甲真菌的分离鉴定及分类地位初探
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摘要
本文综述了甲真菌病的历史和现状，包括甲真菌病的易感因素、甲真菌的取材方法、甲真菌病的诊断和鉴定方法、致病菌菌种的构成与变迁以及甲真菌病的治疗，同时介绍了当前有关甲真菌病流行病学资料的局限性以及我们研究甲真菌病的目的及意义。
本论文第一部分介绍了甲真菌的分离和纯化。从甲病患者病甲中分离出甲碎屑，选用KOH为浮载剂，直接镜检，采用沙堡氏培养基对它们进行分离培养，浓度梯度法和切取菌丝尖端生长法对菌种进行纯化，得到纯化后的菌株16株。
本论文第二部分采用形态学、生理生化特征、DNAG+Cmol％、可溶性蛋白电泳及18S rDNA序列分析等分类技术对所分离的16株甲真菌进行了系统的分类研究，从而初步确定了16株甲真菌的分类地位。
结果表明，Fh_6和W_1分属于红酵母属中的胶红酵母和其它种，FJ_1、Xh_3、F_2、F_1、FJ_2、F_8、FJ_3均属于假丝酵母属，其中FJ_1、Xh_3为季也蒙假丝酵母，F_2、F_1为近平滑假丝酵母，FJ_2为热带假丝酵母，F_8、FJ_3为白假丝酵母；W_2属于隐球酵母属的新型隐球菌。Fh_2属于头孢霉属的顶头孢霉，Fh_3、W_3属于该属的其它种；Fh_5属于曲霉属中的黄曲霉群；菌株Dh_3与Acremonium alternatum关系最近，属于枝顶孢霉属；菌株Fh_4与Pleospora rudis关系最近，初步归入格孢腔菌属。
论文最后一部分初步探讨了甲真菌的动物回接方法。浅部感染真菌的易感动物是豚鼠，但由于实验条件的限制，目前只能用白鼠代替。回接后所有实验菌种在显微镜下均可见有菌丝或菌细胞生长，而阴性对照组则未见生长，同时可以从沙堡氏培养基中再次分离得到该菌，说明这些菌种均可以在动物体内生长繁殖，但是否能真正引起感染，还需要进一步使用易感动物进行确认。另外，根据资料记载，很多浅部真菌并不能在实验动物的皮肤产生明显感染，这也成为了甲真菌动物实验中的一个难题，有待于进一步探讨。
In this thesis, history and present condition of onychomycosis have been introduced which including impressionable factors of onychomycosis , the method of getting specimens , diagnosis and identification of onychomycosis, composing and variance of pathogen and therapy of onychomycosis. At the same time , the epidemiology data's localization of present onychomycosis and the purpose and meaning of our study have been presented .
In the first part of this research , isolation and purity method were introduced. Crumbs of nails were isolated from onychomycosis suffers, KOH was chosen to be solvent , nail fungi were examined by microscopy , cultured in Sabourud, purified by method of concentration grads and method of exsecting cusp of mycelia. Finally, sixteen purified strains were ready.
In the second part, polyphasic taxonomy methods such as morphological method, physiological and biochemical method, DNA G+Cmol%, soluble protein electrophoresis and 18S rDNA sequence analysis were used for systematics of all strains isolated and purified with the method above.
The results showed, Fh6 and Wj belong to Rhodotorula mudlaginosa and Rhodotorula sp, FJ, Xh3, F2, F1, FJ2,F8,FJ3 belong to Candida guilliermondii, Candida parapsilosis, Candida tropicalis and Candida albicans, W2 belongs to Cryptococcus neoformans, Fh2, Fh3, W3 belong to Cephalosporium acremonium and Cephalosporium sp, Fh5 belongs to Aspergillus flavus, Dh3 belongs to Acremonium, Fh4 belongs to Pleospora.
In the last part of this research, elementary animal experiment were processed. Impressionable animals of superficial infected fungi are guinea pigs, but because of restrict of experiment condition, white rats were replaced. After inoculation, all strains were examined by microscopy, hyphae or cells of all strains
were observed, but none was seen in negative comparison. In the same time, originally fungi were isolated again in Sabourud which showed that these fungi could grow and reproduce in these animals, but if they can cause infection or not will be make sure with impressionable animals.
Additional, according to the data, many superficial infected fungi don't arise obvious infection in skin of experimental animals, the status has become problem in animal experiments of nail fungi which await to further study.
本论文第一部分介绍了甲真菌的分离和纯化。从甲病患者病甲中分离出甲碎屑，选用KOH为浮载剂，直接镜检，采用沙堡氏培养基对它们进行分离培养，浓度梯度法和切取菌丝尖端生长法对菌种进行纯化，得到纯化后的菌株16株。
本论文第二部分采用形态学、生理生化特征、DNAG+Cmol％、可溶性蛋白电泳及18S rDNA序列分析等分类技术对所分离的16株甲真菌进行了系统的分类研究，从而初步确定了16株甲真菌的分类地位。
结果表明，Fh_6和W_1分属于红酵母属中的胶红酵母和其它种，FJ_1、Xh_3、F_2、F_1、FJ_2、F_8、FJ_3均属于假丝酵母属，其中FJ_1、Xh_3为季也蒙假丝酵母，F_2、F_1为近平滑假丝酵母，FJ_2为热带假丝酵母，F_8、FJ_3为白假丝酵母；W_2属于隐球酵母属的新型隐球菌。Fh_2属于头孢霉属的顶头孢霉，Fh_3、W_3属于该属的其它种；Fh_5属于曲霉属中的黄曲霉群；菌株Dh_3与Acremonium alternatum关系最近，属于枝顶孢霉属；菌株Fh_4与Pleospora rudis关系最近，初步归入格孢腔菌属。
论文最后一部分初步探讨了甲真菌的动物回接方法。浅部感染真菌的易感动物是豚鼠，但由于实验条件的限制，目前只能用白鼠代替。回接后所有实验菌种在显微镜下均可见有菌丝或菌细胞生长，而阴性对照组则未见生长，同时可以从沙堡氏培养基中再次分离得到该菌，说明这些菌种均可以在动物体内生长繁殖，但是否能真正引起感染，还需要进一步使用易感动物进行确认。另外，根据资料记载，很多浅部真菌并不能在实验动物的皮肤产生明显感染，这也成为了甲真菌动物实验中的一个难题，有待于进一步探讨。
In this thesis, history and present condition of onychomycosis have been introduced which including impressionable factors of onychomycosis , the method of getting specimens , diagnosis and identification of onychomycosis, composing and variance of pathogen and therapy of onychomycosis. At the same time , the epidemiology data's localization of present onychomycosis and the purpose and meaning of our study have been presented .
In the first part of this research , isolation and purity method were introduced. Crumbs of nails were isolated from onychomycosis suffers, KOH was chosen to be solvent , nail fungi were examined by microscopy , cultured in Sabourud, purified by method of concentration grads and method of exsecting cusp of mycelia. Finally, sixteen purified strains were ready.
In the second part, polyphasic taxonomy methods such as morphological method, physiological and biochemical method, DNA G+Cmol%, soluble protein electrophoresis and 18S rDNA sequence analysis were used for systematics of all strains isolated and purified with the method above.
The results showed, Fh6 and Wj belong to Rhodotorula mudlaginosa and Rhodotorula sp, FJ, Xh3, F2, F1, FJ2,F8,FJ3 belong to Candida guilliermondii, Candida parapsilosis, Candida tropicalis and Candida albicans, W2 belongs to Cryptococcus neoformans, Fh2, Fh3, W3 belong to Cephalosporium acremonium and Cephalosporium sp, Fh5 belongs to Aspergillus flavus, Dh3 belongs to Acremonium, Fh4 belongs to Pleospora.
In the last part of this research, elementary animal experiment were processed. Impressionable animals of superficial infected fungi are guinea pigs, but because of restrict of experiment condition, white rats were replaced. After inoculation, all strains were examined by microscopy, hyphae or cells of all strains
were observed, but none was seen in negative comparison. In the same time, originally fungi were isolated again in Sabourud which showed that these fungi could grow and reproduce in these animals, but if they can cause infection or not will be make sure with impressionable animals.
Additional, according to the data, many superficial infected fungi don't arise obvious infection in skin of experimental animals, the status has become problem in animal experiments of nail fungi which await to further study.
引文
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[3] Elewski BE. Onychomycosis: Pathogenesis, Diagosis, and Management. Clinical Microbiology Revinew. 1998; 11(3):415-429
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