【作者】 李昌吉；

【导师】 骆志成；

【作者基本信息】 兰州大学 ， 皮肤病与性病学， 2007， 硕士

【摘要】 手癣(tinea manuum)、足癣(tinea pedis)及是指累及手掌和指间、足跖和趾间的皮肤癣菌感染，甲真菌病(onychomycosis)是指由任何真菌所致的甲感染，是一类常见的真菌感染性疾病。具有传染性，发病率高，容易复发和再感染，虽不危及生命，但对患者的生活质量造成不同程度的影响。手足癣甲真菌病的主要致病菌为红色毛癣菌，其病原菌菌种构成及分布存在地区差异；而且随着人口流动、环境因素变化及治疗干预等，同一地区的菌种也会发生变迁。尚缺乏兰州地区手足癣甲真菌病的发病情况及病原学流行病学的详细资料。目的：1．了解兰州地区手足癣甲真菌病发病情况及病原菌菌种颁布特点，为手足癣甲真菌病的流行病学研究奠定基础，同时为临床抗真菌药物的合理应用提供理论依据。2．探讨PCR结合RFLP的分子生物学技术鉴定皮肤癣菌的特异性和敏感性，以便在手足癣甲真菌病病原真菌的鉴定中推广应用。方法：2006年1月～2006年12月，对兰州大学第二医院皮肤科门诊拟诊为手足癣甲真菌病的患者，分别取其皮屑和／或甲屑做15％KOH直接涂片镜检。采用调查问卷的方式，详细登记镜检阳性患者的相关临床资料。对阳性标本行沙堡葡萄糖琼脂分离培养、鉴定菌种。采用真菌通用引物ITS1、ITS4对手足癣甲真菌病的优势致病菌rDNA的ITS区进行PCR扩增，从分子水平鉴定到红色毛癣菌菌种的水平，经鉴定的红色毛癣菌进一步对其可变区NTS(nontranscribedspacer)区两个串联重复单元TRS(tandemly repetitive subelements)即TRS-1、TRS-2区片段进行了PCR扩增，显示同种不同株红色毛癣菌基因型可能的差异。对基因组DNA采用限制性片段长度多态性(RFLP)分析，用HinfⅠ和TaqⅠ两种限制性内切酶对ITS1、ITS4的扩增产物进行酶切。结果：1.262例真菌镜检阳性的手足癣甲真菌病患者，甲真菌病153例(58.40％)，足癣150例(57.25％)，手癣37例(14.12％)。镜检可见菌丝和／或孢子，以菌丝为主。分离菌株135株(阳性率51.53％)，其中红色毛癣菌107株(占培养结果的79.26％)，白念珠菌17株(占12.59％)，犬小孢子菌9株(占6.67％)，红酵母属及链格孢霉属各1株(各占0.74％)。2．引物ITS1、ITS4在红色毛癣菌均扩增出一约690bp特异性条带；3．TRS—1区TrNTSF-2和TrNTSR-4引物把72株红色毛癣菌共分为3型，呈现明显的种内多态性，在29例受试患者中，6例不同感染部位分离得到的菌株基因型有差异；TRS—2区TrNTSC—1和TrNTSR-1引物扩增出一约500bp特异性条带。4．HinfⅠ、TaqⅠ的酶切片段完全相同。结论：1．兰州地区手足癣甲真菌病的致病菌种比较单一，优势致病菌为红色毛癣菌；2．引物ITS1、ITS4可以把红色毛癣菌特异地鉴定到种的水平，从而使临床快速鉴定该菌成为可能；3．TRS—1区TrNTSF-2和TrNTSR-4引物可以对红色毛癣菌进行种内分型，而TRS—2区TrNTSC-1和TrNTSR-1引物也可以特异地鉴定红色毛癣菌。多部位红色毛癣菌感染患者存在不同型别菌株的感染，提示部分多部位皮肤癣菌感染患者不同部位皮肤癣菌感染可能为多菌株的混合感染。4．内切酶HinfⅠ和TaqⅠ也可以特异地鉴定红色毛癣菌。更多还原

【Abstract】 Background: Tinea manuum et pedis are a kind of dermatophytosis which invades the hand and foot, onychomycosis is defined as the infection of the nail by fungus. Dermatophyte infections of humans are among the most common forms of skin diseases, which may be harmful to the body and decrease people’s life quality. From the previous investigations, it is suggested that the etiologic agents of dermatomycoses not only vary with the site of the infection, but also differ to some extent according to the city, state or country of occurrence. The geographical distribution of the anthropophilic and zoophilic dermatophytes is not static within a district, but dynamic, and subject to change by population shifts due to migration, health habits, standards of living or travel. They also may change with the passage of time. It is well established that an understanding of the prevalence and identity of etiological agents assists in the prevention and treatment of dennatophytoses. However, very few have been published about the prevalence and types of dennatophytoses and what etiologic agents are present in Lanzhou district up to the present.Objective: 1. To investigate the risk factors, incidence and etiological agents of tinea manuum tinea pedis and onychomycosis in Lanzhou district. 2. To inquire into the application of rapid and precise identification for the pathogenic fungus species of T. rubrum by using moluculer methods.Methods: The specimens of clinically suspected patients with dermatomycoses were examined for causative fungi in the Second Hospital of Lanzhou University during January to December of 2006, while the clinical materials of each patient with KOH positive were recorded and analyzed. The total DNAs of 72 isolates of T. rubrum were extracted by using mini-preparation, then the internal transcribed spacer (ITS) region of ribosomal DNA were amplified by using the fungi-universal primers ITS1 (5’-TCCGTAGGTGAACCTGCGG-3’), ITS4 (5’-TCCTCCGCTTATTGATATGC-3’). Moreover, two novel tandem repeat subelements (TRSs), TRS-1 and TRS-2, located in the T. rubrum rDNA nontranscribed spacer (NTS), were amplified from T. rubrum. Then restriction fragment length polymorphisms (RFLP) was performed by three restriction enzymes (Hinf I, Taq I) digesting those production of Polymerase chain reaction (PCR) amplification.Results: 1.One hundred and fifty-three cases are onychomycosis in the 262 patients and107 T. rubrum strains were isolated in the 135 strains of pathogenic fungi. 2. The patterns of 72 strains of T. rubrum were isologous after the primer-pair ITS1 and ITS4 being amplified, and about 690bps were obtained in T. rubrum. 3. The patterns of 72 strains of T. rubrum were isologous after the primer-pair TrNTSC-1 and TrNTSR-1 being amplified, and about 500bps were obtained in T. rubrum. Meanwhile, according to TrNTSF-2 and TrNTSR-4 being amplified, we could separate 3 subgroups within72 strains of T. rubrum. It happened to 6 of the 29 patients that multiple genotypes were involved in T. rubrum on different sites in the same body. 4. The patterns of two restriction enzymes Hinf I and Taq I were respectively identical.Conclusions: 1. T. rubrum was the main dermatophytes isolated on the tinea manuum tinea pedis and onychomycosis of people in Lanzhou district. 2. PCR of ribosomal DNA intergenic spacer regions is a useful method for species identification of common dermatophyte fungi. 3. PCR of TRS-2 in the T. rubrum rDNA nontranscribed spacer (NTS) is a useful method for species identification of T. rubrum, and PCR of TRS-1 in the T. rubrum rDNA nontranscribed spacer (NTS) can differentiate T. rubrum at the level of sub-species, which is valuable for the pathogenic diagnosis and epidemiology survey. The study shows that multiple genotypes are involved in T. rubrum infection on different sites in the same patient. The results suggest that in some cases the infections are due to different strains of fungi. 4. Restriction enzymes Hinf I and Taq I digestion of PCR amplified ITS regions produce unique and easily identifiable fragment patterns for T. rubrum species.更多还原