异质性耐药对结核分枝杆菌表型和基因型耐药检测结果的影响  

Heteroresistance in Mycobacteria tuberculosis is an important factor for the inconsistency between the results of phenotype and genotype drug susceptibility tests

作　　者：高旭[1] 李静[2] 柳清云[1] 沈鑫[2] 梅建[2] 高谦[1] 

机构地区：[1]复旦大学上海医学院教育部医学分子病毒学重点实验室,200032 [2]上海市疾病预防控制中心结核病防治科

出　　处：《中华结核和呼吸杂志》2014年第4期260-265,共6页Chinese Journal of Tuberculosis and Respiratory Diseases

基　　金：国家科技重大专项课题“艾滋病和病毒性肝炎等重大传染病防治”（2013ZX10004903）

摘　　要：目的 研究异质性耐药对MTB基因型耐药检测结果的影响.方法 选择上海市疾病预防控制中心在2009年收集的80株氧氟沙星临床耐药菌株,利用基因测序检测菌株在gyrA和gyrB耐药决定区的基因突变情况,再用分子克隆和荧光定量PCR探针熔解曲线法,分析经基因测序未检测到耐药突变的菌株中是否存在异质性耐药现象.结果 基因测序报告显示,80株氧氟沙星耐药菌株中有15株未发现已知gyrA或gyrB耐药决定区的基因突变.测序图谱及分子克隆和探针熔解曲线法分析结果表明,其中14株菌株含有已知gyrA耐药突变,即存在异质性耐药现象.纳入异质性耐药的菌株后,基因型与表型耐药检测的符合率从81.3％（65/80）提高到98.8％（79/80）.结论 异质性耐药是导致表型和基因型耐药检测结果不符的重要原因.为排除异质性耐药对耐药基因检测的影响,提高基因型与表型耐药检测结果的一致性,需要开发应用更敏感的基因型耐药检测技术,以提高耐药结核病快速检测水平.Objective To investigate the impact of heteroresistance on the results of genotype drug susceptibility test for Mycobacterium tuberculosis （M.tuberculosis）.Methods A total of 80 phenotype ofloxacin-resistant M.tuberculosis isolates obtained from Shanghai Municipal Centers for Disease Control and Prevention were included in the study.The mutations of gyrA and gyrB in each isolate were identified by gene sequencing.Molecular cloning and multiplex real-time PCR melting curve assay were applied to analyze the presence of heteroresistance in those isolates that had showed no mutations by gene sequencing.Results The sequencing results showed that 15 of these 80 isolates had no mutations in gyrA or gyrB.By analyzing the sequencing chromatogram,we found that 7 of the 15 isolates had gyrA mutations.By molecular cloning and melting curve assay,we identified another 7 isolates with low frequency mutations in gyrA that had been previously identified.In total,14 isolates with heteroresistance were detected in these 15 isolates.The consistency between the results of phenotype and genotype drug susceptibility tests was increased from 81.3％ （65/80） to 98.8％ （79/80）.Conclusions Heteroresistance is an important factor for the inconsistency between the results of phenotype and genotype drug susceptibility tests.For improving the rapid detection of drug resistant tuberculosis,novel genotypic drug susceptibility test methods with higher sensitivity should be developed and applied.

关 键 词：分枝杆菌 结核 遗传异质性 耐药快速检测 

分 类 号：R52[医药卫生—内科学]