ABSTRACT
Tuberculosis [TB] control remains an important public health priority for Bulgaria. The population structure of Mycobacterium tuberculosis is clonal and certain genetic families of this species [e.g., Latin-American-Mediterranean [LAM]] have attracted more attention due to their global dissemination and/or particular pathogenic properties, e.g., association with multidrug resistance [MDR]. The aim of this study was to evaluate the prevalence of the M. tuberculosis LAM family in Bulgaria based on the use of different molecular markers. A total of 101 previously spoligotyped M. tuberculosis strains were studied by LAM-specific PCR assay to detect an insertion of IS6110 in the specific genome region. On the whole, clear-cut results were obtained for most strains; spoligotype-based family was reassigned in some of them. At the same time, double bands were amplified in some cases and warrant further validation studies of this method. The higher MDR rate among LAM versus other genotype isolates was observed [P = 0.04]. In conclusion, these results suggest a low [<4%] prevalence rate of LAM in Bulgaria [that is similar to its Balkan neighbors] and highlight the importance of using robust markers for correct detection of the LAM family
ABSTRACT
<p><b>BACKGROUND</b>China is one of the high burden countries of Mycobacterium tuberculosis (TB) infection globally, with high incidence and mortality. We studied the molecular characteristics of rifampin (RIF) and isoniazid (INH) resistant Mycobacterium tuberculosis strains from Beijing, China, in order to find out the genetic marker for rapid detection of specific drug resistance.</p><p><b>METHODS</b>Forty pansusceptible and 81 resistant strains of Mycobacterium tuberculosis isolated from Beijing, China during 2002-2005 were analyzed. The modified rifampin oligonucleotide (RIFO) assay based on reverse line blot hybridization was used to detect mutations in the 81 bp hot-spot region of rpoB gene, which is associated with RIF resistance. The INH resistance associated genes, regulatory region mab-inhA (-15C/T) and structural gene katG S315T were detected by reverse line blot hybridization and PCR-restriction fragment length polymorphism (RFLP) method respectively. All the strains were typed by spoligotying and the Beijing genotype was further subdivided by NTF locus analysis. The distribution of drug resistance associated mutations in the above genes was compared in these groups.</p><p><b>RESULTS</b>Sixty-five (91.5%) of 71 RIF resistant and 52 (92.9%) of 56 multidrug-resistant (MDR, i.e. resistant to at least RIF and INH) strains were found to harbor mutations in the rpoB hot-spot region. No mutation was detected in RIF sensitive strains. The specificity and sensitivity of the modified RIFO assay were 100% and 91.5%, respectively. katG315 AGC>ACC and inhA-15C>T mutations were found in 40 (60.6%) and 10 (15.2%) of 66 INH resistant strains, respectively; 7.6% of INH-resistant strains had mutations in both of these genes. Therefore, a combined use of both katG315 and inhA-15 identified 68.2% of INH-resistant strains. The Beijing genotype accounted for 91.7% of total strains and was further subdivided into "modern" (76.6%) and "ancestral" (23.4%) group. There is no significant difference between "ancestral" and "modern" group in prevalance of drug resistance-associated gene mutations.</p><p><b>CONCLUSIONS</b>The hot-spot region of rpoB gene can be used as genetic marker for detection of RIF resistant strains; a combined use of both katG315 and inhA-15 can improve the detection rate of INH resistant strains; the Beijing genotype is prevalent in Beijing, China; the modified RIFO assay can be a practical tool for rapid detection of RIF resistant and MDR isolates in the routine diagnostic work.</p>