荧光PCR探针熔解曲线法检测结核分枝杆菌对利福平和异烟肼耐药情况的研究

doi:10.3969/j.issn.2096-8493.2020.03.008

摘要/Abstract

摘要：

目的评价荧光聚合酶链反应(polymerase chain reaction, PCR)探针熔解曲线法(简称“熔解曲线法”)检测结核分枝杆菌对利福平和异烟肼耐药的情况。方法对832例涂阳肺结核患者的痰标本进行BACTEC MGIT 960(简称“MGIT 960”)液体培养,结果显示污染35例,培养阴性52例,培养阳性745例,对培养阳性的745份培养物进行菌种鉴定(基因芯片法),其中混合感染7例,非结核分枝杆菌60例,结核分枝杆菌复合群678例;对鉴定为结核分枝杆菌复合群的678例患者标本采用MGIT 960液体培养药物敏感性试验(简称“MGIT 960药敏试验”)和熔解曲线法进行利福平和异烟肼耐药基因检测。熔解曲线法检测对利福平是否耐药的结果无效(无法判断是否耐药)31例,检测对异烟肼是否耐药的结果无效34例,剩余639例利福平和异烟肼同时检测结果有效。以MGIT 960液体药敏试验为参考标准,分析熔解曲线法检测耐药基因的效果。结果以MGIT 960液体药敏试验结果为参考标准,639例患者的样本进行利福平和异烟肼熔解曲线法耐药性检测,对利福平耐药性检测的符合率为96.2%(615/639),敏感度和特异度分别为93.3%(126/135)和97.0%(489/504),阳性预测值和阴性预测值分别为89.4%(126/141)和98.2%(489/498),Kappa值为 0.89;对异烟肼耐药性检测的符合率为93.6%(598/639),敏感度和特异度分别为83.1%(157/189)和98.0%(441/450),阳性预测值和阴性预测值分别为94.6%(157/166)和93.2%(441/473), Kappa值为0.84。结论熔解曲线法对利福平和异烟肼耐药性检测的敏感度高、特异度好,对于早期诊断耐药肺结核有较好的临床意义。

关键词: 结核,抗多种药物性, 聚合酶链反应, 微生物敏感性试验, DNA探针, 微阵列分析, 对比研究, 数据说明,统计

Abstract:

Objective To evaluate the efficacy of fluorescence PCR probe melting curve technique (hereinafter referred to as melting curve technique) in detecting for the resistance of Mycobacterium tuberculosis to rifampicin and isoniazid. Methods Sputum specimens of 832 smear-positive pulmonary tuberculosis patients were cultured in liquid, and the results showed that specimens of 35 cases were contaminated, 52 were culture negative, and 745 were positive. Seven hundred and forty-five positive cultures were then tested with gene chip which found 7 cases of mixed infection, 60 cases of non-mycobacterium tuberculosis, and 678 cases of Mycobacterium tuberculosis complex. The BACTEC MGIT 960 (“MGIT 960”) liquid drug sensitivity test and melting curve method were then performed on specimens of those 678 cases to detect drug resistance of rifampicin and isoniazid. The results of the melting curve method in rifampicin detection were invalid (impossible to tell whether it’s resistant) in 31 cases and isoniazid in 34 cases,for the remaining 639 cases valid results were reported for both rifampicin and isoniazid. Taking MGIT 960 liquid drug sensitivity test method as the golden standard to analyze the performance of melting curve method. Results The rate of concordance, sensitivity, specificity, positive predictive value and negative predictive value to rifampicin detection were 96.2% (615/639), 93.3% (126/135), 97.0% (489/504), 89.4% (126/141) and 98.2% (489/498) respectively. The Kappa value was 0.89. The rate of concordance, sensitivity, specificity, positive predictive value and negative predictive value to isoniazid detection were 93.6% (598/639), 83.1% (157/189), 98.0% (441/450), 94.6% (157/166) and 93.2% (441/473) respectively. The Kappa value was 0.84. Conclusion The melting curve method has high sensitivity and good specificity for detecting rifampicin and isoniazid resistance, thus is of great value for early diagnosis of drug-resistant tuberculosis.

Key words: Tuberculosis,multidrug-resistant, Polymerase chain reaction, Microbial susceptibility tests, DNA probes, Microarray analysis, Comparative study, Data interpretation,statistical

苏碧仪, 周德旺, 马品云, 关平, 谭耀驹. 荧光PCR探针熔解曲线法检测结核分枝杆菌对利福平和异烟肼耐药情况的研究[J]. 结核与肺部疾病杂志, 2020, 1(3): 245-248. doi: 10.3969/j.issn.2096-8493.2020.03.008

SU Bi-yi, ZHOU De-wang, MA Pin-yun, GUAN Ping, TAN Yao-ju. Diagnostic value of melting curve method in detecting resistance of Mycobacterium tuberculosis to rifampicin and isoniazid[J]. Journal of Tuberculosis and Lung Disease, 2020, 1(3): 245-248. doi: 10.3969/j.issn.2096-8493.2020.03.008

图/表 2

参考文献 13

[1]	World Health Organization. Global tuberculosis report 2020. Geneva:World Health Organization, 2020.
[2]	Dean AS, Cox H, Zignol M. Epidemiology of Drug-Resistant Tuberculosis. Adv Exp Med Biol, 2017,1019:209-220. doi: 10.1007/978-3-319-64371-7_11. doi: 10.1007/978-3-319-64371-7_11 URL pmid: 29116637
[3]	Opota O, Mazza-Stalder J, Greub G, et al. The rapid molecular test Xpert MTB/RIF ultra: towards improved tuberculosis diagnosis and rifampicin resistance detection. Clin Microbiol Infect, 2019,25(11):1370-1376. doi: 10.1016/j.cmi.2019.03.021. URL pmid: 30928564
[4]	Machado D, Couto I, Viveiros M. Advances in the molecular diagnosis of tuberculosis: From probes to genomes. Infect Genet Evol, 2019,72:93-112. doi: 10.1016/j.meegid.2018.11.021. doi: 10.1016/j.meegid.2018.11.021 URL pmid: 30508687
[5]	中华医学会结核病学分会临床检验专业委员会. 结核病病原学分子诊断专家共识. 中华结核和呼吸杂志, 2018,41(9):688-695. doi: 10.3760/cma.j.issn.1001-0939.2018.09.008.
[6]	李强, 包训迪, 赵雁林, 等. MTBDRplus V2用于诊断涂阴疑似肺结核患者的效果评价. 中国防痨杂志, 2016,38(7):544-548. doi: 10.3969/j.issn.1000-6621.
[7]	赵冰, 时金艳, 逢宇, 等. 基因芯片结核分枝杆菌耐多药检测在地市级实验室的应用性评估. 中国防痨杂志, 2013,35(9):718-722.
[8]	陈曦, 马玙, 金奇, 等. 耐异烟肼结核分枝杆菌临床分离株耐药相关基因突变研究. 中华结核和呼吸杂志, 2005,28(4):250-253. doi: 10.3760/j:issn:1001-0939.
[9]	Zhang Y, Yew WW. Mechanisms of drug resistance in Mycobacterium tuberculosis: update 2015. Int J Tuberc Lung Dis, 2015,19(11):1276-1289. doi: 10.5588/ijtld.15.0389. doi: 10.5588/ijtld.15.0389 URL pmid: 26467578
[10]	Pang Y, Liu G, Wang Y, et al. Combining COLD-PCR and high-resolution melt analysis for rapid detection of low-level, rifampin-resistant mutations in Mycobacterium tuberculosis. J Microbiol Methods, 2013,93(1):32-36. doi: 10.1016/j.mimet.2013.01.008. URL pmid: 23396215
[11]	胡春梅. 荧光定量PCR探针熔解曲线法在结核分枝杆菌耐药基因检测中的应用. 中国防痨杂志, 2016,38(1):38-41. doi: 10.3969/j.issn.1000-6621. doi: 10.3969/j.issn.1000-6621.2016.01.009 URL
[12]	田际云, 武洁, 李静, 等. 熔解曲线法在结核分枝杆菌药敏试验质量评估中的应用. 中华结核和呼吸杂志, 2015,38(2):105-109. doi: 10.3760/cma.j.issn.1001-0939.
[13]	李桂莲, 万康林. 结核分枝杆菌异烟肼耐药性—— 一个不容忽视的问题. 中国人兽共患病学报, 2019,35(6):475-479. doi: 10.3969/j.issn.1002-2694.2019.00.100.

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