广西贵港地区2009-2012年新生隐球菌和格特隐球菌临床分离株基因分型与毒力特性研究

Study on genotype and virulence of Cryptococcus neoformans and Cryptococcus gattii clinical isolates in Guigang, Guangxi Zhuang Autonomous Region

目的研究广西贵港地区新生隐球菌和格特隐球菌临床分离株，在种、基因型和交配型的构成及毒力特性。方法收集2009--2012年广西贵港地区临床分离的新生隐球菌株20株，PCR扩增转录间隔区（ITS）序列进行分子生物学验证，通过特异性引物进行PCR扩增，鉴定血清型、交配型。利用M13-PCR指纹图谱法和URA5-限制性片段长度多态性分析法，鉴定和验证基因型。表型研究包括37℃生长试验、黑色素生成试验和尿素酶试验。结果20株有19株为新生隐球菌格鲁比变种，血清型为A型，cL交配型，基因型为VNI型。1株为格特隐球菌，属a交配型，基因型VGI。毒力特性研究结果显示，所有临床分离株37℃正常生长，黑色素试验、尿素酶试验阳性。结论广西贵港地区隐球菌致病菌株中绝大多数（95％）为新生隐球菌格鲁比变种、血清型为A型，a交配型，基因型为VNI型，并发现格特隐球菌菌株。

Objective To understand the species, genotypes and mating types of Cryptococcus neoformans and Cryptococcus gattii isolated from clinical samples in Guigang, Guangxi Zhuang Autonomous Region. Methods A total of 20 Cryptococcas strains were isolated from clinical samples in Guigang from 2009 to 2012. The biological identification was conducted by polymerase chain reaction （PCR） to amplify internal transcribed spacer （ITS） sequences. The serotypes and mating types of C. neoformans and C. gattii were identified by PCR with serotype-specific and mating type-specific primers. The genotype was characterized by PCR fingerprinting and URA5 gene restriction fragment length polymorphism （URA5-RFLP）. Phenotype study included growth test at 37℃ , melanin production test and urease test. Results Among the 20 strains, 19 （95%） were identified as C. neoformans varieties （var.） grubii （serotype A, mating type ct, genotype VN I ）, and only 1 was identified as C. gattii （mating type α, genotype VG I ）. The results of virulence test showed that all the strains grew well at 37 ℃ and positive in both urease test and melanin production test. Conclusion C. neoformans var. grubii （serotype A, genotype VN I and mating type α ） was the predominant pathogen causing cryptococcosis in Guigang, and C. gattii strain was also detected.