Objective To evaluate the application of multiple locus variable number tandem repeat analysis (MLVA, 15-locus set)for genotyping of Mycobacterium tuberculosis (MTB) strains of Beijing genotype. Methods Total 72 Beijing genotype MTB strains obtained from Beijing Thoracic Hospital were genotyped by MLVA (15-locus set). The results were compared with that generated from "gold standard"IS6110-RFLP. Results After genotyped by MLVA ( 15-locus set), 72 strains were grouped into 59 types,of which 53 were unique types. The Hunter-Gaston index (HGI) of MLVA ( 15-locus set) was 0.990. The loci QUB-11b, Mtub 21 and QUB-26 were polymorphic in selected Beijing genotype strains. Genotyping by IS6110-RFLP generated 69 types, of which 66 were unique types. The HGI of IS6110-RFLP was up to 0.999, and the MLVA (15-locus set) clustered strains could be further subdivided. Conclusion MLVA(15-locus set) showed better discriminatory ability in Beijing genotype MTB strains, though secondary typing of clustered strains by IS6110-RFLP is needed.

BACKGROUNDChina 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.

METHODSForty 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.

RESULTSSixty-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.

CONCLUSIONSThe 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.

Antitubercular Agents ; pharmacology ; Bacterial Proteins ; genetics ; Catalase ; genetics ; DNA Transposable Elements ; DNA, Bacterial ; analysis ; DNA-Directed RNA Polymerases ; Drug Resistance, Bacterial ; Isoniazid ; pharmacology ; Microbial Sensitivity Tests ; Mutation ; Mycobacterium tuberculosis ; drug effects ; genetics ; Rifampin ; pharmacology