| Background and ObjectiveTrichosporonosis is one kind of mycosis caused by Trichosporon which usually involves hair, nail, skin and visceras. Clinical manifestation of trichosporonosis is complex. In 1970,Watson reported the first case of trichosporonosis in the world, and the first report in China was presented by Yang rongya in 2001. In recent years, the incidence of trichosporonosis have been increasing conspicuously. This fact thought to be related to several factors such as the increasing occurrence of malignant diseases,more and more patients exposed to organ transplantation, immunosuppressive therapies, chemotherapy, broad spectrum antibiotics and invasive medical procedures.There are several of Trichosporon which have the potential to cause human systemic infection.These kinds of Trichosporon have been known as T. cutaneum, T. asahii, T. asteroides, T. mucoides, T. inkin, T. jirovecii, T. dermatis, T. domesticum, T. montevideense, T. japonicum, T. coremiiforme, T. faecalis and T. loubieri. Trichosporon asahii (T. asahii) is considered as the principal etiologic agent of disseminated trichosporosis and non-Candida fungemia. The infection caused by T. asahii may disseminate to many organs, and its diagnose and treatment is comparatively difficult. Since the trichosporosis caused by T. asahii is being found more frequently in clinic, and usually result in the death of immunocompromised hosts, so that enouth attention must be paid to it by clinicians.Sequence polymorphism is an important character of fungi. As an important method for all microorganisms'epidemiologic studies, sequence polymorphism also can be used to assess strains'relationship on genic level. Microbiotic genotyping study is very important for epidemiologic survey and monitoring, understanding and intercepting of the route of transmission and pathogenetic research in community and hospital infection. For the same reason to study gene polymorphism and genotyping in T. asahii. is very vital for epidemiology, pathogenecity and treatment of trichosporonosis. It has been illuminated that there is interspecific gene polymorphism and genetic diversity in Trichosporon. Genotype of rRNA intergenic spacer (IGS) was various in different strains of T.asahii isolated from different areas and countries. Genotype of internal transcribed spacer regions (ITS) is different between the strains of T.asahii separated from epidermis and those from blood and mucous membrane. Meanwhile, genetic diversity and biological characters of T.asahii are various in different samples and different environment. But it is still unknow whether there exists intraspecific gene polymorphism and genetic diversity in T.asahii in China. Otherwise, infection sites, geographical distribution and type of infection and pathogenicity are different in 5 strains of T.asahii isolated from clinical specimens in China. Furthermore it has not been reported that whether they belong to different genotype and whether there is a relation in sites, geographical distribution and their genotype. Restriction fragment length polymorphism(RFLP) is a technique of genic detection and genotyping developed in recent years, and it is one of the most classic methods to analyze organism's genetic structure, which has not been used in genotyping research of T.asahii.For the first time, with the application of RFLP, the intraspecific gene polymorphism of T. asahii in China was studied, and the feasibility of RFLP method in genotyping study of T. asahii was estimated. The research also analyses the genotype of 5 isolates of T.asahii from clinical specimens in China on the basis of their IGS1 regions sequences so as to ascertain their genotype and distributed character. The results of the research will provide important reference evidence for epidemiologic survey and monitoring, understanding and intercepting of route of transmission, pathogenetic research in trichosporonosis infected by T.asahii, and exploring phylogenetic relations, distributing character, and relations in infection sites, geographical distribution, pathogenicity and genotype of T. asahii in China.Method5 isolates of T.asahii from clinical specimens in China were studied, and 1 T.asahii is used as the control group (CBS2479T). Firstly, nuclear DNA was extracted from the cells of all T.asahii strains by the method of glass pearls. Secondly, rRNA IGS1and ITS regions were amplified by using specific primers of PCR, the PCR products were cutted by restriction enzyme Hinfâ… , Hapâ…¡, Mboâ… , Aluâ… , Hhaâ… and Ncoâ… , after that the PCR products were analysed by the method of RFLP. Thirdly, the PCR products of ITS and IGS1 were depurated, cloned and sequenced. Then, the sequences were refered to Genbank and obtained register numbers. Furthermore, comparing sequences by Clustal x in order to find out the mutational point. Finally, in order to decide genotyp of 5 isolates of T.asahii from clinical specimens in China, this research compares with other IGS1 sequences of different genotypes in T.asahii from Genbank to present molecular phylogenetic trees.Results1. ITS and IGS1 regions of all strains were successfully amplified, cloned and sequenced. And the lengths of IGS1 and ITS are respectively 643bp and 541bp. Sequences of ITS of the strains are same. But there are some mutational points in IGS1 regions in some strains of T.asahii. Among them, there are mutational points in 449bp(T C) and 561bp(A T) respectivly in strain BZP07002, in129bp(A G), 178bp(C T), 204bp(T C), 223bp(G A), 451bp(A T) in strain BZP07003 and 333bp(A G) in strain BZP07004.2. There are no conspicuous difference in ITS sequence among all 5 strains of T.asahii cutted by same restriction enzyme. There are three different results in IGS1, among them, BZP07003 by Hapâ…¡a nd BZP07004 by Hhaâ… are difference than others. There are no difference among in all isolates of IGS1 cutted by same restriction enzyme Hinfâ… , Mboâ… , Aluâ… a nd Ncoâ… respectivly.3. Strains of BZP7001, BZP07005 are located in the same arborization of phylogenetic tree, and both of them belong to genotype 1. Whereas, BZP070003 is located in another arborization, which belongs to genotype 4. Strain of BZP07002 and BZP07004 found to belong to 2 new genotypes respectively which are different from any other genotypes. These two new genotypes were named genotype 7 and genotype 8 respectively.Conclusion1. There are intraspecific gene polymorphism and genetic diversity in IGS1 sequences of 5 isolates of T.asahii from clinical specimens in China, gene polymorphism can not be found in ITS sequences. IGS1 regions evolve faster than ITS regions in T.asahii.2. Restriction enzyme Hhaâ… and Hapâ…¡can be used to analyse the intraspecific gene polymorphism of T.asahii.3. The five stains of T.asahii isolated from clinical specimens in China are divided into four genotypes, among them, strains of BZP07001 and BZP07005 belong to genotype 1, strain of BZP07002 belongs to genotype 7, strain of BZP07003 belongs to genotype 4 and strain of BZP07004 belongs to genotype 8. In this research, we discovered T. asahii genotype 4 in China and T. asahii genotype 7 and 8 in the world for the first time.
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