中国东南部地区格特隐球菌的生态学、表型及系统发育学研究

The Cryptococcus gattii outbreak began in 1999 on Vancouver Island is now endemic, causing numerous human and veterinary infections, and has spread to mainland British Columbia, Washington and Oregon in the United States. Most fungi investigators felt compelled to draw attention to the emergence of C.gattii in these regions because of it traditionally been found in tropical and subtropical climates. Currently, several molecular typing methods including PCR fingerprint patterns based on M13 microsatellite DNA, restriction fragment length polymorphism (RFLP) and multilocus sequence typing (MLST) analysis divided these strains into three primarily clonal genotypes: VGIIa(major genotype), VGIIb and VGIIc. Recently, Japan reported a case of cerebral cryptococcoma caused by C.gattii VGIIa (a strain identical to the Vancouver Island outbreak major genotype strain R265) in a patient from Japan who had no recent travel history to known diseaseendemic areas. Likewise, Feng et al. also reported a human infection caused by C. gattii VGIIb strain, the minor genotype strain R272, in Guangdong, and the patient had never been abroad. These research revealed associations between geographic origin and particular genotypes, implying an epidemiologic significance of certain genotypes. Recognizing the urgent need for a standardized globally acceptable typing method, however, the International Society of Human and Animal Mycoses (ISHAM) established the consensus multi-locus typing scheme, including the housekeeping genes CAP59, GPD1, LAC1, PLB1, SOD1, URA5 and the IGS1 region, to facilitate a better understanding of the phylogenetic aspects of the Cryptococcus species complex, comprising Cryptococcus neoformans and Cryptococcus gattii. However, China's study is not yet refer to this technology system suggested by ISHAM. In addition, our previous work showed that large areas of Eucalyptus spp.， Ceratonia siliqua and other trees related to C.gattii were widely planted in Southeast China, and the case of C.gattii infections were continuously diagnosed in these regions in our survey. With the ongoing migration between Chinese and foreigners increased significantly, the possibility of the C.gattii outbreak occur in the future can not be ruled out in China. Despite the ecological, phenotypic and phylogenetic survey is crucial to the prevention of the similar outbreak in our country, however, the Chinese report on C.gattii about the ecological, phenotypic and phylogenetic analysis is rare. It is time to initiate more systematic, prospective efforts, using the new MLST system suggested by ISHAM or the latest whole genome sequence typing (WGST) technology, to uncover the facial mask of the C.gattii isolates in Southeast China. These studies will extend our understanding of whether the Southeast China regions is likely to occur the similar outbreak,and aim to provide solid theoretical basis and technical reserves for our country's public health and safety system.

格特隐球菌是临床上重要的病原真菌，近年来因在北美引发蔓延至今的感染爆发性流行而广受医学专家关注。最近，日本及我国也相继发现了感染与此次流行事件遗传背景高度相似菌株的患者。我们前期工作显示，华东、华南存在大片桉树林等格特隐球菌"生态点"密集的自然环境，近年来又持续发现格特隐球菌感染患者，加之国际间交往日益频繁，存在发生类似爆发性流行事件的可能，而既往我国在此领域的研究甚少，环境分离菌株的报道几为空白。因此，我们拟引入目前"国际人与动物真菌学学会"推荐的"MLST"、WGST 等技术体系，系统开展我国东南地区格特隐球菌临床和环境株的分离、收集、保藏等工作，研究其表型、基因型特征及之间的相关性，探索该地区环境株的自然"生态点"时空分布规律及其与临床株之间的起源与进化关系，为临床隐球菌病的治疗提供参考依据，亦为预防未来我国发生类似的格特隐球菌爆发流行及公共卫生安全体系提供亟需的理论基础与技术储备。

格特隐球菌是临床上重要的病原真菌，近年来因在北美引发蔓延至今的感染爆发性流行而广受医学真菌学、感染病学及流行病专家关注。最近，日本也发现了感染与此次流行事件遗传背景高度相似菌株的患者。我们前期工作显示，华东、华南存在大片桉树林等格特隐球菌“生态点”密集的自然环境，近年来又持续发现格特隐球菌感染患者，加之国际间交往日益频繁，存在发生类似爆发性流行事件的可能，而既往我国在此领域的研究甚少，环境分离菌株的报道几为空白。. 因此，我们利用最新的“国际人与动物真菌学学会”推荐的“多位点序列”及全基因组序列分析等技术体系，自2013年1月至2015年6月，在我国东南部地区系统开展了格特隐球菌感染的多中心流行病学监测和环境菌株的分离、收集、保藏等工作，2014 年6月在湖北施恩、2015 年10月在四川成都分别发现 1例格特隐球菌感染患者，显示我国的格特隐球菌感染亦有从热带、亚地区扩散到温带地区的趋势。先后采集环境标本1230份，分别于2013 年 6月30日、 2014年9月26日、 2014年10月6日分别在昆明、珠海、广州分离到阳性的格特隐球菌环境株，初步揭示了格特隐球菌在我国自然环境中 的小生境的时空分布特征，即海拔100-1800米左右、年平均温度>15度、相对湿度约70-80%的蓝桉树林中。 . 基于多基因序列分析的系统发育学研究表明，华东地区格特隐球菌种群结构以VGI基因型为主，华南地区以VGI及VGIIb型为主，且两个地区间的菌株都未发生过重组现象，提示我国格特隐球菌主要以克隆性无性繁殖方式为主，与引起爆发流行的北美地区不同；基于全基因组数据的系统发育学研究进一步提示，分离自我国的格特隐球菌相互之间存在紧密亲缘关系，可能由同一种群的菌株进化而来。而且分离自我国的格特隐球菌遗传多态性低于澳洲、北美等国家，但与分离自印度等亚洲国家的格特隐球菌相互之间存在较紧密亲缘关系，提示中国乃至全球的格特隐球菌可能随着桉树在全球的引种经历过一次播散过程。另外，分离自中国的格特隐球菌毒力较低，未测出有性交配现象，且尚未发现对两性霉素B等一线抗真菌药物有耐药趋势。

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