Rapid molecular identification of imported mycoses in Japan

加上日本国际化程度的大幅提高，一些日本人有机会出国旅游或工作。然而，经常有报道称，他们中的一些人患有东亚(泰国、中国、印度尼西亚)、美国、巴西等国特有的严重真菌地方性疾病。这些感染被称为输入性霉菌病：球孢子菌病、组织胞浆菌病、乳头状青霉病和副球孢子菌病。由于这些病原体(3类)的使用非常危险，临床实验室需要简单安全的鉴定和诊断系统，本项目旨在建立简单、安全的输入性真菌感染病原体(卡氏假单胞菌、玛氏假单胞菌、巴西假单胞菌和新生弧菌)的分子鉴定体系。1)通过ITS区(ITS_1-5.8S rDNA-ITS_2)序列分析，对29个不同地理位置的致病菌株进行了系统发育分析，发现ITS区包含了足够的信息来解决真菌菌株之间的系统发育关系。2)根据ITS区序列信息，设计了一条新的扩增418个碱基的聚合酶链式反应(PCR)引物，用于巴西巴氏拟青霉的分子鉴定。3)它的区域分析使我们能够将所有的新生隐孢子虫变种划分为四个地理群。4)证实了本课题组研制的乳房革兰氏假丝酵母菌的聚合酶链式反应引物对的有效性，为临床实验室的分子鉴定提供了一种新的方法。

Coupled with the large increase in Japanese internationalization, a number of Japanese people have had opportunities to go abroad for oversea travel or work purposes. However, frequently it has been reported that some of them had suffered from serious fungal endemics peculiar to the countries such as East Asia (Thailand, China, Indonesia), USA, Brazil and others. These infections are called as imported mycoses : coccidioidomycosis, histoplasmosis, penicilliosis mameffei and paracoccidioidomycosis. Since these causative agents are very dangerous to handle (class 3), simple and safe identification and diagnostic systems are requested in clinical laboratory.In this project, preparation of simple and safty molecular identification system for the etiological agents of these imported fungal infection (H.casulatum, P.mameffei, P.brasiliensis and C.neoformans) was aimed. 1) Phylogeny of twenty nine geographically diverse H.capsulatum isolates representing the three varieties, var.capsulatum, duboisii and farciminosum were evaluated using (ITS) region (ITS1-5.8S rDNA-ITS2) sequence analyses, and we found that the ITS region contained sufficient information to resolve the phylogenetic relationship among the fungal isolates. 2) Based on the ITS region sequence information, a new PCR primer (designed to amplify 418 bp) was prepared for the molecular identification of P.brasiliensis. 3) ITS region analysis of allowed us to distinguish all isolates of C.neoformans var.gattii into four geographic groups. 4) Usefulness of PCR primer pairs for P.mameffei (developed by our research group) was also confirmed.Thus newly prepared PCR primer pairs for each etiological agent of imported mycoses in Japan were simple and found to be useful for the molecular identification in clinical laboratory.