Molecular characterization of multidrug-resistant isolates of mycobacterium tuberculosis from patients in Punjab, Pakistan

Globally, Pakistan with population of around 180 million moved up two spots to 6th in the list of countries with the highest number of tuberculosis (TB) patients in the world according to WHO report (2011). Global TB control is further challenged by drug-resistant TB. For controlling the spread and better understanding of Mycobacterium tuberculosis (M. tb) isolates circulating in this region, it is important to explore the characteristics of these strains. In this study 71 isolates were tested for drug sensitivity and 41 out of 71 were found to be resistant. Out of these 41 isolates, 21 (51%) were resistant to isoniazid (INH) and rifampicin (RIF) and remaining have different drug resistance patterns. Genes associated with drug resistance like ahpC, katG and inhA for INH, rpoB for RIF, rrs and rpsL for streptomycin (STR) and embB for ethambutol (EMB) resistant isolates were checked for mutation using PCR amplification and sequencing. Out of 27 RIF resistant strains, 22 have mutation affecting four amino acid codons of rpoB; 531 (52%), 516 (15%), 526 (7.0%) and 512 (7.0%). In codon 512 two isolates showed insertion of GCC. These insertions were novel and observed for the first time in Pakistani isolates. The overall sensitivity of mutation detection in rpoB gene was 88.8% and specificity was 100%. Out of 24 INH resistant isolates, 19 (79 %) had S315T mutation in katG while 5 isolates had no mutation in this region. Thus, the overall sensitivity of mutation detection in katG gene was 79 % and specificity was 100%. These 5 isolates were further checked for mutation in promoter region of inhA and ahpC. In 2 isolates there was G to A transition at -88 of the promoter region of ahpC while 3 isolates had C to T transition at point -15 for inhA promoter region. Out of 14 STR resistant isolates, 7 isolates showed the mutation K88M, K88R and K43R in the gene rpsL while no mutation was found in the gene rrs in STR resistant strains. Six (67%) out of 9 EMB resistant isolates showed the mutation M306I while no mutation was found in 3 isolates. The mutations observed in this study are similar to the mutations commonly present worldwide. However, our results have shown one novel mutation in rpoB gene that can be considered for improving molecular methods used for the detection of multidrug-resistant strains. Furthermore wide range of screening is needed to characterize drug resistant strains prevalent in this region.