应用MALDI-TOF-MS联合分离胶法快速鉴定阳性血培养标本的初步研究

Preliminary study on identification of positive blood culture specimens by MALDI-TOF-MS combined with separation gel method

目的运用基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)联合分离胶法快速鉴定血培养阳性标本,探讨快速鉴定法的符合率。方法运用分离胶法提取阳性血培养瓶中富集的菌落,应用MALDITOF-MS对经前处理的血培养阳性标本进行直接鉴定,鉴定结果与传统的VITEK-2Compact全自动细菌分析仪鉴定结果进行比较,若有不符,以16SrRNA序列分析结果作为金标准。结果在实验的200株阳性血培养标本中,革兰阳性菌的鉴定符合率达79.5%;革兰阴性菌鉴定符合率达85.7%;真菌的鉴定符合率达20.0%。由此可见,革兰阴性菌的鉴定符合率高于革兰阳性菌,二者鉴定符合率均远高于真菌。两种及两种以上细菌混合感染的阳性标本直接鉴定还存在一定困难,其直接鉴定方式还需进一步探讨。应用两种鉴定方法得出不同结果的12株阳性标本,16SrRNA序列分析结果均与VITEK-2Compact全自动细菌分析仪鉴定结果一致。结论MALDI-TOF-MS联合分离胶促凝管直接检测血培养阳性标本,对血流感染中主要病原菌的鉴定符合率较高,且迅速简便,此方法可将细菌更快地鉴定到种属,使临床能够进行早期治疗,有效控制菌血症的病死率和院内感染。

Objective To evaluate the direct identification of microorganisms from blood culture by a separation gel tube-based matrix-assisted laser desorption/ionization-time of flight mass spectrometry(MALDI-TOF-MS)and its identification rate.Methods The bacteria were enriched and purified directly from blood culture bottle by separation gel tube,and then the bacteria were identified by MALDI-TOF-MS,and the cultured pure colonies were identified by VITEK-2 Compact automated microbial analysis system(VITEK-2 Compact).The identification of bacterial isolates by MALDI-TOF-MS was compared with that of traditional biochemical testing,and discrepancies were resolved by gene sequencing.Results A total of 200 strains of positive blood cultures in the test specimen,gram-positive bacteria identification coincidence rate of 79.5%;gram-negative bacteria identification coincidence rate was 85.7%;fungal identification coincidence rate of 20.0%.Thus,gram-negative bacteria identification coincidence rate is higher than gram-positive bacteria,the identification coincidence rate is much higher than fungi.More than two and the positive samples direct identification of bacteria mixed infection still exist certain difficulties,its direct identification methods had yet to be explored.The results of 16SrRNA sequence analysis of 12 positive samples with different results obtained by the two methods are consistent with those obtained by VITEK-2 Compact automatic bacteria analyzer.Conclusion This separation gel tube-based MALDI-TOF-MS for the direct identification of blood culture pathogens is established.The identification rate of common pathogens in bloodstream infection by MALDI-TOF-MS is higher compared with that by traditional culture identification processes.Application of this method can be faster identification of bacteria species,make the clinical ability of early treatment,effective control of the mortality of bacteremia and nosocomial infection.