吡嗪酰胺耐药性结核分枝杆菌的噬菌体检测技术研究

目的 建立噬菌体生物扩增法（PhaB）快速测定吡嗪酰胺耐药性，并探讨其在结核分枝杆菌吡嗪酰胺耐药性测定中的应用价值。方法 应用建立的PhaB测定108株结核分枝杆菌临床分离株的吡嗪酰胺耐药性，并与绝对浓度法的药敏结果进行比较，对不符合的菌株进行最低抑菌浓度（MIC）测定和序列测定分析。结果 PhaB检测吡嗪酰胺耐药性的最佳测定条件为pH值5．5、药物浓度200μg／ml、37℃作用48h。用绝对浓度法检测108株结核分枝杆菌临床分离株，其中吡嗪酰胺敏感33株、耐药75株；用PhaB检测该108株结核分枝杆菌临床分离株，若以噬菌斑减少95％为判断标准。则吡嗪酰胺敏感32株，耐药76株。2种方法检测均为敏感的为28株，均为耐药的为71株，符合率为91．7％；2种方法检测结果不符的为9株，其中5株的PhaB结果与MIC结果相符，4株的结果不符，测序结果表明9株中有7株的PhaB结果与测序结果相符。如以绝对浓度法药敏结果为判断标准，PhaB检测吡嗪酰胺耐药性的敏感性为94．7％，特异性为84．8％，阳性预测值为93．4％，阴性预测值为87．5％，准确性为91．7％。结论 PhaB测定吡嗪酰胺耐药性简便、快速，3d即可获得药敏结果，可作为吡嗪酰胺耐药性的快速筛选方法。

A study of the bacteriophage-based assay for the detection of pyrazinamide resistance in Mycobacterium tuberculosis

OBJECTIVE: To set up and evaluate the method of phage amplified biologically assay (PhaB) in rapid detection of pyrazinamide (PZA) resistance. METHODS: The PhaB assay was developed and applied in detecting PZA resistance in 108 clinical isolates of Mycobacterium tuberculosis and the results were compared with those of the absolute concentration method. The minimum inhibitory concentration (MIC) was detected for all discrepancy isolates. RESULTS: The results showed that the optimal detecting condition was pH 5.5, PZA 200 microg/ml and 48 h. Of the 108 strains of Mycobacterium tuberculosis, 28 strains were PZA-susceptible and 80 strains were PZA-resistant detected by PhaB; while 32 strains were PZA-susceptible and 76 strains were PZA-resistant by absolute concentration method. Twenty-eight of the 108 strains were PZA-susceptible and 71 were PZA-resistant by the two methods. The concordant isolates of determination of PZA resistance were 99 by the two methods and the concordance rates was 91.7%. There were 9 strains in discordant isolates, of which 7 were the same with MIC method and gene chip in drug susceptibility. If the results of absolute concentration method was the gold standard, the sensitivity, specificity, positive and negative predictive values, as well as accuracy of PhaB assay was 94.7%, 84.8%, 93.4%, 87.5% and 91.7% respectively. CONCLUSION: The PhaB assay can be used as a rapid screening method for detection of drug susceptibility of PZA in clinical isolates of Mycobacterium tuberculosis.