Mulit-drug resistant tuberculosis (MDR-TB) is caused by Mycobacterium tuberculosis when it is resistant to isoniazid and rifampicin with or without being resistant to any other first line drug. Mycobacterium tuberculosis is rod shaped aerobic bacteria. There are more then 50 species of Mycobacteria. Resistance to rifampicin is caused by mutations in rpoB gene which forms the beta subunit of RNA polymerase. Due to mutations in rpoB gene, rifampicin losses its affinity to bind RNA polymerase and the bacteria becomes resistant. MDR-TB is more dangerous than tuberculosis as former is treated by less effective and more expensive drugs. Also MDR-TB takes longer duration of treatment. So it is needed to study the pattern of mutations of rpoB gene in Mulit-drug resistant Mycobacterium tuberculosis and to identify any new mutations which can contribute towards rifampicin resistance.
1080 sputum samples were included in the study. Sputum samples were cultured and tested for drug sensitivity on Lowenstein Jenson (LJ) medium. DNA was extracted from the colonies on LJ medium. After PCR, the product was purified and sequenced. The mutations analysis was performed by comparing the wild type rpoB gene H37RV with the sequence of rpoB gene of our present MDR-TB isolates.
In our study we found mutation On codon 531, Mutation was observed in 6 strains ( 35%), which was of one type in which Serine was converted into Leucine . On codon 516, mutation was observed in 3 strains (18%), which was of two types in which Aspartic acid was converted into Valine and in second mutation Aspartic acid was converted into Tyrosine. On codon 512, mutation was observed in 1 strains ( 6%) in which Serine was converted into Isoleucine. On codon 533, mutation was also observed in 1 strain ( 6%). Mutation was of one type in which Leucine was converted into Proline. On codon 528, mutation was observed in 1 strain ( 6%) in which Arginine was converted into Arginine. On codon 533, mutation was observed in 1 strain ( 6%) in which Leucine is converted into Proline.
By studying and identifying mutations in rpoB gene in strains of our geographical region, we will be able to make better policies in rapid diagnosis and appropriate chemotherapy. That may contribute in controlling growing epidemic of tuberculosis in Pakistan. The result of present study have concluded that the molecular techniques can be use as rapid tool for the diagnosis and identification of MDR-TB in clinical isolates of MTB.