一种联合MALDI-TOF MS直接鉴定阳性血培养瓶方法改进及与Sepsityper Kit试剂盒法、SELTERS法和血清分离胶法的比较

目的评价直接使用基质辅助激光解析电离飞行时间质谱(MALDI-TOF MS)联合Sepsityper Kit试剂盒法(简称试剂盒法)、SELTERS法和血清分离胶法(简称分离胶法)鉴定阳性血培养瓶血中细菌的符合率,并对SELTERS方法进行改进,以缩短样本处理时间。方法对656例临床血培养阳性标本,应用试剂盒法、SELTERS方法或分离胶法处理后,直接使用质谱仪快速鉴定菌株,同时进行传统培养,比较分析二者之间的差异。结果656例血培养阳性标本共分离出626株单种菌感染和30株多种菌感染标本。MALDI-TOF MS联合试剂盒法、SELTERS法或分离胶法可在1 h内快速鉴定血培养阳性标本。在单种细菌感染中,MALDI-TOF MS联合试剂盒法直接鉴定革兰阳性菌的种、属符合率分别是66.8%(141/211)、21.3%(45/211),革兰阴性菌的种、属符合率分别是97.1%(367/378)、0.8%(3/378),真菌的种、属符合率分别是32.4%(12/37)、0.0%(0/37);MALDI-TOF MS联合SELTERS法直接鉴定革兰阳性菌的种、属符合率分别是66.8%(141/211)、21.3%(45/211),革兰阴性菌的种、属符合率分别是96.3%(364/378)、2.4%(9/378),真菌的种、属符合率分别是32.4%(12/37)、2.7%(1/37);MALDI-TOF MS联合分离胶法直接鉴定革兰阳性菌的种、属符合率分别是51.2%(108/211)、20.9%(44/211),革兰阴性菌的种、属符合率分别是93.4%(353/378)、1.6%(6/378),真菌的种、属符合率分别是13.5%(5/37)、2.7%(1/37);MALDI-TOF MS联合改良SELTERS法直接鉴定革兰阳性菌的种、属符合率分别是59.1%(13/22)、18.2%(4/22),革兰阴性菌的种、属符合率分别是88.5%(23/26)、3.8%(1/26),真菌的种、属符合率分别是0.0%(0/2)、50.0%(1/2)。而对于多种菌感染的血培养瓶,3种方法鉴定率均较低。结论MALDI-TOF MS联合试剂盒法、SELTERS法或分离胶直接鉴定阳性血标本中的病原菌,其结果可在1 h内获得,并与传统培养结果相比具有较高的符合率。但是这些方法检测更快速、操作更简便,同时改良SELTERS法样本处理时间缩短,成本降低,且符合率与前3种方法没有区别。这4种方法均能满足临床快速诊断和及时有效抗菌治疗的需求,临床可根据自身情况选择。

An improved method for direct identification of positive blood culture flasks in combination with MALDI|TOF MS and comparison with Sepsityper Kit method, SELTERS method and serum separation gel method

Objective To evaluate the coincident rates of direct detection of blood culture bottle with matrix-assisted laser desorption ionization time-of-flight mass spectrometry(MALDI-TOF MS) combined with Sepsityper Kit method(hereinafter referred to as kit method), SELTERS method and serum separation gel method(hereinafter referred to as separation gel method), and improve the SELTERS method to shorten sample processing time. Methods 656 clinical specimens with positive blood culture results were treated with the kit method, SELTERS method or separation gel method. Then the strains were directly identified using mass spectrometry, and conventional culturing was carried out at the same time. The difference between the two methods was compared. Results A total of 626 strains of single-bacterial infections and 30 strains of multiple-bacterial infections were isolated. MALDI-TOF MS combined kit method, SELTERS method or separation gel method were able to quickly identify blood culture-positive specimens within 1 hour. For single bacterial infections, the species and genus coincidence rates of Gram-positive bacteria identified with the MALDI-TOF MS combined kit method were 66.8%(141/211) and 21.3%(45/211), those of Gram-negative bacteria were 97.1%(367/378) and 0.8%(3/378), while those of fungi were 32.4%(12/37) and 0%(0/37). With MALDI-TOF MS combined with SELTERS, the species and genus coincidence rates of Gram-positive bacteria were 66.8%(141/211) and 21.3%(45/211), those of Gram-negative bacteria were 96.3%(364/378) and 2.4%(9/378), and those of fungi were 32.4%(12/37) and 2.7%(1/37). With MALDI-TOF MS combined with separation gel method, the species and genus coincidence rates of Gram-positive bacteria were 51.2%(108/211) and 20.9%(44/211), those of Gram-negative bacteria were 93.4%(353/378) and 1.6%(6/378), and those of fungi were 13.5%(5/37) and 2.7%(1/37). Using MALDI-TOF MS combined with modified SELTERS method, the species and genus coincidence rates of Gram-positive bacteria were 59.1%(13/22) and 18.2%(4/22), those of Gram-negative bacteria were 88.5%(23/26) and 3.8%(1/26), while those of fungi were 0%(0/2) and 50%(1/2). For the blood culture bottles infected with multiple bacteria, the identification rates of the three methods were low. Conclusion MALDI-TOF MS combined with kit method, SELTERS method or separation gel can directly identify the pathogens in positive blood samples within 1 hour, with higher coincidence rate than conventional culturing, and are faster and easier to operate. The improved SELTERS method can shorten the time for sample processing and lower the cost, with a similar coincidence rate as the above-mentioned three methods. All the four methods can meet the needs of rapid clinical diagnosis and prompt and effective antibacterial therapy.