Mutations in the rpob gene of Mycobacterium tuberculosis identified by sequencing method

Abstract

Objective: To identify the mutation in the rpoB gene of Mycobacterium tuberculosis (MTB), using by sequencing method from pulmonary specimens of presumptive TB patients belonging to the districts of Tamil Nadu. Methods: A total of 8697 clinical specimens of presumptive MTB patients were collected from various districts of Tamil Nadu. Smear microscopy was performed by light emitting diode fluorescent microscopy and all the smear positive samples were tested using line probe assay (LPA) to detect the percentage of drug resistance pattern and to identify the missing mutation in LPA by the sequencing method. Results: Among 4897 smear positives subjected to LPA method; 407 (8.3%) MTB was not detected and 16 (0.3%) showed invalid result; 4473 (91.4%) strains showed MTB positive; 3695 (82.6%) were sensitive for both rifampicin (RIF) and isoniazid (INH) drugs; 502 (11.2%) were resistance for INH; 73 (1.6%) resistant for RIF; 203 (4.5%) were resistance for RIF and INH. Totally, 52 (1.2%) strains results cannot be confirmed by LPA and reported as sensitive for RIF, because of the faint and the missing bands in both wild type and mutation. These strains were sequenced and 39 (75%) strains showed resistant to RIF. Conclusion: Hence LPA may be the molecular technology for the rapid, feasible and reliable method for the detection of multidrug resistant mutation but few confusion bands cannot be reported as resistance, which should be confirmed by either conventional phenotypic drug susceptibility testing or by sequencing method.