MALDI-TOF MS直接靶板微滴生长法对耐碳青霉烯类肠杆菌的快速鉴别诊断价值

Value of rapid identification of carbapenem-resistant Enterobacteriaceae using MALDI-TOF MS-based direct-on-target microdroplet growth assay

目的:探讨基质辅助激光解析电离飞行时间质谱(matrix-assisted laser desorption ionization time-of-flight mass spectrometry,MALDI-TOF MS)直接靶板微滴生长法(direct-on-target microdroplet growth assay,DOT-MGA)对快速鉴别耐碳青霉烯类肠杆菌(carbapenem-resistant Enterobacteriaceae,CRE)的价值。方法:收集32株大肠埃希菌和28株肺炎克雷伯菌,将菌液与含有单种抗菌药物(亚胺培南、美罗培南或厄他培南)的肉汤在靶板上混合作为检测孔,与不加抗菌药物的肉汤混合作为生长对照孔,(35±1)℃分别孵育3 h、4 h、5 h和6 h后,根据VITEK MS系统能否成功鉴定靶板上的待测菌,判断其是否对碳青霉烯类药物耐药。采用微量肉汤稀释法检测菌株的最小抑菌浓度,与DOT-MGA结果进行比较。结果:肺炎克雷伯菌孵育至4 h时,生长对照有效率为92.9%,DOT-MGA检测3种碳青霉烯类药物的灵敏度、特异度、阳性预测值和阴性预测值均为100.0%。大肠埃希菌孵育至5 h时,生长对照有效率为100%,DOT-MGA检测亚胺培南、美罗培南和厄他培南的特异度及阳性预测值均为100.0%,灵敏度依次为85.0%、70.0%和95.0%,阴性预测值依次为80.0%、66.7%和92.3%。肺炎克雷伯菌DOT-MGA结果与微量肉汤稀释法完全一致(Kappa=1),大肠埃希菌则较好(厄他培南0.93,亚胺培南0.81)和一般(美罗培南0.64)。结论:MALDI-TOF MS鉴别CRE快速、准确、操作简便,特别是检测耐碳青霉烯类肺炎克雷伯菌的准确性高,可为临床早期的抗菌药物靶向精准治疗提供帮助。

Objective:To explore the value of matrix-assisted laser desorption ionization time-of-flight mass spectrometry(MALDI-TOF MS)-based direct-on-target microdroplet growth assay(DOT-MGA)for rapid identification of carbapenem-resistant Enterobacteriaceae(CRE).Methods:Totally,32 Escherichia coli and 28 Klebsiella pneumoniae isolates were collected in this study.The microorganisms were mixed with and without imipenem,meropenem,or ertapenem in nutrient broth on MALDI-TOF MS target directly as detecting spots and growth controls.These mixtures were incubated at(35±1)℃for 3 h,4 h,5 h and 6 h,respectively.Using VITEK MS,CRE were distinguished if the microorganisms could be identified successfully.At the same time,minimal inhibitory concentration(MIC)of these strains was determined by broth microdilution method,and the results were compared with those of DOT-MGA.Results:For Klebsiella pneumoniae,sensitivity,specificity,positive,and negative predictive values of three carbapenems were 100%after 4 h incubation with 92.9%validity of growth controls.For Escherichia coli,validity of growth controls was 100%after 5 h incubation.At this time-point,specificity and positive predictive value of imipenem,meropenem,and ertapenem were 100%,sensitivity was 85.0%,70.0%and 95.0%,negative predictive value was 80.0%,66.7%and 92.3%,respectively.The results of Klebsiella pneumoniae DOT-MGA were consistent with the microbroth dilution method(Kappa=1),while the results of Escherichia coli were better(ertapenem 0.93,imipenem 0.81)and normal(meropenem 0.64).Conclusions:Using MALDI-TOF MS-based DOT-MGA to identify CRE is rapid,accurate,and easy to operate,especially for carbapenem-resistant Klebsiella pneumoniae(CRKP),which can assist clinicians to select the most appropriate antibiotics for early treatment.