MUTATIONS IN THE RPOB GENE OF MYCOBACTERIUM TUBERCULOSIS IDENTIFIED BY SEQUENCING METHOD
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.
Keywords: Line probe assay, Sequencing, Mutation, Multidrug resistant tuberculosis.
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Table 1: Total presumptive MDR cases tested, smear positive was diagnosed by LPA and the drug susceptibility results
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Strains show the faint band or the missing bands in WT8 and also missing band in specific probe MUT 3
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MDR: Multidrugâ€‘resistant, LPA: Line probe assay, MTB: Mycobacterium tuberculosis, RIF: Rifampicin, INH: Isoniazid
Table 2: 52 Strains were sequenced and the amino acid changes were observed in 39 strains, i.e., out of 52 strains 32 strains show the resistant in 531bp region and 7 strains were resistant in 526bp region in rpoB gene with 3 strains misread
Total number of resistance
Total number of sensitive
Total number of misread
Additionally amino acid changes in 7 strains were found in 526bp region
Fig. 4: rpoB mutation-specific probes: MUT D516V, H526Y, H526D, S531L and rpoB wild type probes: WT1 to WT8
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