四种实验室检测技术对利福平和异烟肼耐药性检测的临床价值

doi:10.3969/j.issn.1000-6621.2019.02.009

摘要/Abstract

摘要：

目的探讨微孔板法、实时荧光PCR熔解曲线技术(简称“熔解曲线法”)、多色巢式实时荧光定量PCR技术(简称“Xpert 法”)和罗氏药物敏感性试验(L-J药敏试验,简称“比例法”)用于快速筛查耐多药结核病(MDR-TB)的临床价值。方法从医院信息系统(hospital information system,HIS)连续收集2014年7月至2018年3月重庆市公共卫生医疗救治中心收治的确诊为结核病,且具有微孔板法、比例法、熔解曲线法、Xpert 法检测MTB对利福平、异烟肼耐药性诊断结果的2792例患者资料;其中微孔板法、比例法采用阳性分离菌株检测,熔解曲线法和Xpert 法采用患者标本直接检测。纳入同时具有微孔板法和比例法耐药性检测结果的1488例患者作为研究对象,其中341例行微孔板法+比例法+Xpert法检测利福平耐药性,87例行微孔板法+比例法+熔解曲线法检测利福平耐药性,66例行微孔板法+比例法+熔解曲线法检测异烟肼耐药性。以比例法为标准,采用SPSS 13.0软件分别计算微孔板法、熔解曲线法、Xpert法检测利福平和(或)异烟肼耐药性的敏感度、特异度、符合率、Kappa值等。结果以比例法为标准,微孔板法、Xpert法、熔解曲线法检测利福平耐药性的敏感度、特异度、阳性预测值、阴性预测值、符合率、Kappa值分别为97.2%(731/752)、96.9%(713/736)、96.9%(731/754)、97.1%(713/734)、97.0%(1444/1488)、0.94,97.2%(140/144)、94.9%(187/197)、93.3%(140/150)、97.9%(187/191)、95.9%(327/341)、0.92,97.1%(33/34)、84.9%(45/53)、80.5%(33/41)、97.8%(45/46)、89.7%(78/87)、0.79;微孔板法和熔解曲线法检测异烟肼耐药性的敏感度、特异度、阳性预测值、阴性预测值、符合率、Kappa值分别为94.8%(751/792)、95.7%(667/697)、96.3%(751/780)、94.2%(667/708)、97.9%(1418/1448)、0.91, 97.3%(36/37)、86.2%(25/29)、90.0%(36/40)、96.2%(25/26)、92.4%(61/66)、0.84。结论微孔板法、熔解曲线法、Xpert 法检测利福平和(或)异烟肼耐药性均具有较高的敏感度和特异度,适合快速筛查耐多药结核病;微孔板法还能获得各药物最低药物浓度,为临床用药剂量的选择提供参考依据。

关键词: 分枝杆菌,结核, 微生物敏感性试验, 实验室技术和方法, 结核,抗多种药物性, 敏感性与特异性, 对比研究

Abstract:

Objective To explore the clinical value of micropore-plate method, real-time fluorescent PCR melting curve technology (melting curve method), multicolor nested real-time fluorescence PCR detection technology (Xpert method) and Roche drug sensitivity test (L-J drug sensitivity test, referred to as proportional method) in rapid screening multidrug-resistant tuberculosis (MDR-TB). Methods From the hospital information system of Chongqing Public Health Medical Center during July 2014 to March 2018, patients who were diagnosed as TB and had the rifampicin and/or isoniazid resistance data by the Micropore-plate, melting curve, Xpert and/or proportional method were screened. The micropore-plate and proportional method were conducted by using positive isolates, and the melting curve and Xpert method were conducted using patient specimens. A total of 1488 patients with both micropore-plate and proportional drug resistance test results were included in the study. Among them, 341 patients underwent micropore-plate + proportional + Xpert methods to detect rifampicin resistance, and 87 patients underwent micropore-plate + proportional + melting curve methods to detect rifampicin resistance, and 66 cases were tested by micropore-plate + proportional + melting curve methods for isoniazid resistance. Taking the proportional method as the standard, SPSS 13.0 software was used to calculate the sensitivity, specificity, coincidence rate and Kappa value of rifampicin and/or isoniazid resistance by micropore-plate, melting curve and Xpert method. Results Taking the proportional method as the standard, the sensitivity, specificity, positive predictive value, negative predictive value, coincidence rate and Kappa value of rifampicin resistance test were 97.2% (731/752), 96.9% (713/736), 96.9% (731/754), 97.1% (713/734), 97.0% (1444/1488) and 0.94 by micropore-plate method, 97.2% (140/144), 94.9% (187/197), 93.3% (140/150), 97.9% (187/191), 95.9% (327/341) and 0.92 by Xpert method, and 97.1% (33/34), 84.9% (45/53), 80.5% (33/41), 97.8% (45/46), 89.7% (78/87) and 0.79 by melting curve method, respectively. The sensitivity, specificity, positive predictive value, negative predictive value, coincidence rate and Kappa value of isoniazid resistance test were 94.8% (751/792), 95.7% (667/697), 96.3% (751/780), 94.2% (667/708), 97.9% (1418/1448) and 0.91 by micropore-plate method and 97.3% (36/37), 86.2% (25/29), 90.0% (36/40), 96.2% (25/26), 92.4% (61/66) and 0.84 by melting curve method. Conclusion Micropore-plate, melting curve and Xpert method have high sensitivity and specificity in detecting rifampicin and/or isoniazid resistance, which are suitable for rapid screening of MDR-TB. The Micropore-plate method also shows the minimum drug concentration of each drug, which provides a reference for the selection of clinical dosage.

Key words: Mycobacterium tuberculosis, Microbial sensitivity tests, Laboratory techniques and procedures, Tuberculosis,multidrug-resistant, Sensitivity and specificity, Comparative study

王鹏森,周刚,蒙昌平,蒋明英,孙强中,叶嗣宽,周刊,黄正谷,廖传玉,张汇征,罗明,杨国强,陈玉,李晓旭,李同心. 四种实验室检测技术对利福平和异烟肼耐药性检测的临床价值[J]. 中国防痨杂志, 2019, 41(2): 169-175. doi: 10.3969/j.issn.1000-6621.2019.02.009

Peng-sen WANG,Gang ZHOU,Chang-ping MENG,Ming-ying JIANG,Qiang-zhong SUN,Si-kuan YE,Kan ZHOU,Zheng-gu HUANG,Chuan-yu LIAO,Hui-zheng ZHANG,Ming LUO,Guo-qiang YANG,Yu CHEN,Xiao-xu LI,Tong-xin LI. Clinical diagnostic value of four laboratory techniques in detecting drug resistance to rifampicin and isoniazid[J]. Chinese Journal of Antituberculosis, 2019, 41(2): 169-175. doi: 10.3969/j.issn.1000-6621.2019.02.009

图/表 2

参考文献 27

[1]	World Health Organization . Global tuberculosis report 2018. Geneva:World Health Organization, 2018.
[2]	van Cutsem G, Isaakidis P, Farley J , et al. Infection control for drug-resistant tuberculosis: early diagnosis and treatment is the key. Clin Infect Dis, 2016,62 Suppl 3: S238-243. doi: 10.1093/cid/ciw012 URL
[3]	Abdel-Rahman SM, Erfan D, Abdel-Latif W , et al. Evaluation of Sensititre ^® MYCOTB panel for the susceptibility testing of Mycobacterium tuberculosis to first and second lines anti-tuberculosis drugs . Clinical Medicine and Diagnostics, 2016,6(1):13-19. doi: 10.4236/ojcd.2016.62003 URL
[4]	Heysell SK, Pholwat S, Mpagama SG , et al. Sensititre MycoTB plate compared to Bactec MGIT 960 for first- and second-line antituberculosis drug susceptibility testing in Tanzania: a call to operationalize MICs. Antimicrob Agents Chemother, 2015,59(11):7104-7108. doi: 10.1128/AAC.01117-15 URL
[5]	Yu X, Ma YF, Jiang GL , et al. Sensititre ^® MYCOTB MIC plate for drug susceptibility testing of Mycobacterium tuberculosis complex isolates . Int J Tuberc Dis, 2016,20(3):329-334. doi: 10.5588/ijtld.15.0573 URL
[6]	中华人民共和国国家卫生和计划生育委员会.WS 196—2017 结核病分类. 2017 -11-09.
[7]	中华人民共和国国家卫生和计划生育委员会.WS 288—2017 肺结核诊断. 2017 -11-09.
[8]	中国防痨协会基础专业委员会. 结核病诊断实验室检验规程.北京: 中国教育文化出版社, 2006.
[9]	赵雁林, 王黎霞, 成诗明 , 等. 分枝杆菌分离培养标准化操作程序及质量保证手册.北京: 人民卫生出版社, 2013.
[10]	郭苏珊, 林健雄, 郭金镇 , 等. BACTEC MGIT 960用于结核分枝杆菌直接药敏试验的研究. 中国病原生物学杂志, 2014,9(10):946-948,952.
[11]	史祥, 尹洪云, 沙巍 , 等 . 三种检测技术对肺结核辅助诊断价值的研究. 中国防痨杂志, 2018,40(2):173-176.
[12]	Gallo JF, Pinhata JMW, Saraceni CP , et al. Evaluation of the BACTEC MGIT 960 system and the resazurin microtiter assay for susceptibility testing of Mycobacterium tuberculosis to second-line drugs. J Microbiol Methods, 2017,139:168-171. doi: 10.1016/j.mimet.2017.06.007 URL
[13]	Lee J, Armstrong DT, Ssengooba W , et al. Sensititre MYCOTB MIC plate for testing Mycobacterium tuberculosis susceptibility to first- and second-line drugs. Antimicrob Agents Chemother, 2014,58(1):11-18. doi: 10.1128/AAC.01209-13 URL
[14]	Chazel M, Marchandin H, Keck N , et al. Evaluation of the SLOMYCO Sensititre ^® panel for testing the antimicrobial susceptibility of Mycobacterium marinum isolates . Ann Clin Microbiol Antimicrob, 2016,15:30. doi: 10.1186/s12941-016-0145-1 URL
[15]	郑扬, 夏辉, 赵雁林 . TREK Sensititre ^® MYCOTB检测结核分枝杆菌对一、二线抗结核药物的敏感性研究 . 中国防痨杂志, 2015,37(6):597-602.
[16]	胡春梅, 郭晶, 严虹 , 等. 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.1000-6621.2016.01.009
[17]	柳清云, 罗涛, 李静 , 等. 双通道实时荧光PCR熔解曲线法检测结核分枝杆菌药物耐药相关基因突变. 中华检验医学杂志, 2013,36(1):63-67.
[18]	王峰, 崔运勇, 胡思玉 , 等. 实时聚合酶链反应熔解曲线法快速检测耐多药结核分枝杆菌. 中华结核和呼吸杂志, 2011,34(12):888-893. doi: 10.3760/cma.j.issn.1001-0939.2011.12.003
[19]	Raoot A, Dev G . Evaluate “rifampicin resistance” as surrogate marker for rapid detection of MDR-TB using Real-Time PCR directly on FNAC samples of tuberculous lymphadenitis. BJMMR, 2015,9(5):1-8.
[20]	欧维正, 厉宁, 蒙俊 , 等. Hain和基因芯片技术在结核分枝杆菌异烟肼耐药性检测中的应用评价. 中国病原生物学杂志, 2016,11(12):1062-1065.
[21]	Shao Y, Peng H, Chen C , et al. Evaluation of GeneXpert MTB/RIF for detection of pulmonary tuberculosis at peripheral tuberculosis clinics. Microb Pathog, 2017,105:260-263. doi: 10.1016/j.micpath.2017.02.040 URL
[22]	Pang Y, Dong H, Tan Y , et al. Rapid diagnosis of MDR and XDR tuberculosis with the MeltPro TB assay in China. Sci Rep, 2016,6:25330. doi: 10.1038/srep25330 URL
[23]	Cheng S, Cui Z, Li Y , et al. Diagnostic accuracy of a molecular drug susceptibility testing method for the antituberculosis drug ethambutol: a systematic review and meta-analysis. J Clin Microbiol, 2014,52(8):2913-2924. doi: 10.1128/JCM.00560-14 URL
[24]	Ismail NA, Ismail MF, Noor SS , et al. Genotypic detection of rpoB and katG gene mutations associated with rifampicin and isoniazid resistance in Mycobacterium tuberculosis isolates: A local scenario (Kelantan). Malays J Med Sci, 2016,23(1):22-26.
[25]	Kawulur HSI, Ngili Y . Study in silico and characterization of mutations in rpoB and katG genes in patients of tuberculosis in Jayapura, Papua province, Indonesia. Der Pharmacia Lettre, 2016,8(14):85-91.
[26]	Isakova J, Sovkhozova N, Vinnikov D , et al. Mutations of rpoB, katG, inhA and ahp genes in rifampicin and isoniazid-resistant Mycobacterium tuberculosis in Kyrgyz Republic. BMC Microbiol, 2018,18(1):22. doi: 10.1186/s12866-018-1168-x URL
[27]	Martin IW, Dionne K, Deml SM , et al. Automated broth-based systems versus the MYCOTB plate for antimicrobial susceptibility testing of the Mycobacterium tuberculosis complex: challenges in interpretation. Diagn Microbiol Infect Dis, 2018,91(1):38-41. doi: 10.1016/j.diagmicrobio.2018.01.002 URL