兰州地区手足癣甲真菌病的病原学研究
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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。
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.
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。
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.
引文
1. Haneke E, Roseeuw D. The scope of onychomycosis: epidemiology and clinical feature. Int J Dermatot. 1999, 38 (Suppl 2): 7-12.
2．吴绍熙，廖万清，郭宁如，李春阳，毛玲娥，张宏，曾凡钦，李锡儒，封绍奎，李若瑜，石玉秀，郑岳臣，冉玉平，王家俊，喻楠，谭升顺，江致德．中国致病真菌10年动态流行病学研究．临床皮肤科杂志．1999，28(1)：1-5．
3．李若瑜，万喆，周祖得，李冬梅，王端礼．北京医科大学1992至1994年致病真菌种类分析．中华皮肤科杂志．1995，28(3)：188-189．
4．张雅洁，张桂梅，熊峰，李放娟，郭棱棱．深圳地区五年皮肤真菌病及致病真菌种类的分布．中华皮肤科杂志．2002，35(5)：401．
5．吴绍熙，刘维达，郭宁如，廖万清，李若瑜，王家俊，章强强，席丽艳，冉玉平，赖维．2002-2003年中国部分地区甲真菌病致病菌流行病学调查报告．临床皮肤科杂志．2005，34(9)：632-633．
6．程培华，廖耀玲，高秋恩，王端礼．桂北地区浅部真菌病病原菌分离培养观察．中华皮肤科杂志．1996，29(1)：62-63．
7．胡志敏，江萍，董碧麟，徐续玲，曾志良，董玉洁，周小勇，孙曾拯．武汉地区甲真菌病的致病菌及菌种变迁分析．中国皮肤性病学杂志．2006，20(10)：600-601．
8. Cheng S, Chong L. A prospective epidemiological study on tinea pedis and onychomycosis in HongKong. Chin Med J (Engl). 2002, 115 (6): 860-865.
9. Vella Zahra L, Gatt P, Boffa MJ, Mifsud E, Scerri L, Vella Briffa D, Pace JL. Characteristics of superficial mycoses in Malta. Int J Dermatol. 2003, 42: 265-271.
10. Shibaki H, Shibaki A. Analysis of dermatophyte flora at a private clinic in Sapporo during the period 1992 to 2001. Jpn J Med Mycol. 2003, 44: 209-216.
11. Buchvald J, Simal Jakova M. Epidemiology of dermatomycoses in Slovakia. Epidemiol Microbiol Imunol. 2002, 51 (1): 71-73.
12. Kuklova I, Kucerova H. Dermatophytoses in Prague, Czech Republic, between 1987 and 1998. Mycoses. 2001, 44: 493-496.
13. Tan Hiok-Hee. Superficial fungal infections seen at the National Skin Centre, Singapore. Nippon Ishinkin Gakkai Zasshi. 2005; 46 (2):77-80.
14．牛桃香，骆志成，石磊，武三卯，李文竹，周晓黎，薛晓云．兰州地区浅部真菌病及病原菌种类分析．中国皮肤性病学杂志．2004，18(4)：219-221．
15．蒋霞，骆志成，李文竹，周晓黎，牛桃香．136例股癣发病情况及菌种分析．中国麻风皮肤病杂志，2005，21(12)：953-954．
16．王恩文，骆志成，李文竹，杜华，曹庚．兰州地区105例头癣的发病情况及其病原菌种分析．中国麻风皮肤病杂志，2006，22(12)：993-995．
17．成晓茹，吴爱军，张建秀，陈清，于娜沙，王红，俞守义．红色毛癣菌随机扩增DNA多态性分型研究．中华皮肤科杂志，2001，34(6)：448-449．
18．陈辉，刘维达，沈永年．多部位红色毛癣菌感染分离菌株DNA分型研究．中华皮肤科杂志，2005，38(12)：732-734．
19．陈辉，李筱芳，刘维达，沈永年，吕桂霞，胡素泉，徐宏斌．股癣患者致病菌与足趾间分离皮肤癣菌的同源性分析．中国麻风皮肤病杂志，2006，22(11)：909-911．
20. Yazdanparast A., Jackson C J, Barton RC, Evans EGV. Molecular strain typing of Trichophyton rubrum indicates multiple strain involvement in onychomycosis. Br J Dermatol 2003, 148: 51-54.
21. Gupta AK, Kohli Y, and Summerbell RC. Variation in Restriction Fragment Length Polymorphisms among Serial Isolates from Patients with Trichophyton rubrum Infection. J Clin microbiol, 2001, 39: 3260-3266.
22. Rad MM, Colin Jackson C, Barton RC, Evans EGV. Single strains of Trichophyton rubrum in cases of tinea pedis. J Med Microbiol, 2005, 54: 725-726.
23. Roseeuw D. Achille foot screening project: preliminary results of patients screened by dermatologists. J Eur Acad Dermatol Venerol. 1999, 12 (Suppl 1): S6-S9.
24．中华医学会皮肤性病学分会．门诊就诊患者足部真菌感染的调查．中华皮肤科杂志．2002(35)：404-405．
25．卢洁，卢伟．足部真菌病情况调查．中国皮肤性病学杂志．2002(16)：387-388
26．甲真菌病调查协作组．甲真菌病患者生活质量调查研究．中华皮肤科杂志．2001，34(5)：364-366．
27. Bramono K, Budimulja U. Epidermiology of onychomycosis in Indonesia: data obtained from three individual studies. Jpn J Med Mycol. 2005; 46:171-176.
28. Roberts DT. Prevalence of dermatophyte onychomycosis in the United kingdom: Results of an omnibus survey. Br J Dermatol. 1992, 126 (Suppl 39): 23-27.
29. Sais G. Jucglà A, PeyrI J. Prevalence of dermatophyte onychomycosis in Spain: a cross-sectional study. Br J Dermatol. 1995, 132 (5): 758-761.
30．陈光仍，张莉．浅部真菌病致病菌株分析．中华皮肤科杂志，1996，29(5)：365．
31．张雅洁，王立阳，张卓，张桂梅，李欢梓，吴文中，翁翊，饶宗建．深圳地区甲真菌病病原菌流行病学的多中心研究．中国皮肤性病学杂志．2003，17(1)：28—29．
32．赖维，曾凡钦，卢念祖，曾仁山，候显增，杨健，刘次伟，佟菊贞，徐广坤．广州地区甲真菌病病原菌流行病学的多中心研究．中国皮肤性病学杂志．2001，15(2)：86—87．
33．甲真菌病流行病学调查协作组．甲真菌病致病菌的调查研究．中华皮肤科杂志．1996，29(1)：61—62．
34. Liu D, Coloe S, Baird R. Molecular determination of dermatophy fungi using the arbitraritly primed polymerase chain reaction. Br J Dematol, 1997, 137: 351-355.
35. Jackson CJ, Barton RC, Relly SL, Evans EG.. Strain idetification of Trichophyton rubrum by specific amplification of subrepeat element in the ribosomal DNA nontranscribed spacer. J Clin Microbiol, 2000, 38 (12): 4527-4534.
36. De Hoog GS, Bowman B, Graser Y, Haase G, El Fari M, Gerrits van den Ende AH, Melzer-Krick B, Untereiner WA.. Molecular phylogeny and taxonomy of medically important fungi. Med Mycol. 1998; 36 (Suppl 1): 52-56.
37. Jackson CJ, Barton RC, Evans EGV. Species identification and strain differentiation of dermatophyte fungi by analysis of ribosomal DNA intergenic spacer regions. J Clin Microbiol. 1999, 37 (4): 931-936.
38. Harmson D, Schwinn A, Brocker EB, Frosch M. Moleculer differention of dermatophyte fungi. Mycoses, 1999, 42 (1-2):67-70.
39．李若瑜，李东明，余进，刘伟，冀朝辉，王端礼．分子生物学技术在病原真菌鉴定和真菌感染诊断中的应用．北京大学学报(医学版)，2004，36(5)：536-539．
40. Hershkovitz MA, Lewis LA. Deep-Level Diagnostic Value of the rDNA-ITS Region. Mol Biol, 1996, 13 (9): 1276-1295.
41．李民，龙丰，兰和魁，陈慧，章强强，王家俊，任大明．PCR-RFLP鉴别临床常见的皮肤癣茵．中国皮肤性病学杂志，2002；16(3)：158-160．
42. Aguirre LD, Hurst SF, Choi JS, Shin JH, Hinrikson HP, Morrison CJ. Rapid differentiation of Aspergillus species from other medically important opportunistic molds and yeasts by PCR-enzyme immunoassay. J Clin Microbiol, 2004, 42 (8): 3495-3504.
43．孔祥明，林俊萍，王雅坤，陈洪铎．用PCR—RFLP进行申克孢子丝菌的分子生物学鉴定．中国皮肤性病学杂志，2004；18(12)：705-707．
44．王英，刘维达，顾军，赵敬军，陶苏江，吕桂霞．PCR结合基因扫描分析技术对凡种深部真菌病致病菌的快速检测及鉴定．中华皮肤科杂志，2003，36(8)：436-438．
45．余进，冀朝晖，李若瑜．常见致病性念珠菌的PCR—RFLP鉴定研究．中华皮肤科杂志，2002，35(2)：131-133．
46．李劲松，宋诗铎，祁伟，魏殿军．常见深部念珠菌感染快速基因诊断．中国感染控制杂志，2004，3(3)：198-201．
47. Mochizuki T, Tanabe H, Kawasaki M, Ishizaki H, Jackson CJ. Rapid identification of Trichophyton tonsurans by PCR-RFLP analysis of ribosomal DNA regions. J Dermatol Sci. 2003, 32 (1): 25-32.
48. Graser Y, Fari M EL, Presber W, Sterry W, Tietz H-J. Identification of common dematophytes using polymerase chain reaction. Br J Dermatol, 1998, 138: 576-582.
49. Summerbell RC, Haughland RA. rRNA gene internal transcribed spacer 1 and 2 sequences of asexual, anthropophilic dermatophytes related to Trichophyton rubrum. J Clin Microbiol. 1997, 37: 4005-4011.
50. Innis MA, Gelfland DH, Sninsky JJ. PCR protocol: a guide to methods and applications. SAN Diego Academic Press. 1990, 315-322.
51．樊建峰，李恒进，索继江，索继江，万喆．红色毛癣菌种内分型．军医进修学院报，2004，25(3)：225-226．
52．李民，王家俊，章强强，李莉，兰和奎，任大明．用聚合酶莲反应进行红色毛癣菌种内分型．中华皮肤科杂志，2002，35(5)：355-357．
53．卢圣栋．现代分子生物学实验技术．中国协和医科大学出版社，1999(第二版)：71．
54．张宏，吴绍熙．真菌分类学中的分子生物学技术点评．国外医学皮肤性病学分册，1997，23(4)：215—219．
55．钱骏，查国章，余平．白色念珠菌基因分型研究进展．国外医学生理、病理科学与临床分册，1998：18(2)：177-179．
56．刘洁，雷鹏程，应建明，高子芬．PCR—RFLP用于甲真菌病病原菌诊断和鉴别．中国皮肤性病学杂志，2001；15(6)：364-367．
57. Shin JH, Sung JH, Park SJ, Kim JA, Lee JH, Lee DY, Lee ES, Yang JM. Species identification and strain differentiation of dermatophyte fungi using polymerase chain reaction amplification and restriction enzyme analysis. J Am Acad Dermatol. 2003, 48(6): 857-865.
58．李乔，刘维达，杨国玲，李朝军．红色毛癣菌的基因分型研究．中华皮肤科杂志，2002，35(5)：352-354．
59. Nishio K, Kawasaki M, Ishizaki H. Phylogeny of the genera Trichophyton using mitochondrial DNA analysis. Mycopathologia, 1992,117: 127-132.
60. Mochizuki T, Uehara M, Menon T, Ranganathan S. Minipreparation of total cellular DNA is useful as an alternative molecular marker of mitochondrial DNA for the identification of Trichophyton mentagrophytes and T. rubrum. Mycoses, 1996, 39(1-2): 31-35.
61. Liu D, Coloe S, Baird R, Pederson J. Rapid Mini-Preparation of Fungal DNA for PCR. J Clin Microbiol, 2000, 38: 471.
2．吴绍熙，廖万清，郭宁如，李春阳，毛玲娥，张宏，曾凡钦，李锡儒，封绍奎，李若瑜，石玉秀，郑岳臣，冉玉平，王家俊，喻楠，谭升顺，江致德．中国致病真菌10年动态流行病学研究．临床皮肤科杂志．1999，28(1)：1-5．
3．李若瑜，万喆，周祖得，李冬梅，王端礼．北京医科大学1992至1994年致病真菌种类分析．中华皮肤科杂志．1995，28(3)：188-189．
4．张雅洁，张桂梅，熊峰，李放娟，郭棱棱．深圳地区五年皮肤真菌病及致病真菌种类的分布．中华皮肤科杂志．2002，35(5)：401．
5．吴绍熙，刘维达，郭宁如，廖万清，李若瑜，王家俊，章强强，席丽艳，冉玉平，赖维．2002-2003年中国部分地区甲真菌病致病菌流行病学调查报告．临床皮肤科杂志．2005，34(9)：632-633．
6．程培华，廖耀玲，高秋恩，王端礼．桂北地区浅部真菌病病原菌分离培养观察．中华皮肤科杂志．1996，29(1)：62-63．
7．胡志敏，江萍，董碧麟，徐续玲，曾志良，董玉洁，周小勇，孙曾拯．武汉地区甲真菌病的致病菌及菌种变迁分析．中国皮肤性病学杂志．2006，20(10)：600-601．
8. Cheng S, Chong L. A prospective epidemiological study on tinea pedis and onychomycosis in HongKong. Chin Med J (Engl). 2002, 115 (6): 860-865.
9. Vella Zahra L, Gatt P, Boffa MJ, Mifsud E, Scerri L, Vella Briffa D, Pace JL. Characteristics of superficial mycoses in Malta. Int J Dermatol. 2003, 42: 265-271.
10. Shibaki H, Shibaki A. Analysis of dermatophyte flora at a private clinic in Sapporo during the period 1992 to 2001. Jpn J Med Mycol. 2003, 44: 209-216.
11. Buchvald J, Simal Jakova M. Epidemiology of dermatomycoses in Slovakia. Epidemiol Microbiol Imunol. 2002, 51 (1): 71-73.
12. Kuklova I, Kucerova H. Dermatophytoses in Prague, Czech Republic, between 1987 and 1998. Mycoses. 2001, 44: 493-496.
13. Tan Hiok-Hee. Superficial fungal infections seen at the National Skin Centre, Singapore. Nippon Ishinkin Gakkai Zasshi. 2005; 46 (2):77-80.
14．牛桃香，骆志成，石磊，武三卯，李文竹，周晓黎，薛晓云．兰州地区浅部真菌病及病原菌种类分析．中国皮肤性病学杂志．2004，18(4)：219-221．
15．蒋霞，骆志成，李文竹，周晓黎，牛桃香．136例股癣发病情况及菌种分析．中国麻风皮肤病杂志，2005，21(12)：953-954．
16．王恩文，骆志成，李文竹，杜华，曹庚．兰州地区105例头癣的发病情况及其病原菌种分析．中国麻风皮肤病杂志，2006，22(12)：993-995．
17．成晓茹，吴爱军，张建秀，陈清，于娜沙，王红，俞守义．红色毛癣菌随机扩增DNA多态性分型研究．中华皮肤科杂志，2001，34(6)：448-449．
18．陈辉，刘维达，沈永年．多部位红色毛癣菌感染分离菌株DNA分型研究．中华皮肤科杂志，2005，38(12)：732-734．
19．陈辉，李筱芳，刘维达，沈永年，吕桂霞，胡素泉，徐宏斌．股癣患者致病菌与足趾间分离皮肤癣菌的同源性分析．中国麻风皮肤病杂志，2006，22(11)：909-911．
20. Yazdanparast A., Jackson C J, Barton RC, Evans EGV. Molecular strain typing of Trichophyton rubrum indicates multiple strain involvement in onychomycosis. Br J Dermatol 2003, 148: 51-54.
21. Gupta AK, Kohli Y, and Summerbell RC. Variation in Restriction Fragment Length Polymorphisms among Serial Isolates from Patients with Trichophyton rubrum Infection. J Clin microbiol, 2001, 39: 3260-3266.
22. Rad MM, Colin Jackson C, Barton RC, Evans EGV. Single strains of Trichophyton rubrum in cases of tinea pedis. J Med Microbiol, 2005, 54: 725-726.
23. Roseeuw D. Achille foot screening project: preliminary results of patients screened by dermatologists. J Eur Acad Dermatol Venerol. 1999, 12 (Suppl 1): S6-S9.
24．中华医学会皮肤性病学分会．门诊就诊患者足部真菌感染的调查．中华皮肤科杂志．2002(35)：404-405．
25．卢洁，卢伟．足部真菌病情况调查．中国皮肤性病学杂志．2002(16)：387-388
26．甲真菌病调查协作组．甲真菌病患者生活质量调查研究．中华皮肤科杂志．2001，34(5)：364-366．
27. Bramono K, Budimulja U. Epidermiology of onychomycosis in Indonesia: data obtained from three individual studies. Jpn J Med Mycol. 2005; 46:171-176.
28. Roberts DT. Prevalence of dermatophyte onychomycosis in the United kingdom: Results of an omnibus survey. Br J Dermatol. 1992, 126 (Suppl 39): 23-27.
29. Sais G. Jucglà A, PeyrI J. Prevalence of dermatophyte onychomycosis in Spain: a cross-sectional study. Br J Dermatol. 1995, 132 (5): 758-761.
30．陈光仍，张莉．浅部真菌病致病菌株分析．中华皮肤科杂志，1996，29(5)：365．
31．张雅洁，王立阳，张卓，张桂梅，李欢梓，吴文中，翁翊，饶宗建．深圳地区甲真菌病病原菌流行病学的多中心研究．中国皮肤性病学杂志．2003，17(1)：28—29．
32．赖维，曾凡钦，卢念祖，曾仁山，候显增，杨健，刘次伟，佟菊贞，徐广坤．广州地区甲真菌病病原菌流行病学的多中心研究．中国皮肤性病学杂志．2001，15(2)：86—87．
33．甲真菌病流行病学调查协作组．甲真菌病致病菌的调查研究．中华皮肤科杂志．1996，29(1)：61—62．
34. Liu D, Coloe S, Baird R. Molecular determination of dermatophy fungi using the arbitraritly primed polymerase chain reaction. Br J Dematol, 1997, 137: 351-355.
35. Jackson CJ, Barton RC, Relly SL, Evans EG.. Strain idetification of Trichophyton rubrum by specific amplification of subrepeat element in the ribosomal DNA nontranscribed spacer. J Clin Microbiol, 2000, 38 (12): 4527-4534.
36. De Hoog GS, Bowman B, Graser Y, Haase G, El Fari M, Gerrits van den Ende AH, Melzer-Krick B, Untereiner WA.. Molecular phylogeny and taxonomy of medically important fungi. Med Mycol. 1998; 36 (Suppl 1): 52-56.
37. Jackson CJ, Barton RC, Evans EGV. Species identification and strain differentiation of dermatophyte fungi by analysis of ribosomal DNA intergenic spacer regions. J Clin Microbiol. 1999, 37 (4): 931-936.
38. Harmson D, Schwinn A, Brocker EB, Frosch M. Moleculer differention of dermatophyte fungi. Mycoses, 1999, 42 (1-2):67-70.
39．李若瑜，李东明，余进，刘伟，冀朝辉，王端礼．分子生物学技术在病原真菌鉴定和真菌感染诊断中的应用．北京大学学报(医学版)，2004，36(5)：536-539．
40. Hershkovitz MA, Lewis LA. Deep-Level Diagnostic Value of the rDNA-ITS Region. Mol Biol, 1996, 13 (9): 1276-1295.
41．李民，龙丰，兰和魁，陈慧，章强强，王家俊，任大明．PCR-RFLP鉴别临床常见的皮肤癣茵．中国皮肤性病学杂志，2002；16(3)：158-160．
42. Aguirre LD, Hurst SF, Choi JS, Shin JH, Hinrikson HP, Morrison CJ. Rapid differentiation of Aspergillus species from other medically important opportunistic molds and yeasts by PCR-enzyme immunoassay. J Clin Microbiol, 2004, 42 (8): 3495-3504.
43．孔祥明，林俊萍，王雅坤，陈洪铎．用PCR—RFLP进行申克孢子丝菌的分子生物学鉴定．中国皮肤性病学杂志，2004；18(12)：705-707．
44．王英，刘维达，顾军，赵敬军，陶苏江，吕桂霞．PCR结合基因扫描分析技术对凡种深部真菌病致病菌的快速检测及鉴定．中华皮肤科杂志，2003，36(8)：436-438．
45．余进，冀朝晖，李若瑜．常见致病性念珠菌的PCR—RFLP鉴定研究．中华皮肤科杂志，2002，35(2)：131-133．
46．李劲松，宋诗铎，祁伟，魏殿军．常见深部念珠菌感染快速基因诊断．中国感染控制杂志，2004，3(3)：198-201．
47. Mochizuki T, Tanabe H, Kawasaki M, Ishizaki H, Jackson CJ. Rapid identification of Trichophyton tonsurans by PCR-RFLP analysis of ribosomal DNA regions. J Dermatol Sci. 2003, 32 (1): 25-32.
48. Graser Y, Fari M EL, Presber W, Sterry W, Tietz H-J. Identification of common dematophytes using polymerase chain reaction. Br J Dermatol, 1998, 138: 576-582.
49. Summerbell RC, Haughland RA. rRNA gene internal transcribed spacer 1 and 2 sequences of asexual, anthropophilic dermatophytes related to Trichophyton rubrum. J Clin Microbiol. 1997, 37: 4005-4011.
50. Innis MA, Gelfland DH, Sninsky JJ. PCR protocol: a guide to methods and applications. SAN Diego Academic Press. 1990, 315-322.
51．樊建峰，李恒进，索继江，索继江，万喆．红色毛癣菌种内分型．军医进修学院报，2004，25(3)：225-226．
52．李民，王家俊，章强强，李莉，兰和奎，任大明．用聚合酶莲反应进行红色毛癣菌种内分型．中华皮肤科杂志，2002，35(5)：355-357．
53．卢圣栋．现代分子生物学实验技术．中国协和医科大学出版社，1999(第二版)：71．
54．张宏，吴绍熙．真菌分类学中的分子生物学技术点评．国外医学皮肤性病学分册，1997，23(4)：215—219．
55．钱骏，查国章，余平．白色念珠菌基因分型研究进展．国外医学生理、病理科学与临床分册，1998：18(2)：177-179．
56．刘洁，雷鹏程，应建明，高子芬．PCR—RFLP用于甲真菌病病原菌诊断和鉴别．中国皮肤性病学杂志，2001；15(6)：364-367．
57. Shin JH, Sung JH, Park SJ, Kim JA, Lee JH, Lee DY, Lee ES, Yang JM. Species identification and strain differentiation of dermatophyte fungi using polymerase chain reaction amplification and restriction enzyme analysis. J Am Acad Dermatol. 2003, 48(6): 857-865.
58．李乔，刘维达，杨国玲，李朝军．红色毛癣菌的基因分型研究．中华皮肤科杂志，2002，35(5)：352-354．
59. Nishio K, Kawasaki M, Ishizaki H. Phylogeny of the genera Trichophyton using mitochondrial DNA analysis. Mycopathologia, 1992,117: 127-132.
60. Mochizuki T, Uehara M, Menon T, Ranganathan S. Minipreparation of total cellular DNA is useful as an alternative molecular marker of mitochondrial DNA for the identification of Trichophyton mentagrophytes and T. rubrum. Mycoses, 1996, 39(1-2): 31-35.
61. Liu D, Coloe S, Baird R, Pederson J. Rapid Mini-Preparation of Fungal DNA for PCR. J Clin Microbiol, 2000, 38: 471.