| China was second highest TB burden countries only next to India. Though modern TB control program based on Directly Observed Treatment (DOTS) has been implementing since the 1990s, the incidence rate decreases slowly at a rate of 1%. It's estimated that there are still about 2.58 million TB cases of all forms nationwide (WHO,2007)[1].China is also a hotspot area of drug-resistant TB. About 130,000 new MDR-TB cases were reported in China in 2006, which account for 25% of the global burden[2]. As the use of fluoroquinolones increases, a high proportion of fluoroquinolone resistant tuberculosis was detected in a hospital in Guangzhou [3]. The problem with drug-resistant TB especially those with fluoroquinolone resistance has become new challenge for public health in China[4].According to surveillance data, the problem with drug-resistant TB is also severe in Eastern China [5-7]. It deserves investigating the epidemic of drug-resistant TB as well as relevant risk factors especially those with fluoroquinolone resistance. We conducted this study in three counties (or districts), Laiyang in Shandong Province, Yinzhou in Zhejiang Province and Jiading in Shanghai, in Eastern China, attempting to describe the drug resistant profile and transmission of TB in this area, by conventional epidemiological study design combined with the molecular methods.Study Objectives:to describe the drug-resistant profile to 1st line anti-TB drugs and major member of fluoroquinolone (Ofloxacin) in TB patients in Eastern China; to analyze the impact of population's demographic characteristics, clinical characteristics, previous treatment history and domestic migration on the epidemic of drug-resistant TB; to analyze the association between drug resistance phenotype and the mutations in hotspot genes; to discuss the potential mechanisms for drug-resistant TB epidemic in selected areas, so as to provide the basis for blocking the transmission of TB and controlling the drug-resistant TB epidemic.The methods used in this study and main results are described as follows:1. A cross-sectional study on the epidemic of 1st line anti-TB drug-resistant TB was conducted in the three study sites in Eastern China. Three hundred and six newly diagnosed pulmonary TB patients with Mycobacterium tuberculosis complex (MTB) successfully cultured were recruited during a period of 12 months from 2008 to 2009. The proportion method was used for drug susceptibility test for the 4 1st-line anti-TB drugs: Rifampicin, Isoniazid, Streptomycin and Ethambutol. The drug-resistant phenotypes were successfully acquired from 302 MTB strains. The overall drug resistant rate was 16.6% and the overall rate for MDR-TB was 3.3%. The overall primary drug resistance rate was 16.7%(48/288), and the rate for primary MDR-TB is 3.1%(9/288). The overall acquired resistance rate was 14.3%(2/14) in the 14 previously treated TB cases. No MDR-TB case was found in previously treated TB cases. The primary drug resistance rates for the 4 1st-line drugs were:11.8% for Streptomycin,8.0% for Isoniazid,4.2% for Rifampicin and 2.1% for Ethambutol, respectively. The contribution of primary treated TB was observed to the epidemic of drug-resistant TB in selected areas,The overall drug resistance rate of patients in Yinzhou (24.3%) was significantly higher than that of patients in Jiading (8.8%, OR=3.32,95% CI:1.45-7.58,P=0.004); The overall drug resistance rate in Laiyang was higher than Jiading, too (16.5% vs 8.8%, OR=2.30,95% CI:0.80-6.63, P>0.05). The drug resistance rate among domestic migrant patients (17.3%) was higher than the local patients (15.8%) with no statistical significance (χ2=0.13, P=0.72). No statistical difference was observed in the patients with different socio-demographic (sex, age) characteristics or treatment history.Direct sequencing was used to detect the mutation in the drug resistance-determining regions in rpoB gene to Rifampicin, katG and inhA gene to Isoniazid and rpsL and rrs gene to Streptomycin. Totally 90.9%(10/11) of Rifampicin-resistant strains had mutations at codon 531,526,511 of rpoB gene, respectively representing 63.6%,27.3% and 9.1% of Rifampicin-resistant mutations. Totally 69.6%(16/23) of Isoniazid resistance in MTB strains was due to the mutations at codon 315 of katG gene (52.2%), as well as codon 15 of inhA genes (21.7%), with 1 strain had mutation at both codon 315 of katG gene and codon 15 of inhA genes. A total of 44.1% (15/34) of Streptomycin-resistant strains had mutaions at codon 43 of rpsL gene, while rrs gene mutation occurred in 1 strain (2.9%). Our results show that mutations on codons of rpoB, katG and inhA, rpsL gene are the main mechanisms for MTB resistance to Rifampicin, Isoniazid and Streptomycin, respectively. The sensitivity and specificity for diagnosing MDR-TB by detecting mutations in the Rifampicin resistance-determining region of rpoB gene are 100% and 99.2%, relatively, which indicate that mutations in the Rifampicin resistance-determining region of rpoB gene are good indicators for MDR-TB, which could be used as rapid diagnosis tool for MDR-TB.2. The cross-sectional study on the prevalence and distribution of fluoroquinolone-resistant TB. The susceptibility to Ofloxacin was successfully detected on 297 MTB strains, among which 8 (2.7%) were resistant to the Ofloxacin. The relatively low proportion of Ofloxacin resistance in TB patients in the study region indicates that fluoroquinolones have their therapeutic value in treating MDR-TB in this region. The risk for patients older than 60 to acquire Ofloxacin resistance was significantly higher than patients younger than 60 (9.4% vs 1.2%; OR: 8.4,95%CI:1.9-36.2,P=0.006), indicating that TB patients older than 60 are high risk population for fluoroquinolone resistance.The fluoroquinolone resistance-determining region of gyrA gene was sequenced with 3 strains with fluoroquinolone resistance-related mutation found in the 8 Ofloxacin resistant strains, indicates that mutation on gyrA gene is important mechanism for fluoroquinolone resistant MTB.3. Genotypes of MTB strains and risk factors for clustering. 7 loci MIRU-VNTR genotyping assay was applied in the genotyping of the 293 MTB strains with DNA extracted, getting the genotypes of 287 strains. There were 15,10,21 strains forming 7,5,9 clusters in Yinzhou, Jiading and Laiyang respectively, with the clustering proportion of 14.9%, 10.1% and 24.1% in each site. Totally,46 strains were clustered in 21 clusters, with an average of 2-3 strains in each cluster. The relatively small proportion of clustering of TB patients in the study region indicates that the incidence of TB is mainly the result of reactivation of previously infected MTB, while recent transmission accounts for a low percentage of the incidence.Younger patients were more likely to be clustered (the proportion of clustering in patients younger than 40 vs patients older than 60:18.1% vs 10.0%, OR=3.15,95%CI: 1.05-9.47,P=0.04; the proportion of clustering in patients between 40 and 60 years old vs patients older than 60:15.0% vs 10.0%, OR=2.11,95%CI:0.64-7.00, P>0.05). Patients residing in the center of the county/district had higher risk of clustering, compared with patients living in the countryside (OR=2.29,95%CI:1.12-4.69, P=0.02).The reason for the relatively higher potential for the younger patients and patients living in the center of the county/district to be clustered might be that they are more socially active and have more chances of contacting TB patients. Nineteen point three percent of drug resistant strains were found in clusters, a little higher than the drug-sensitive strains (12.9%), with no statistical significance (drug-resistant strains/drug-sensitive strains:OR:1.30,95%CI:0.60-2.82, P=0.51), which indicates that the effect of recent transmission on generating drug-resistant TB is equivalent to drug-sensitive TB. The socio-demographic and medical characteristics were compared between the index cases and the secondary cases in the clusters. No statistically significant differences were found between different sex, age, place patients living, sputum smear result or previous treatment history. No genotype was simultaneously observed between the local patients and the domestic migrant patients, which may be the results of the independent living and working environments of the two subgroups. Of the 46 patients in the 21 clusters,20 (44.5%) patients in 13 (61.9%) clusters were found and responded. Epidemiological links were identified in 2 clusters (15.4%, 2/13), which indicates that causal contact might contribute a lot to the recent transmission of TB.
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