Study On The Molecular Epidemiology Of Drug Resistant Tuberculosis And The Association Between EmbB Codon 306 Mutations And Drug Resistance In Mycobacterium Tuberculosis

Tuberculosis(TB) remains a serious problem of public health worldwide and in China.China has the world's second largest TB epidemic with more than 4.5 million cases of TB,and 130,000 death of TB every year.Furthermore,almost half of population are already infected with Mycobacterium tuberculosis(M.tb) in China.The emergence of drug resistant TB,especially multidrug resistant(MDR) TB and extensively drug resistant(XDR) TB,pose a substantial threat to TB control programmes,and become an urgent issue to be solved.In this study,a retrospective analysis was performed on all pulmonary TB patients reported in Shanghai,China during 2000-2006 to determine the prevalence, trends and risk factors of drug resistant TB.Based on the epidemiologic study, genotyping was used to investigate the cause of drug resistance among relapse TB eases.Furthermore,a cross-sectional study of the drug susceptibility patterns of clinical isolates of M.tb and the distribution of the embB codon 306(embB 306) mutations of a subset of these isolates were performed to investigate whether embB 306 mutations are associated with EMB resistance and/or broad drug resistance.Of 8,419 pulmonary TB patients with drug susceptiblity testing available in Shanghai during 2000-2006,16.6%had resistance to any first-line antituberculosis drug and 4.0%had MDR.The percentage of TB patients with resistance to any first-line antituberculosis drug and MDR significantly increased during 2000-2003(p =0.01 and p=0.005,respectively).After improvements in the TB control program, the percentage of patients with MDR significantly decreased from 5.9%in 2004 to 4.2%in 2006(p=0.04),suggesting that improvements in the TB control programme prevented further increases in drug resistance and MDR.Overall,72.5%of patients with resistance to any first-line antituberculosis drug and 59.8%of patients with MDR did not report a history of previous TB treatment,suggesting recent transmission of drug resistant Mycobacterium tuberculosis may be a major cause of drug resistant TB and MDR TB in this setting.Pulmonary TB patients who had previous TB treatment, were 30-59 years old,were urban migrants,and resided in an urban area were more likely to have resistance to any first-line antituberculosis drug and M-DR.To determine the cause of drug resistance(primary or acquired drug resistance), we genotyped the initial and subsequent drug-resistant clinical isolates of M.tb collected from relapse TB patients during 1999-2004 in Shanghai,China.In 13 (72.2%) of all 18 relapse patients with drug resistance,isolates from the two tuberculosis episodes showed different MIRU pattern,indicating vast majority of patients had primary drug resistance due to transmission of a drug-resistant strain of M.tb.Only 27.8%(5/18) had acquired drug resistance because of a poor treatment regimen or nonadherence to an adequate regimen.These findings highlight the urgency of increasing efforts to interrupt the transmission of drug-resistant tuberculosis in communities and facilities in Shanghai,China.Ethambutol(EMB) is commonly used as one of the first-line drugs for anti-tuberculosis therapy.Previous studies showed that strains with EMB-resistance were always resistant to other anti-TB drugs.To further investigate wether this phenomenon is widespread,we analyzed the drug susceptibility patterns of clinical isolates of M.tb during 1999-2005 in Shanghai.Among all 104 EMB-resistant isolates, only 2.9%were mono-resistant to EMB.Moreover,the number of EMB-resistant isolates increased stepwise as the number of other first-line anti-tuberculosis drugs with resistance increased,indicating the association between EMB-resistance and resistance to other anti-TB drugs.To investigate the association between embB 306 mutations and EMB resistance and/or broad drug resistance,we selected a total of 162 clinical isolates for embB306 mutations screening.An association was observed between embB306 mutations and broad drug resistance in our study.None of the pan-susceptible isolates contained embB306 mutations,while 28.7%(31/108) of the isolates resistant to at least one anti-tuberculosis drug contained a mutation at embB306(p＜0.0001).Moreover,we also observed a strong association between embB306 mutants and resistance to increasing numbers of anti-tuberculosis drugs. The percentage of mutants in drug resistant isolates increased stepwise with the number of drugs resistant.In addition,the proportion of embB306 mutants among MDR strains(35.3%) was significantly higher than the proportion of embB306 mutants among non-MDR strains(7.4%,p＜0.0001).Furthermore,the specificity of embB 306 locus for MDR-TB detection is 92.6%,the positive predictive value is 77.4%and the negative predictive value is 66.4%,suggesting that embB306 locus can be considered a candidate marker for the rapid detection of MDR-TB and XDR-TB.