MPT64在MTB利福平和异烟肼耐药性检测方法中的应用

Application of MPT64 in detecting rifampicin and isoniazid resistance in Mycobacterium tuberculosis

目的建立一种以MPT64为指标的结核分枝杆菌(MTB)耐药性检测的快速方法,探讨MPT64在MTB利福平(RFP)和异烟肼(INH)耐药性检测中的应用。方法应用胶体金免疫层析法(GICA)分别检测MTB敏感株和耐多药株在含(观察组)与不含(对照组)RFP、INH的Middlebrook 7H9液体培养基上培养3、7、10 d的培养液中的MPT64,通过凝胶成像仪白光源拍照进行条带灰度分析(Image Jab Software 3.0),比较敏感株和耐多药株间的灰度比值的差异,依据受试者工作特征(ROC)曲线下面积(AUC)分析确定RFP、INH耐药的灰度比值界值;以比例法检测结果为金标准,评价新建方法的准确度、灵敏度、特异度、阳性预测值和阴性预测值。结果对照组中,随着培养时间的延长,12株MTB敏感株和11株耐多药株的灰度比值增大;观察组中,敏感株在不同的培养时间的灰度比值没有显著变化,而耐多药株则明显增大。3、7、10 d敏感菌与耐多药菌在药物培养基中MPT64检测灰度比值差异均有统计学意义(P均<0.05)。依据观察组灰度比值分别绘制以MPT64指示的RFP和INH耐药性检测的ROC曲线,两者的3 d AUC分别为0.84和0.77,灰度比值界值均为0.05;7、10 d AUC均为1.00,7 d的灰度比值界值分别为0.23与0.20;10 d的灰度比值界值分别为0.49和0.48。GICA检测MPT64用于34株MTB的RFP、INH耐药性鉴定的准确度分别为97%和94%,灵敏度分别为93%和86%,特异度均为100%,阳性预测值均为100%,阴性预测值分别为95%和91%。结论 GICA检测MPT64于MTB培养7 d时能准确检测抗结核药物RFP、INH耐药性,MPT64可作为MTB药敏试验的有效检测指标。

Objective To establish a rapid method for detecting drug resistance of Mycobacterium tuberculosis(MTB) by using an indicator of MPT64 and to evaluate the application of MPT64 in detecting rifampicin(RFP) and isoniazid(INH) resistance of MTB. Methods The expression levels of MPT64 in the Middlebrook 7H9 liquid medium containing(observation group) and non-containing(control group) RFP and INH, separately cultured by MTB sensitive strains and multidrug-resistant strains for 3, 7, 10 days were detected by using gold immunotomographic assay(GICA). The grayscale of the experimental strip was analyzed by photographs with white light source of gel imaging instrument(Image Lab Software 3.0). The grayscale ratio was compared between the sensitive and multidrug-resistant strains. The cutoff value of the grayscale ratio for RFP and INH resistance was determined based on the analysis of the area under the receiver operating characteristic(ROC) curve(AUC). The accuracy, sensitivity, specificity, positive and negative prediction values of this new method were evaluated with the proportional test results as the gold standard. Results With the prolongation of culture time, the grayscale ratios of 12 MTB sensitive strains and 11 multidrug-resistant strains were increased in the control group. The grayscale ratios of sensitive strains at different culture times did not change significantly in the observation group, whereas those of the multidrugresistant strains were significantly increased. The MPT64 grayscale ratios of sensitive and multidrug-resistant strains in the drug medium significantly differed at 3, 7 and 10 days(all P < 0.05). According to the grayscale ratio in the observation group, the ROC curves of the RFP and INH resistance detection were plotted with the indicator of MPT64. On day 3, the AUCs of RFP and INH were 0.84 and 0.77, and the grayscale ratios were both 0.05. On day 7 and day 10, the AUCs were both 1.00. On day 7, the grayscale ratios were 0.23 and 0.20. On day 10, the grayscale ratios were 0.49 and 0.48. The accuracy of GICA in detecting RFP and INH drug-resistance of 34 MTB was 97% and 94%, 93% and 86% for the sensitivity, 100% and 100% for the specificity, 100% and 100% for the positive prediction value, and 95% and 91% for the negative prediction value, respectively. Conclusions MPT64 detection by GICA can accurately and rapidly identify the RFP and INH resistance on day 7 of MTB culture. MPT64 can be used as an effective detection indicator for MTB drug sensitivity.