DNA sequence analysis of rpoB gene mutations in rifampicin-resistant Mycobacterium tuberculosis.

摘要

Multidrug-resistant tuberculosis (MDR-TB) is an increasing health problem, which contributes to mortality from tuberculosis (TB). Rifampicin (RIF) resistance is considered a marker for MDR-TB. Resistance to RIF results from mutation in the gene encoding the beta subunit of RNA polymerase (rpoB) of the mycobacterial tuberculosis (MTB) complex. The aim of this study was to assess the presence of RIF-resistant TB strains using the BACTEC 460 TB system and to determine the pattern of rpoB gene mutations in those strains by line probe assay in combination with DNA sequence analysis. The study was carried out on 60 patients from Egypt with pulmonary TB [date not given]. Sputum samples were cultured in the BACTEC 460 system. Positive TB cultures were subjected to antimycobacterial susceptibility testing by BACTEC 460 and agar proportional susceptibility test. RIF-resistant strains were further evaluated by line probe assay for the presence of rpoB mutations and DNA sequence analysis for detection of type of mutation. All sputum samples yielded growth by both conventional and BACTEC 460 system. Of the isolated TB bacilli, 20.0, 20.7, 6.7 and 3.3% were resistant to RIF, isoniazid (INH), ethambutol and streptomycin, respectively. Twenty percent were MDR-TB. The line probe assay had 100% sensitivity and 95.8% specificity. The most common mutations detected by DNA sequencer were detected within the 69-bp hypervariable region of the rpoB gene, mostly His 526Tyr and Ser531 Leu mutations. From this study, we could conclude that the most frequent drug resistance of TB was for RIF and INH. The most frequent sequence mutations were detected within the 69-bp hypervariable region of the rpoB gene as His526Tyr and Ser531 Leu mutations. The line probe assay was a simple, reliable and rapid screening tool for early detection and characterization of RIF-resistant Mycobacterium tuberculosis strains..