华东农村地区利福平耐药结核病的分子流行病学特征研究

Population-based molecular epidemiologic study of rifampicin-resistant tuberculosis in rural area of eastern China

目的描述华东农村地区利福平耐药结核分枝杆菌（结核菌）的耐药相关分子特征和成簇性规律。方法以浙江省和江苏省两个农业县结核病防治所一年内所有登记的结核菌检测阳性肺结核病例为研究对象，收集痰标本、培养分离菌株，并进行问卷调查；对培养获得的结核菌株采用比例法进行药敏试验。对获得的65株利福平耐药结核菌株进行rpoB基因利福平耐药决定区和katG基因异烟肼耐药相关热点区测序，采用间隔区寡核苷酸分型（spoligotyping）方法识别北京株，采用IS6110限制性内切酶多态指纹技术（RFLP）对分离株进行基因分型和成簇性分析。结果92％（60／65）的利福平耐药菌株在利福平耐药决定区531位（57％）、526位（29％）或516位（11％）发生突变，82％（49／60）的rpoB基因突变菌株为耐多药结核菌（MDR—TB）；spoligotyping识别出54株（83％）北京家族菌株；IS6110-RFLP发现共有24株（37％）利福平耐药菌株成簇，形成11个簇，提示簇中病例的结核菌近期传播。所有成簇菌株都为MDR—TB。共有7个簇，簇内菌株具有不完全相同的利福平耐药相关的rpoB基因突变。分析可能与成簇性相关的因素，初治病例在菌株成簇患者中的比例高于在菌株非成簇患者巾所占的比例（初治／复治：OR=3．342，95％CI：1．081～10．32）。结论在其他耐药结核菌（如异烟肼耐药结核菌）中进一步产生对利福平的选择性耐药和生长，很有可能是利福平耐药结核病流行的基础，而既往较长的不规则治疗史也为利福平耐药的发生和传播提供条件。

Objective To describe the drug resistance-related molecular characterization and clustering feature of rifampiein-resistant （RIFt） M.tuberculosis （M.tb） in rural area of eastern China. Methods All patients diagnosed as RIFr M.tb in Deqing and Guanyun county during one year period from 2004 to 2005 were included in the study. By proportion method of drug susceptibility test, 65 isolates were identified resistant to rifampicin and regarded as the studied strains. Hotspots of rpoB gene and katG gene were detected by direct DNA sequencing. Beijing genotype M.tb strains were identified by spoligotyping. IS6110-RFLP （IS6110 restriction fragment length polymorphism） and clustering analysis were performed on all RIFr M.tb isolates available. Results The mutations in 81 bp rifampicin-resistance determination region（RRDR） of the rpoB gene were observed among 60 （92%） RIFt M.tb isolates, with mutation in locus 531 observed in the majority of RIFt isolates （37/ 65 ）. 49 （ 82% ） of the 60 isolates were multidrug resistant TB （MDR-TB）, which were referred to as resistant to both RIF and isoniazid （INH）. Through spoligotyping, 54（83%） isolates were identified as Beijing genotype strains. In clustering analysis of IS6110-RFLP, 24 isolates were grouped into 11 clusters, suggesting that the recent transmission of M.tb did exist among patients. Regarding the drug resistance profile in clusters, all the isolates in clusters were also MDR-TB. 7 clusters contained isolates carrying different mutations were related to RIF-resistance. Multivariate analysis showed the proportion of new cases in clustered patients is higher than that in the un-clustered patients （new/ previously treated： OR= 3.342; 95% CI： 1.081-10.32）. Conclusion The acquisition of rifampicin resistance in M.tb was more likely to be resulted from the selective growth of RIFr M.tb in the specific drug resistant M.tb such as isoniazid-reisistant M.tb. Previous elongated irregular treatment might favor the epidemic of RIFr M.tb.