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Objective To study the genotypes of representative Mycobacterium tuberculosis (M.tuberculosis) strains from China with spacer oligonucleotide typing (spoligotyping),and to investigate the prevalence of different genotypes TB in China,and analyse the relationship between genotype and drug resistance.Methods 4017 clinical isolates were collected by Chinese Center for Disease Control and Prevention from 2007 to 2008 in 31 provinces in China according to sampling principle of epidemiology.Drug susceptibility testing was performed using proportion method,and spoligotyping was chosen to carry out genotyping of these M.tuberculosis.In addition,chi-square test was used to compare the differences among the detection rate of different genotypes.Results Among the 4017 M.tuberculosis isolates,2500 ( 62.2％ ) isolates belonged to Beijing genotype.The percentage of Beijing genotypes in the northern of China was higher than that in the southern of China ( 76.5％ vs.53.2％,x2 =219.69,P ＜ 0.05 ),while T1 genotypes were more common in the southern China,compared with that in northern China ( 13.3％ vs.4.3％,x2 =219.69,P ＜ 0.05 ).The differences were statistically significant.The proportions of Rifampinresistant (21.7％ vs.21.7％ ),Ofloxacin-resistant (4.9％ vs.2.4％ ) and Multidrug-resistant ( 11.3％vs.7.4％ ) isolates among Beijing genotype strains were significantly higher than those among non-Beijing strains (x2 =22.10,14.42 and 14.83,respectively,P ＜ 0.05 ).Conclusions Beijing genotype was still predominant epidemic genotypes.The percentage of Beijing genotype showed difference between distinct areas,and the percentage of Beijing genotypes in northern China was higher than that in southern China.Beijing genotype strains reveal correlation with Rifampin-resistance,Ofloxacin-resistance and Multidrug-resistance.
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Objective To evaluate a rapid biochip system for the determination of muhidrugresistant tuberculosis (MDR-TB) in Mycobacterium tuberculosis isolates. MethodsA total of 1 186 clinical strains, including 800 rifampin (RFP) resistant isolates, 797 isoniozid (INH)resistant isolates, 791 MDR-TB and 380 susceptible strains, were selected from Beijing Chest Hospital, Shanghai Pulmonary Hospital and Guangzhou Chest Hospital respectively using stratified sampling method. Biochips were used to detect loci of rpoB 511 (T→C), 513 (A→C, C→A), 516 (G→T, A→T, A→G) , 526 (C→T, C→G, A→T, A→G), 531 (C→T, C→G), 533 (T→C), katG 315 ( G→C, G→A) and inhA -15 (C→T). Absolute concentration drug susceptibility test of RFP and INH were performed to serve as the gold standard to calculate susceptibility, specificity and overall concordance of biochip test. All polymerase chain reaction (PCR) products were sequenced to confirm the mutations. ResultsThe concordances between the biochip system and absolute concentration drug susceptibility test were 93.7％ ( 1 108/1 183 ) for RFP, 83. 8％(994/1 186) for INH and 82.4％ (975/1 183) for MDR-TB. Compared with absolute concentration drug susceptibility test, the biochip method displayed a sensitivity of 92. 0％ (733/797) and 77. 4％ (617/797)and a specificity of 97. 2％ (375/386) and 96. 9％ (377/389) for RIF and INH, respectively. For MDR-TB, the biochip system reached a sensitivity of 74. 6％ ( 588/788 ) and a specificity of 98.0％ ( 387/395 ).Among rpoB mutants, mutations were mostly detected at codon 531[64. 5％ (480/744)]. In stains with mutations in katG or inhA, 77.4％ ( 487/629 ) had mutation at codon 315 ( TCG ) of katG only. The sequencing results had a high concordance with that of the biochip method. There were slight differences in 5 strains, among which one strain was detected by biochip as katG 315(G→C) mutant, but was identified by sequencing as wild type, and mutation types other than those detected by the biochip were confirmed in the other 4 strains by sequencing. Conclusion This biochip system is adapted for extensive application in clinical diagnosis, as it allows fast and reliable detection of resistance to isoniazid and rifampin in tuberculosis clinical isolates.
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Objective To evaluate the usefulness of mycolic acid for identification of Mycobacterium species using SMIS. Methods One hundred and eighteen clinical Mycobacterium isolates collected from Beijing Tuberculosis and Thoracic Tumor Research Institute through whole year of 2007 were analyzed. The 118 isolates contain 25 isolates of Mycobacterium tuberculosis and 93 non tuberculosis Mycobacterium identified by PNB method. Mycolic acid analysis using SMIS is evaluated for identification of a broad range of Mycobacteria in comparison with 16S rDNA , 16-23S rDNA ITS sequencing to measure the concordance rate and agreement, and verify the concordance rate and agreement among results of mycolic acid, sequencing and PNB in identifying Mycobacterium tuberculosis and non tuberculosis Mycobacterium. Results The concordance rate between mycolic acid method analysis and DNA sequencing is 92% ( 108/118), of which concordance rate in Mycobacterium tuberculosis complex and non tuberculosis Mycobacterium are 95% (35/37) and 90% (73/81) respectively, agreement of both is great( agreement Kappa value is 0. 96). Through retrospective analysis, the concordance of results between SMIS and PNB method analysis is 90% (106/118)and agreement is well( agreement Kappa value is 0. 73 ), the concordance of results between sequencing and PNB method analysis is also 90% ( 106/118 ) and agreement is well (agreement Kappa value is 0. 74 ),despite the identification results of 11 isolates by PNB method are discordant. Conclusion Mycolic acid analysis by SMIS enables rapid identification of a broad range of clinical Mycobacterium species, which could play an important role in polyphasic identification of Mycobacterium species.