Multi-drug resistant gene mutation analysis in Mycobacterium tuberculosis by molecular techniques

Monika Sultana; Mohammad Mamun Alam; Somen Kumar Mistri; S. M.  Mostafa Kamal; Chowdhury Rafiqul Ahsan; Mahmuda Yasmin

doi:10.18502/ijm.v16i4.16304

Monika Sultana Department of Microbiology, University of Dhaka, Dhaka-1000, Bangladesh
Mohammad Mamun Alam Department of Microbiology, University of Dhaka, Dhaka-1000, Bangladesh
Somen Kumar Mistri Department of Microbiology, University of Dhaka, Dhaka-1000, Bangladesh
S. M. Mostafa Kamal National Tuberculosis Reference Laboratory (NTRL), Dhaka-1207, Bangladesh
Chowdhury Rafiqul Ahsan Department of Microbiology, University of Dhaka, Dhaka-1000, Bangladesh
Mahmuda Yasmin Department of Microbiology, University of Dhaka, Dhaka-1000, Bangladesh

DOI: https://doi.org/10.18502/ijm.v16i4.16304

Keywords: Drug susceptibility testing; GeneXpert assay; Multidrug-resistant tuberculosis; Multidrug-resistant; Mycobac- teria; Pulmonary tuberculosis

Abstract

Background and Objectives: Rifampicin (RIF) and isoniazid (INH), two most potent antibiotics, are prescribed to cure tuberculosis. Mycobacterium tuberculosis, the causative agent of multidrug-resistant tuberculosis (MDR-TB), is resistant to these first-line drugs. Here, two molecular techniques were demonstrated such as PCR sequencing-based and GeneXpert assay for rapidly identifying MDR-TB.

Materials and Methods: Pulmonary samples (sputum) were collected from 55 MDR-TB suspected patients from the Na- tional Tuberculosis Reference Laboratory (NTRL), Dhaka where the research work was partially accomplished and con- tinued in the department of Microbiology, University of Dhaka, Bangladesh. We strived for sequencing technique as well as GeneXpert assay to identify mutations in rpoB and katG genes in MTB strains and sputum directly. Culture-based drug susceptibility testing (DST) was performed to measure the efficacy of the molecular methods employed.

Results: When analyzed, rpoB gene mutations at codons 531 (54.54%), 526 (14.54%), and 516 (10.91%) were found by sequencing in 80% of the samples. Nucleotide substitution at katG315 (AGC→ACC) was spotted in 16 (76.19%) out of 21 samples. When comparing the sequencing results with DST, sensitivity and specificity were investigated to determine drug-resistance (rifampicin-resistance were 98 and 100% whereas isoniazid-resistance were 94 and 100% respectively). Additionally, as a point of comparison with DST, only 85.45% of RIF mono-resistant TB cases were accurately evaluated by the GeneXpert assay.

Conclusion: This research supports the adoption of PCR sequencing approach as an efficient tool in detecting MDR-TB, counting the higher sensitivity and specificity as well as the short period to produce the results.