RESUMO
OBJECTIVE: This is the new comparative geogenetic molecular evolution research of M. tuberculosis in Iran and Belarus. Thus, we researched the genetic patterns of samples collected in the first survey of anti-tuberculosis drug-resistance by gene coding of RNA polymerase as part of the international project of on tuberculosis. METHOD: DNA extraction and amplification of rpoB gene was performed. All PCR products of gene were sequenced using the Amersham auto sequencer. For analysing phenogram has been demonstrated by method UPGMA and Neighbour-Joining. Clinical isolates (70/473) were analyzed by using sequencing gene rpoB and genotyped by program DNAMAN and MEGA. RESULTS: The all data were compared with the international database of national center for biotechnology information website. Multi drug resistant of tuberculosis patient (MDR-TB) was 92% in never treated and 8% in previously treated. Mutations in rpoB gene and katG genes were showed in 95% and 84% of the MDR isolates, respectively. Two clusters were found to be identical by the four different analysis methods, presumably representing cases of recent transmission of MDR tuberculosis. The other isolates are divided in Iran into 2 groups: group A - similar to the Eastern strains (China, Taiwan) and group B - strains of another genotype. And 3 groups in Belarus: group A - Strains of the first group are more similar to the standard European and Eastern ones China and Taiwan) which diverged in the last 10 years (Genetic evolution rate), i.e. they are relatively new ones, and that is confirmed by the mutations, group B - Strains of the second group diverged earlier; they are older than the strains of the first group (16 years old- time and rate of evolution) and group C - Strains of the third group are similar to European strains and only circulate in Brest region. They are grouped separately on the phenogram and became prevalent in Iran (they are called Iranian residential strains and also is genetic analogy between group A from Iran and Belarusian isolates. CONCLUSION: This research gives a first result on genetic evolution of the M. tuberculosis strains distributing in the Iran and Belarus during the first survey of anti-tuberculosis drug-resistance and is homologies between groups A from Iran with group A from Belarus. It may aid in the creation of a national database that will be a valuable support for further studies.
RESUMO
The aim of this study was to investigate the significance of multiple-mutations in the katG gene, predominant nucleotide changes and its correlation with high level of resistance to isoniazid in Mycobacterium tuberculosis isolates that were randomly collected from sputa of 42 patients with primary and secondary active pulmonary tuberculosis from different geographic regions of Iran. Drug susceptibility testing was determined using the CDC standard conventional proportional method. DNA extraction, katG gene amplification, and DNA sequencing analysis were performed. Thirty four (80%) isolates were found to have multiple-mutations (composed of 2-5 mutations) in the katG gene. Increased number of predominant mutations and nucleotide changes were demonstrated in codons 315 (AGC-->ACC), 316 (GGC-->AGC), 309 (GGT-->GTT) with a higher frequency among patients bearing secondary tuberculosis infection with elevated levels of resistance to isoniazid (MIC ≥ 5-10 µg/mL). Furthermore, it was demonstrated that the combination of mutations with their predominant nucleotide changes were also observed in codons 315, 316, and 309 indicating higher frequencies of mutations among patients with secondary infection respectively. In this study, 62% (n= 21) of multi-mutated isolates found to have combination of mutations with predominant nucleotide changes in codons 315 (AGC-->ACC), 316 (GGC-->GTT), 309 (GGT-->GGT), and also demonstrated to be more frequent in isolates of patients with secondary infections, bearing higher level of resistance to isoniazid (≥ 5-10 µg/mL).
