实验室检测结核分枝杆菌对吡嗪酰胺耐药性的方法学评价

Methodology evaluation of determining the drug resistance of Mycobacterium tuberculosis to pyrazinamide

目的评估MGIT 960药物敏感性检测法、吡嗪酰胺酶(PZase)活性检测法和pncA基因序列分析法在检测结核分枝杆菌对吡嗪酰胺(PZA)耐药性中的应用价值。方法应用上述3种方法对上海市疾病预防控制中心2014年收集的207株结核分枝杆菌临床分离菌株进行PZA耐药性检测,比较3种方法的耐药性检测结果。结果在207株结核分枝杆菌对PZA的耐药性检测结果中有38株菌3种方法检测结果不一致,对其进行重复性试验,结果显示13株MGIT 960药物敏感性检测法结果为耐药的菌株重复性试验有10株转为敏感,且这10株菌株pncA基因序列分析结果均为敏感。以3种方法结果完全一致或2种方法结果一致的PZA耐药性检测结果为参考结果,MGIT 960药物敏感性检测法、PZase活性检测法和pncA基因序列分析法的敏感性分别为94.8%、100.0%和94.8%,特异性分别为93.3%、83.9%和98.0%。与参考结果的一致性Kappa值分别为0.85、0.74和0.93。结论 MGIT 960药物敏感性检测法和pncA基因序列分析法在检测结核分枝杆菌对PZA的耐药性上具有较好的一致性。MGIT960药物敏感性检测法存在假阳性,建议对MGIT 960药物敏感性检测法检测PZA耐药的样本进行pncA基因序列分析,若二者结果不符则进行MGIT 960药物敏感性重复性试验,以提高PZA耐药性检测的准确性。

Objective To compare the performance of MGIT 960 system,pyrazinamidase(PZase)activity assay and pncA gene sequencing for the determination of Mycobacterium tuberculosis drug resistance to pyrazinamide(PZA).Methods The drug resistance to PZA of 207 Mycobacterium tuberculosis isolates from Shanghai Municipal Center for Disease Control and Prevention in 2014 were determined by the 3 methods.Results There were 38 isolates with inconsistent results by the 3 methods.After repeated experiments,10 of 13 isolates reported as PZA being resistant by MGIT 960 system proved susceptible.When the results were consistent in 2 methods or more than 2 methods as reference results,the sensitivities of MGIT 960 system,PZase activity assay and pncA gene sequencing were 94.8%,100.0%and 94.8%,respectively.The specificities of MGIT 960 system,PZase activity assay and pncA gene sequencing were 93.3%,83.9%and 98.0%,respectively.Compared with the reference results,the Kappa values of MGIT 960 system,PZase activity assay and pncA gene sequencing were 0.85,0.74 and 0.93,respectively.Conclusions The performance of MGIT 960 system and pncA gene sequencing shows consistency with the reference results.There exists false positive results for PZA drug resistance determination by MGIT 960 system.Mycobacterium tuberculosis isolates report as PZA being resistant by MGIT 960 system,which should be determined by pncA gene sequencing.If the 2 methods have inconsistent results,repeated experiments should be performed for improving the accuracy of PZA drug resistance determination.