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中国防痨杂志 ›› 2023, Vol. 45 ›› Issue (6): 583-588.doi: 10.19982/j.issn.1000-6621.20230086

• 论著 • 上一篇    下一篇

荧光PCR探针熔解曲线技术检测痰标本结核分枝杆菌利福平耐药性的研究

王嫩寒, 田丽丽, 陈双双, 赵琰枫, 樊瑞芳, 陈昊, 任怡宣, 张洁, 易俊莉, 杨新宇, 于兰, 丁北川, 李传友(), 代小伟()   

  1. 北京市疾病预防控制中心结核病实验室,北京 100035
  • 收稿日期:2023-03-23 出版日期:2023-06-10 发布日期:2023-06-06
  • 通信作者: 李传友, E-mail:lichuanyou@ccmu.edu.cn;代小伟,E-mail: happydaixw@126.com
  • 基金资助:
    北京市疾病预防控制中心自管课题(JKS2021-06)

Study on the detection of rifampicin resistance of Mycobacterium tuberculosis in sputum samples using fluorescence PCR probe melting curve technique

Wang Nenhan, Tian Lili, Chen Shuangshuang, Zhao Yanfeng, Fan Ruifang, Chen Hao, Ren Yixuan, Zhang Jie, Yi Junli, Yang Xinyu, Yu Lan, Ding Beichuan, Li Chuanyou(), Dai Xiaowei()   

  1. Department of Tuberculosis Laboratory, Beijing Center for Disease Prevention and Control, Beijing 100035, China
  • Received:2023-03-23 Online:2023-06-10 Published:2023-06-06
  • Contact: Li Chuanyou,E-mail:lichuanyou@ccmu.edu.cn;Dai Xiaowei,E-mail: happydaixw@126.com
  • Supported by:
    Beijing Center for Disease Prevention and Control Fund(JKS2021-06)

摘要:

目的: 评价荧光PCR探针熔解曲线(fluorescent polymerase chain reaction probe melting curve,MeltPro)技术检测肺结核患者痰标本中结核分枝杆菌耐药性的临床应用价值。方法: 收集2021年5月至2022年5月北京市疾病预防控制中心结核病门诊收治的GeneXpert MTB/RIF检测阳性的初治肺结核患者的痰标本,对每份标本同时进行涂片镜检、分枝杆菌分离培养、MeltPro技术利福平耐药性检测。分析痰标本荷菌量对MeltPro技术耐药性检测结果的影响;培养阳性菌株行利福平比例法药物敏感性试验(简称“药敏试验”),比较比例法药敏试验、GeneXpert MTB/RIF和MeltPro技术检测利福平耐药率的差异;并以培养阳性菌株利福平比例法药敏试验结果为参考标准,评价MeltPro技术耐药性检测的效能。结果: 研究共收集结核分枝杆菌阳性合格痰标本158份(例),经分枝杆菌分离培养获得阳性菌株130株。MeltPro技术检测痰标本结核分枝杆菌利福平耐药的成功率为79.1%(125/158),且随着痰涂片结果等级的升高,检测成功率由“阴性”的59.3%(32/54)上升至“+++”标本的100.0%(18/18);同时,随着GeneXpert MTB/RIF检测结核分枝杆菌等级的升高,检测成功率也由“极低”的64.5%(20/31)上升至“高”的92.9%(26/28),各等级间差异均有统计学意义(H=22.271,P=0.001;H=15.637,P=0.001)。MeltPro技术的利福平耐药检出率为17.6%(22/125),GeneXpert MTB/RIF方法的利福平耐药检出率为12.7%(20/158),比例法药敏试验的利福平耐药检出率为13.1%(17/130),三者比较差异无统计学意义(χ2=1.537,P=0.464)。以培养阳性菌株比例法药敏试验结果为参考标准,MeltPro技术和GeneXpert MTB/RIF方法检测利福平耐药的敏感度、特异度、阳性预测值、阴性预测值、Kappa值分别为100.0%(17/17)、94.6%(88/93)、77.3%(17/22)、100.0%(88/88)、0.845和100.0%(17/17)、97.3%(110/113)、85.0%(17/20)、100.0%(110/110)、0.906。结论: MeltPro技术检测肺结核患者痰标本结核分枝杆菌利福平耐药性的敏感度和特异度均较高,可快速准确筛查患者利福平耐药情况,但在基层实验室推广应用时需注意痰标本的质量。

关键词: 分枝杆菌, 结核, 聚合酶链反应, 抗药性, 敏感性与特异性

Abstract:

Objective: To evaluate the application value of the fluorescence PCR probe melting curve (MeltPro) technique in the detection of rifampicin-resistance of Mycobacterium tuberculosis (MTB) in sputum samples. Methods: Sputum samples of primary treated pulmonary tuberculosis (PTB) with positive GeneXpert MTB/RIF from Beijing Center for Disease Control and Prevention between May 2021 and May 2022 were collected. These sputum samples were tested using smear, mycobacterium isolation, and rifampicin-resistance by MeltPro technique. Drug sensitivity test (DST) of rifampicin was used for culturing positive strains, the differences in the detection rate of rifampicin resistance using DST, GeneXpert MTB/RIF, and MeltPro technique were compared. The proportional DST results of rifampicin were used as a standard to evaluate the drug-resistance detection efficiency of MeltPro technique. Results: A total of 158 qualified positive MTB sputum samples were collected, after mycobacterium culture, 130 strains were got. The success rate of the detection by MeltPro technique for rifampicin in sputum samples was 79.1% (125/158). As the grade of sputum smear results increased, the success rate of MeltPro technique increased from 59.3% (32/54, negative) to 100.0% (18/18, positive, grade +++); the success rate also increased with the increase of MTB grade detected by GeneXpert MTB/RIF, from 64.5% (20/31, extremely low) to 92.9% (26/28, high), the differences between each grade were statistically significant (H=22.271, P=0.001; H=15.637, P=0.001). The detection rates of rifampicin resistance using MeltPro technique, GeneXpert MTB/RIF method and proportional DST were 17.6% (22/125), 12.7% (20/158) and 13.1% (17/130), respectively. There was no statistically significant difference between the three methods (χ2=1.537, P=0.464). Based on the results of proportional DST, the sensitivity, specificity, positive predictive value, negative predictive value, Kappa of MeltPro technique in the detection of rifampicin were 100.0% (17/17), 94.6% (88/93), 77.3% (17/22), 100.0% (88/88), 0.845, respectively, and were 100.0% (17/17), 97.3% (110/113), 85.0% (17/20), 100.0% (110/110), 0.906, respectively of GeneXpert MTB/RIF in the detection of rifampicin. Conclusion: The MeltPro technique has high sensitivity and specificity in detecting rifampicin resistance of MTB in sputum samples of PTB, and can quickly and accurately screen the rifampicin resistance; attention should be paid to the quality of sputum samples when promoting the application in grassroots laboratories.

Key words: Mycobacterium tuberculosis, Polymerase chain reaction, Drug resistance, Sensitivity and specificity

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