuberculosis

Abstract

The spread of multidrug resistant tuberculosis (MDR-TB) due to emergence of multidrug resistant M. tuberculosis isolates has increased worldwide and reached epidemic proportions in many countries. The emergence of MDR-TB has become a major threat for TB control in Bangladesh. MDR-TB is defined as resistant to at least rifampicin and isoniazid, which are the backbone of short-course chemotherapy for tuberculosis. Mutations in the 81-bp core region of rpoB were reported to be responsible for resistance in at least 95% of isolates. Resistance to isoniazid is due to mutations at one of two sites, in either the katG or inhA genes. The culture-based methods for detection of M. tuberculosis infection and drug susceptibility testing (DST) usually take more than 1 month due to the slow growth of this bacterium. The use of molecular methods for the identification of mutations in the resistance genes may offer the means for rapid screening of the drug resistance among the M. tuberculosis isolates and initiation of early treatment. In this study, we evaluated rapid detection of multidrug resistant strains using the molecular techniques from MTB strains and sputum directly. For this purpose, a total of 55 sputum samples were collected from 55 patients of National Tuberculosis Reference Laboratory (NTRL), Mohakhali, Dhaka- 1212, Bangladesh. Samples were tested for microscopic examination, conventional culture method, drug susceptibility testing, Gene Xpert assay and PCR amplification followed by DNA sequencing. All 55 samples (36 from MTB strain, 19 from direct sputum) were subjected to rpoB gene amplification and 21 samples (12 from MTB strain, 9 from direct sputum) for katG gene amplification by PCR. Then the samples were subjected to DNA sequencing for rpoB and katG gene mutation analysis. RIF-associated mutations were detected in (44/55) samples (80%), of which, mutations at codons 531 (54.54%), 526 (12.73%) and 516 (9.09%) were observed. Sixteen samples (76.19%) out of 21 had isoniazid associated mutations at codon 315 in the katG gene. Considering conventional DST as gold standard, sensitivity and specificity of DNA sequencing for the detection of Rif- resistance were 98% and 100%; whereas isoniazid resistance were 94% and 100%. Comparison between Xpert MTB/RIF and DST results showed that eight samples were sensitive to rifampicin by Genexpert assay, but not on DST. The reason may be due to the selection of resistant strains in the antibiotic added media for a prolonged incubation period. Therefore, falsepositive results of rifampicin resistance were observed in DST.