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

Clinical diagnostic value of four laboratory techniques in detecting drug resistance to rifampicin and isoniazid

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SPSS13.0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。

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.