PCR荧光探针技术对结核分枝杆菌复合群及利福平耐药性检测性能的评价

Evaluation of performance of PCR fluorescent probe method for detecting Mycobacterium tuberculosis complex and rifampicin resistance

目的:评价PCR荧光探针技术检测痰液样本中结核分枝杆菌复合群(Mycobacterium tuberculosis complex,MTBC)和利福平耐药性效果,为进一步开展“结核分枝杆菌复合群及利福平耐药核酸检测试剂盒”多中心临床试验提供可行性研究数据。方法:采用前瞻性研究方法,参照入组标准收集2021年12月至2022年8月上海市3家结核病定点医院肺科门诊就诊的205例初诊疑似肺结核患者的3份痰液样本(即时痰、夜间痰、晨痰),分别进行痰涂片、BACTEC MGIT 960(MGIT 960)、GeneXpert MTB/RIF(Xpert)和PCR荧光探针法检测,评价PCR荧光探针法对痰液样本中MTBC及利福平耐药性的检测效能。另对随机选取同期上海市疾病预防控制中心结核病实验室菌株库中保存的96株MTBC临床菌株(包括利福平表型耐药菌株47株和利福平表型敏感菌株49株)分别进行PCR荧光探针法和Xpert检测利福平耐药性,分析两种检测方法对利福平耐药相关基因突变的检出情况。结果:205例疑似肺结核患者中,PCR荧光探针法对MTBC的检出率为22.44%(46/205),与MGIT 960[21.95%(45/205)]、Xpert[20.98%(43/205)]和痰涂片[16.10%(33/205)]的差异均无统计学意义(χ^(2)=0.014,P=0.904;χ^(2)=0.129,P=0.719;χ^(2)=2.650,P=0.104)。以临床诊断为参照标准,PCR荧光探针法、Xpert、MGIT 960和痰涂片检测MTBC的敏感度(95%CI)分别为48.10%(36.93%~59.56%)、48.10%(36.93%~59.56%)、50.63%(39.23%~61.97%)和40.51%(29.79%~52.15%),特异度(95%CI)分别为93.65%(87.47%~97.12%)、96.03%(90.52%~98.53%)、96.03%(90.52%~98.53%)和99.21%(95.01%~99.96%),一致率分别为76.10%(156/205)、77.56%(159/205)、78.54%(161/205)和76.59%(157/205),Kappa值分别为0.453、0.482、0.507和0.446。PCR荧光探针法检测痰液样本中MTBC的涂阴和极低级别阳性样本的检出率[10.73%(19/177)]与Xpert检测结果[8.47%(15/177)]的差异无统计学意义(χ^(2)=0.793,P=0.373)。PCR荧光探针法在检出MTBC的46份样本中同时检出6份样本发生利福平耐药基因突变,突变率为13.04%(6/46);而Xpert在检出MTBC的43份样本中仅同时检出1份样本发生利福平耐药基因突变,突变率为2.33%(1/43)。在96株临床菌株中,两种分子检测方法均检测到56株菌株发生利福平耐药基因突变,除2株探针突变类型不一致外,其他探针突变类型均一致,且多发生在probe E(529~533)和FAM通道(531/533突变)。结论:以临床诊断为参照标准,PCR荧光探针法检测MTBC的效能与MGIT 960、Xpert和痰涂片基本相当,且与Xpert检测MTBC临床菌株的利福平耐药性效果一致,认为PCR荧光探针法不仅是一种等同于Xpert检测功能的新技术,还具有操作简单、价格低于Xpert、真正实现一体化和全封闭等优势。建议在临床推广过程中进一步优化PCR荧光探针法以提高MTBC检出率,并开展多中心临床试验验证研究。

Objective:To evaluate the efficacy of PCR fluorescent probe method for detecting Mycobacterium tuberculosis complex(MTBC)and rifampicin resistance in sputum samples,and to provide feasibility data for further development of a multi-center clinical trial of MTBC and rifampicin resistance nucleic acid detection kit.Methods:Using prospective research method,three sputum specimens(immediate sputum,night sputum and morning sputum)from each of 205 patients with suspected pulmonary tuberculosis who met the enrollment criteria and were initially diagnosed at the pulmonary outpatient clinics of three designated tuberculosis hospitals in Shanghai from December 2021 to August 2022 were collected,and sputum smears,BACTEC MGIT 960(MGIT 960),GeneXpert MTB/RIF(Xpert)and PCR fluorescent probe method were performed separately to evaluate the efficacy of PCR fluorescence probe method for detecting MTBC and rifampicin resistance in sputum samples.In addition,96 clinical strains identified as MTBC(47 rifampicin-resistant strains and 49 rifampicin-sensitive strains)stored in the tuberculosis laboratory of Shanghai Center for Disease Control and Prevention were randomly selected to be tested for rifampicin resistance by PCR fluorescent probe method and Xpert,then rifampicin resistance-related gene mutations identified by the two methods were analyzed.Results:Among 205 patients with suspected pulmonary tuberculosis,the MTBC detection rate by PCR fluorescent probe assay was 22.44%(46/205),which was not statistically different from MGIT 960(21.95%(45/205);χ^(2)=0.014,P=0.904),Xpert(20.98%(43/205);χ^(2)=0.129,P=0.719),and sputum smear(16.10%(33/205);χ^(2)=2.650,P=0.104).Based on clinical diagnosis,the sensitivities(95%CI)of PCR fluorescent probe method,Xpert,MGIT 960 and sputum smear for the detection of MTBC were 48.10%(36.93%-59.56%),48.10%(36.93%-59.56%),50.63%(39.23%-61.97%)and 40.51%(29.79%-52.15%),the specificities(95%CI)were 93.65%(87.47%-97.12%),96.03%(90.52%-98.53%),96.03%(90.52%-98.53%)and 99.21%(95.01%-99.96%),the coincidence rates were 76.10%(156/205),77.56%(159/205),78.54%(161/205)and 76.59%(157/205),and the Kappa values were 0.453,0.482,0.507 and 0.446,respectively.There was no significant difference between MTBC detection rate of PCR fluorescent probe method(10.73%(19/177))and Xpert(8.47%(15/177);χ^(2)=0.793,P=0.373)on testing smear-negative and extremely low-grade positive samples.Six rifampicin resistance gene mutations out of 46 MTBC were detected by PCR fluorescence probe method,and the mutation rate was 13.04%(6/46),while only 1 gene mutation out of 43 MTBC was detected by Xpert,and the mutation rate was 2.33%(1/43).Among 96 clinical strains,56 strains were found to have rifampicin resistance gene mutation by both methods.Except for mutation types of two probes,the mutation types of all other probes were consistent,and most of them occurred in probe E(529-533)and FAM channel(531/533 mutation).Conclusion:Based on clinical diagnosis,the efficacy of PCR fluorescent probe method in detecting MTBC is basically equivalent to MGIT 960,Xpert and sputum smear,and its efficacy is consistent with Xpert in detecting rifampicin resistance of MTBC clinical strains.It is considered that PCR fluorescent probe method is not only a new technology equivalent to Xpert detection function,but also has the advantages of operation simplicity,lower price,real one-piece and fully enclosed testing platform.It is suggested that PCR fluorescent probe method should be further optimized in the process of clinical popularization to improve detection rate of MTBC,and multi-center clinical trials should be carried out to verify the study.