DNA Sequencing and bacieriophage based technique for rapid detection of rifampicin resistant mycobacterium tuberculosis

ABSTRACT

The drug resistant Mycobacterium tuberculosis [M TB] is an emerging problem of great importance to public health worldwide. This has stressed the need for development of more rapid techniques to accurately identify mycobacteria and drug resistant strains This study was done in an attempt to find a rapid and reliable method for identification of M. TB, and to study the genetic mutation in rifampicin resistance. Thirty sputum samples were subjected to identification of M. TB using conventional methods including Ziehli-Neelsen [ZN] stain and Lowenstein Jensen [LJ culture in comparison to the new rapid FAST plaque TB [bacteriophage based assay] which was used also to detect rifampicin resistance. The rifampicin resistant M. TB were subjected to PCR implication of rpoB gene followed by automated DNA sequencing. The phage technique identified 14/30 [46.7%] and when compared to LJ culture which was able to detect 19/30 [63.3%], the sensitivity was 73.7% and the specificity was 100%. Z.N. stain detected acid fast bacilli [AFB] in 10/30 ['33.3%]. Among the 20 smear negative samples the phage technique was able to detect 4 out of 9 cases identified by LJ culture with 44.4% sensitivity and 100% specificity. Among the rifampicin resistant M. TB, the highest percentage of point mutation was detected at codon 531[40%] and codon 526 [40%] followed by codon 516 [20%]. In conclusion the phage technique could be used in conjunction with sputum smear microscopy to detect additional cases which would be missed with smear alone. Rifampicin resistant M. TB is mostly associated with rpoB gene mutations