全基因组测序预测结核分枝杆菌对氟喹诺酮类药物耐药性的应用价值研究  

Application evaluation of whole genome sequencing in predicting drug resistance to fluoroquinolones of Mycobacterium tuberculosis

作　　者：贺文从 谭云洪 刘彬彬 赵雁林[3] 刘向祎[1] He Wencong;Tan Yunhong;Liu Binbin;Zhao Yanlin;Liu Xiangyi(Department of Clinical Laboratory,Beijing Tongren Hospital,Capital Medical University,Beijing100730,China;Hunan Provincial Chest Hospital,Tuberculosis Control Institution of Hunan Province,Changsha410013,China;National Tuberculosis Reference Laboratory,Chinese Center for Disease Control and Prevention,Beijing102206,China)

机构地区：[1]首都医科大学附属北京同仁医院检验科,北京100730 [2]湖南省胸科医院检验科,长沙410013 [3]中国疾病预防控制中心结核病预防控制中心,北京102206

出　　处：《中华检验医学杂志》2024年第11期1299-1305,共7页Chinese Journal of Laboratory Medicine

基　　金：首都医科大学附属北京同仁医院科研基金种子基金(2023-YJJ-ZZL-026);国家重点研发项目(2022YFC2305200)。

摘　　要：目的评估全基因组测序(WGS)预测结核分枝杆菌对氟喹诺酮类药物耐药性的应用价值,并建立其耐药基因突变与耐药水平的量化关系。方法以中国疾病预防控制中心结核病预防控制中心国家结核病参比实验室保存的2013—2020年不同耐药模式的296株结核病耐药监测菌株为研究对象,采用SensititreTM MYCOTBI微孔板和WGS技术,检测结核分枝杆菌对氧氟沙星和莫西沙星的表型及基因型药物敏感性。以表型药敏结果作为金标准,计算WGS预测氟喹诺酮类药物敏感性的敏感度、特异度和一致性等指标。对耐药突变位点与耐药水平的分布进行汇总分析。采用配对χ^(2)检验检验比较两种检测方法检出率的差异,P<0.05为差异有统计学意义。结果296株不同耐药模式的结核分枝杆菌包括196株利福平耐药,50株其他耐药菌株和50株全敏感菌株。WGS共检测到81株结核分枝杆菌携带氟喹诺酮类药物耐药相关突变,其中以gyrA94、90及91密码子突变为主。WGS预测氧氟沙星的敏感度、特异度和一致性(Kappa值)分别为86.5%、98.1%和0.87。WGS预测莫西沙星的敏感度、特异度和一致性分别为80.0%、99.5%和0.83。表型药敏试验对氧氟沙星耐药检出率与WGS的耐药检出率差异无统计学意义(30.1%比27.4%,χ^(2)=3.06,P=0.08)。表型药敏试验对莫西沙星的耐药检出率(33.8%,100/296)高于WGS的耐药检出率(27.4%,81/296),差异有统计学意义(χ^(2)=15.43,P<0.01)。耐药突变位点与耐药水平的分布分析结果表明,不同耐药突变位点对应的最低抑菌浓度不同,其中多位点组合突变、gyrAD94G、gyrAD94Y、gyrAD94N主要对应高水平耐药,而gyrAD94A、gyrAA90V、gyrAS91P主要对应低水平耐药。结论WGS对氟喹诺酮类药物耐药性的预测有较好的敏感度、特异度和一致性,并可在一定程度上预测其耐药水平的高低。Objective To assess the utility of whole-genome sequencing(WGS)in predicting Mycobacterium tuberculosis resistance to fluoroquinolones(FQs)and to establish a quantitative relationship between resistant gene mutations and resistance levels.Methods A total of 296 drug-resistant tuberculosis surveillance strains with various resistance profiles,preserved by the National Tuberculosis Reference Laboratory of the Tuberculosis Prevention and Control Center at the Chinese Center for Disease Control and Prevention between 2013 and 2020,were included as study subjects.The Sensititre™MYCOTBI microplate method and WGS were used to assess the phenotypic and genotypic drug sensitivity of Mycobacterium tuberculosis to ofloxacin and moxifloxacin.Sensitivity,specificity,and concordance(Kappa value)of WGS in predicting fluoroquinolone sensitivity were calculated using phenotypic drug susceptibility testing(DST)results as the gold standard.A summary analysis was conducted on the distribution of drug resistance mutation sites and resistance levels.The pairedχ^(2)test was used to compare the detection rates between the two methods,with P<0.05 indicating statistical significance.Results Among the 296 Mycobacterium tuberculosis strains with different resistance profiles,196 were rifampicin-resistant,50 were resistant to other drugs,and 50 were fully sensitive.WGS identified 81 strains carrying FQs resistance-related mutations,primarily at gyrA codons 94,90,and 91.Sensitivity,specificity,and consistency(Kappa value)of WGS in predicting ofloxacin resistance were 86.5%,98.1%,and 0.87,respectively.For moxifloxacin resistance prediction,these values were 80.0%,99.5%,and 0.83,respectively.There was no statistically significant difference between the phenotypic DST and WGS detection rates for ofloxacin resistance(30.1%vs 27.4%,χ^(2)=3.06,P=0.08).However,the phenotypic DST detection rate for moxifloxacin resistance(33.8%,100/296)was significantly higher than that of WGS(27.4%,81/296)(χ^(2)=15.43,P<0.01).Analysis of the distribution of res

关 键 词：分枝杆菌 结核 氟喹诺酮类药 耐药性 基因 全基因组测序 

分 类 号：R446.5[医药卫生—诊断学] R52[医药卫生—临床医学]