Detection of mutations in the rpoB gene of rifampicin-resistant Mycobacterium tuberculosis strains inhibiting wild type probe hybridization in the MTBDR plus assay by DNA sequencing directly from clinical specimens

• Published in: BMC microbiology Vol. 20; no. 1; p. 284
• Main Authors: Sinha, Pallavi, Srivastava, G N, Tripathi, Rajneesh, Mishra, Mukti Nath, Anupurba, Shampa
• Format: Journal Article
• Language: English
• Published: England BioMed Central Ltd 16-09-2020; BioMed Central; BMC
• Subjects: Assaying; Deoxyribonucleic acid; Diagnostic systems; DNA; DNA probes; DNA sequencing; Drug resistance; Genes; Genetic aspects; Genetic research; Genetic screening; Genetic testing; Genotype & phenotype; Genotype MTBDRplus; Genotypes; Hybridization; Line probe assays; Mutation; Mycobacterium tuberculosis; Nucleotide sequence; Reference standard; Rifampin; RopB gene; RpoB protein; Sequence analysis; Strains (organisms); Tuberculosis; Genotype MTBDRplus; Mycobacterium tuberculosis; Reference standard; Line probe assays; DNA sequencing
• ISSN: 1471-2180; 1471-2180
• DOI: 10.1186/s12866-020-01967-5

The potential of genetic testing for rapid and accurate diagnosis of drug-resistant Mycobacterium tuberculosis strains is vital for efficient treatment and reduction in dissemination. MTBDR plus assays rapidly detect mutations related to drug resistance and wild type sequences allied with susceptibility. Although these methods are promising, the examination of molecular level performance is essential for improved assay result interpretation and continued diagnostic development. Therefore this study aimed to determine novel mutations that were inhibiting wild type probe hybridization in the Line probe assay by DNA sequencing. Using data collected from Line Probe assay (GenoType MTBDRplus assay) the contribution of absent wild type probe hybridization to the detection of rifampicin resistance was assessed via comparison to a reference standard method i.e. DNA sequencing.
Sequence analysis of the rpoB gene of 47 MTB resistant strains from clinical specimens showed that 37 had a single mutation, 9 had double mutations and one had triple mutations in the ropB gene.
The absence of wild type probe hybridization without mutation probe hybridization was mainly the result of the failure of mutation probe hybridization and the result of the novel or rare mutations. Additional probes are necessary to be included in the Line probe assay to improve the detection of rifampicin-resistant Mycobacterium tuberculosis strains.