| To investigate the prevalence and the risk factors of drug-resistant tuberculosis in Tianjin; to identify the Beijing genotype Mycobacterium tuberculosis and describe the association of Beijing genotype Mycobacterium tuberculosis and drug resistance and other characters; to research the drug-resistance genotype of four anti-tuberculosis drugs and the mutated codons and their association with the corresponding drug resistance phenotype, drug resistance level and Beijing genotype; to detect the expression of drug efflux pump genes and analyze their association with drug resistant phenotype and drug resistance related mutated patterns.Methods:1A cross-sectional study was conducted to describe the drug-resistant tuberculosis prevalence in Tianjin:The subjects were all culture-positive pulmonary tuberculosis patients newly diagnosed or retreated in Tianjin Tuberculosis Control Center and10districs or county tuberculosis dispensaries from January2008to June2009. The proportion method recommended by WHO/IUATLD was used for testing the strains’ susceptibility to rifampicin, isoniazid, streptomicin and ethambutol.2Bivariate and multiple unconditional logistic regression were performed to identified factors associated with multidrug-resistant tuberculosis and other drug-resistant tuberculosis.3Multiplex PCR was performed to identify Beijing genotype of the above subjects’ first isolated strains.4DNA sequencing the genes of the above subjects’first isolated strains was applied to investigate the drug-resistance genotype, the most common mutated codons and their association with the corresponding drug resistance phenotype, drug resistance level and Beijing genotype. The genes sequenced were katG, inhA, oxyR-ahpC and ndh associated with isoniazid resistance, rpoB associated with rifampicin resistance, rpsL and rrs associated with streptomycin resistance, embB, embC and embA associated with ethambutol resistance.5Real-time RT-PCR was used to assess the expressions of putative drug efflux pump genes Rv1258c, Rv1410c, Rv2136c, Rv0783c and Rv2136c in71mycobacterium tuberculosis strains from Tianjin Tuberculosis Control center in2007.Results:1656subjects were included,568were new tuberculosis cases and88were previously treated. The prevalence of overall resistance among all cases was26.98%(177),24.47%(139) among new cases,43.18%(38) among previously treated cases. The prevalence of MDR-TB among all cases was6.25%(41),3.70%(21) in new cases,22.73%(20) among previously treated cases.2The independent predictors of multidrug-resistant tuberculosis were age group (30-49years old/≥50years old, OR=3.000,95%CI:1.121-8.024), the numbers of abnormal lung fields (6/1-5, OR=3.153,95%CI:1.187-8.378), previous treatment history (OR=6.984,95%CI:3.250-15.009), supervisors except doctors and family members (OR=2.787,95%CI:1.152-6.741). No associations were found between any factors and the other drug-resistant tuberculosis.3Six hundreds of656strains were Beijing genotype, and Beijing genotype strains were more prevalent in population from Tianjin (χ2=4.26, p=0.039) and age≤55years old (χ=4.03,p=0.045). No association was found between Beijing genotype and drug resistance.4Of110isoniazid resistant isolates,54(49.09%) had mutations in the katG with315ACC(Thr) for responsible for42.73%of isoniazid resistance,27(24.55%) had mutation of inhA-15Câ†'T, and13(11.82%) had mutations in the oxyR-ahpC, no mutation was found in ndh. In all,94of110isoniazid resistant isolates carried mutations. Mutations in katG315were related to combination resistance of isoniazid and other drugs. Forty six out of51rifampicin resistant isolates carried the mutations within the81-bp rifampicin resistance determining region (RRDR) of the rpoB gene, and the most frequent mutations were at codon531(50.00%),526(23.08%), and516(5.77%), respectively. Mutations of rpoB were related to combination resistance of rifampicin and streptomycin and/or isoniazid. Of116streptomycin resistant isolates,85(73.27%) had mutations in rpsL,17(14.66%) had mutations in rrs. Mutations in rpsL were related to Beijing genotype. Of33ethambutol resistant isolates,9(27.27%) had mutations in embB,1(3.03%) had mutations in embC, no mutations was found in embA.5The expression of drug efflux pump gene Rv1258c of all isolates was negative. Compared to pan-sensitive isolates, Rv1410c showed higher expression in streptomycin (t=2.18, p=0.034), isoniazid (t=2.20,p=0.032), isoniazid+streptomycin (t=2.43, p=0.021),≥2any of four drugs (F=3.60,p=0.018) resistant isolates; Rv2136c showed higher expression in isoniazid resistant isolates (t=2.03,p=0.047) and multidrug-resistant isolates (t=2.22, p=0.037). The expression of Rv0783c in rifampicin resistant isolates with rpoB531mutations was higher than those with wild type of rpoB531(t=2.84,p=0.016).Conclusions:1Age and clinic characters (abnormal lung fields, previous treatment history) and supervisors were related to drug resistance, more interventions should be taken on such characters of tuberculosis patients.2Beijing genotype mycobacterium tuberculosis was prevalent in Tianjin China and population with age≥55years old. No association was found between Beijing genotype and drug resistance.3Mutations in drug related genes were the major mechanism leading to drug resistance. And this study will provide clues for choosing a suitable molecular biology method based on gene mutations testing for diagnosing drug susceptibility of mycobacterium tuberculosis strains in Tianjin, China. Mutations in rpsL were more frequent in Beijing genotype stains.4It was concluded that the expressions of Rv1410c and Rv2136c could be targets of molecular methods for diagnosing drug resistance. The expression of Rv0783c in rifampicin resistant isolates was associated with mutation rpoB531. |
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