新型隐球菌的分子分型及联合药敏实验初步研究

Preliminary study on molecular typing of Cryptococcus neoformans and combined test for drug sensitivity

目的 比较2种不同新型隐球菌分型方法的差异及评价舍曲林与氟康唑体外联合药敏实验作用关系。方法 选择2018年—2022年不同标本来源的10株新型隐球菌作为实验菌株,采用MALDI-TOF MS质谱技术萃取法进行菌种鉴定及同源性分析;同时采用PCR扩增7种管家基因进行MLST分型,采用棋盘微量肉汤稀释法测定舍曲林与氟康唑对新型隐球菌的联合药敏实验。结果 同源分析显示MALDI-TOF VITEK MS蛋白组学与核酸的MLST分型有较好的一致性,皮肤来源的一株菌亲缘关系最远。MSLT分型检测共检出5种型别,分别为ST5、ST53、ST264、ST289、ST662,其中以ST5(6/10)为主。药敏结果显示10株新型隐球菌对舍曲林和氟康唑的部分联合药敏指数(FICI)为0.037 5~1,表现为协同相加关系。结论 MALDI-TOF MS同源分析可作为较好的菌株低成本快捷可靠的分型补充工具,杭州地区新型隐球菌MLST分型以ST5为主。舍曲林与氟康唑联合药敏实验为临床抗隐球菌脑膜炎联用方案提供一定的实验室依据。

Objective This paper aims to compare the differences of two different typing methods for Cryptococcus neoformans and evaluate the relationship between sertraline and fluconazole in vitro combined test for drug sensitivity.Methods Ten strains of Cryptococcus neoformans from different specimens from 2018 to 2022 were selected as experimental strains.Matrix-assisted laser desorption ionization time-of-flight mass spectrometry(MALDI-TOF MS)was used to identify the strains and analyze their homology.Seven housekeeping genes were amplified by PCR for MLST typing.The combined drug susceptibilities of sertraline and fluconazole to Cryptococcus neoformans were determined by chessboard microbroth dilution method.Results Homology analysis showed that the MALDI-TOF VITEK MS proteomics was in good agreement with the multilocus sequence typing(MLST)of the nucleic acid,and the strain from skin was the most distant related.A total of 5 types were detected by MSLT typing,which were ST5,ST53,ST264,ST289 and ST662;ST5(6/10)was the main type in the surveyed areas.Drug susceptibility results showed that the partial combined drug susceptibility index(FICI)of the 10 strains of Crypto⁃coccus neoformans to sertraline and fluconazole was within 0.0375-1,showing a synergistic additive relationship.Conclu⁃sion MALDI-TOF MS for homological analysis can be used as a low-cost,rapid and reliable supplementary tool for strain typing.MLST typing of Cryptococcus neoformans in Hangzhou area was mainly ST5.The drug sensitivity test of sertraline and fluconazole provides a laboratory basis for the combined regimen of clinical anti-cryptococcal meningitis.