微量MIC检测判断结核分枝杆菌药敏的方法学研究

目的 建立用微量MIC值判断结核分枝杆菌药物敏感性的方法 ,并进行初步临床应用评价.方法 采用液体培养基MIC测定技术,在96孔U型板中进行检测,以60株已知药敏结果 的本院菌株库保存菌株作为试验菌株, 根据Bactec MGIT结果采用ROC曲线分析建立微量MIC药敏判断界值,最后用80株连续时间段内收集的结核分枝杆菌临床分离株对微量MIC药敏判断界值进行验证和修正.结果 微量MIC药敏法的最佳接种菌量为10~(-3)mg/ml数量级,7～10 d即可报告结果 ,链霉素、异烟肼、利福平、乙胺丁醇、氧氟沙星、阿米卡星和卷曲霉素的最佳判断界值分别为2、0.25、1、2、1、4和4 μg/ml,其敏感度分别为93.5%、97.7%、93.5%、84.4%、95.8%、91.7%和88.9%, 特异度分别为95.6%、94.6%、100.0%、93.8%、92.9%、95.6%和91.5%.其准确性分别达95.0%、96.3%、97.5%、90.0%、93.8%、95.0%和91.3%. 结论 微量MIC药敏检测方法 具有快速、准确、低成本和操作简单的优点,具有良好的推广应用前景,适合在我国各级结核病专业收治机构使用.

Methodological research of MIC microtiter susceptibility testing for Mycobacterium tuberculosis

Objective To establish of MIC in microtiter as drug susceptibility testing for Mycobacterium tuberculosis(microwell-MIC-test)and evaluate clinical application of this method in Mycobacterium tuberculosis.Methods 60 Mycobacterium tuberculosis clinical isolates which were stored in Shanghai Pulmonary Hospital and whose drug susceptibility status of Bactec MGIT was known were as control.MICs of these 60 Mycobacterium tuberculosis strains were detected in liquid culture in 96-well plate.According to the results of Bactec MGIT.the best cut-off value were chosen by ROC analysis.The drug susceptibility of 80 Mycobacterium tuberculosis clinical isolate strains which were collected continuously from tuberculosis patients in our hospital were detected by Microwell-MIC-test.Bactec MGIT method was used at the sanle time to evaluate and amend the cut-off valne.Results The best concentration of Mycobacterium tuberculosis in microwell is 10~(-3) mg/ml.The best results were read after 7-10 d The best cut-off value of drug susceptibility testing of isoniazid,streptomycin,rifampicin,ethambutol,ofloxacin,amikacin,capreomycin were 2μg/ml,0.25μg/ml,1 μg/ml,2μg/ml,1 μg/ml,4μg/ml and 4μg/ml,respectively,and the seusitivities were 93.5%,97.7%,93.5%,84.4%,95.8%,91.7% and 88.9% respectively,the specificities were 95.6%, 94.6%,100.0%,93.8%,92.9%,95.6% and 91.5% respectively.The accuracies were 95.0%,96.3%,97.5%,90.0%,93.8%,95.0% and 91.3%respectively.Conlusion The drug susceptibility testing method of microwell-MIC-test is accurate,rapid,low-cost and simple and worthy in application in tuberculosis hospital of resource limited countries like China.