血培养瓶增菌法快速鉴定和快速药物敏感性试验可行性分析

Primary exploration of rapid identification and drug sensitivity test of cerebrospinal fluid culture by blood culture enrichment method

目的探讨用血培养瓶增菌法培养脑脊液样本,将报阳样本直接离心富集细菌后,采用激光解吸电离飞行时间质谱(MALDI-TOF MS)进行快速鉴定和快速药物敏感性试验的可行性。方法收集2018年12月—2019年7月华山医院脑脊液样本1 446例,随机抽取345例采用血培养瓶增菌法培养,1 101例采用常规浓缩集菌培养法培养,比较2组样本阳性率。分别采用快速法(BD凝胶分离血清管富集细菌)和常规法(转种过夜孵育获取纯细菌)对血培养瓶增菌法阳性样本进行质谱鉴定和体外药物敏感性试验,分析菌种鉴定符合率和体外药物敏感性试验结果一致率。结果与常规浓缩集菌培养比较,血培养瓶增菌后样本阳性率明显增高(χ^(2)=60.889,P=0.000)。48例血培养瓶增菌法阳性样本中,快速法与常规法菌种鉴定符合率为89.80%,2种鉴定方法差异无统计学意义(χ^(2)=0.116,P=0.990)。快速法与常规法体外药物敏感性试验结果比较,革兰阴性菌除微量肉汤稀释法的替加环素外,其他方法和抗菌药物的一致率均为100%;革兰阳性菌除纸片扩散法的红霉素、微量肉汤稀释法的红霉素和庆大霉素、仪器法的克林霉素和庆大霉素一致率略差外,其他抗菌药物一致率均为100%。隐球菌快速药物敏感性分类一致率及标准一致率为100%。以纯细菌微量肉汤稀释法为标准,快速微量肉汤稀释法和快速仪器法的标准一致率分别为98.56%和97.83%。结论用血培养瓶增菌法进行脑脊液培养,可在不延长报阳时间的情况下提高脑脊液样本培养阳性率和难培养菌的检出率,采用BD凝胶离心直接取菌的快速鉴定和药物敏感性试验方法快速、准确,可大大缩短临床报告时间,及时为临床用药提供实验室依据。

Objectives To explore the feasibility of rapid identification and drug sensitivity test by matrixassisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF MS)after enrichment of bacteria in cerebrospinal fluid(CSF)culture with blood culture bottle.Methods A total of 1446 CSF samples from Huashan Hospital affiliated to Fudan University from December 2018 to July 2019 were collected.Of them,345 samples were randomly cultured by blood culture bottle enrichment method,and the other 1101 samples were cultured by conventional enrichment method.The positive rate of 2 groups were compared.The positive samples of blood culture bottle enrichment method were identified by MALDI-TOF MS and carried out drug sensitivity experiment using the rapid method(BD gel was used to separate serum tubes and enrich bacteria)and conventional method(transfer overnight to obtain pure bacteria).The coincidence rate of identification and in vitro drug sensitivity test were analyzed.Results Compared with the routine culture,the positive rate of CSF blood culture bottle enrichment method increased significantly(χ^(2)=60.889,P=0.000).In 48 positive samples of CSF blood culture bottle enrichment method,the coincidence rate of rapid method and routine method was 89.80%,and there was no statistical significance between the 2 methods(χ^(2)=0.116,P=0.990).In the comparison of rapid and routine in vitro drug sensitivity experiment,for Gram-negative bacteria,except tigacycline with microdilution broth method had a low classification consistency,the consistent rates of other methods and antibacterial drugs were 100%;the consistent rates of erythromycin with Kirby-Bauer method,erythromycin and gentamycin with microdilution broth method,and clindamycin and gentamycin with instrument method were slightly poor,but the consistent rates of other antibacterial drugs were 100%.The consistent rates of classification and standard of cryptococcus rapid drug sensitivity were 100%.Based on pure bacteriummicrodilution broth method,the standard consistent rates of rapid microdilution broth method and rapid instrument method were 98.56%and 97.83%,respectively.Conclusions The positive rate of CSF blood culture bottle enrichment method and the detection rate of refractory bacteria can be improved without prolonging the time of positive report.The rapid identification and drug sensitivity experiment of direct centrifugation by using BD gels can greatly shorten the clinical report time and provide a rapid and accurate basis for clinical medication.