犬小孢子菌生物学性状及基因分型研究
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摘要
犬小孢子菌(Microsporum canis)归属于半知菌亚门-丝孢菌纲-丝孢菌目-丛梗孢科-小孢子菌属,为发外型亲动物型皮肤癣菌,存在于自然界及猫,狗,兔等动物毛皮上,可通过动物—动物,动物—人的途径传染。研究表明,犬小孢子菌可导致头癣,体癣和少数甲癣以及个别少见真菌病,一直是导致国内外浅部真菌病特别是头癣的重要病原菌。近年来,随着人们生活水平的提高和饲养宠物之风的盛行,头癣的发病率居高不下。而犬小孢子菌作为头癣中白癣和脓癣的主要致病菌,对头癣感染及传染起着重要的作用。而且,犬小孢子菌传染性强,在一定条件下还可在特定人群中出现暴发及引发泛发性浅部真菌病。综上所述可见对其研究的必要性。
目的:1.分析本地犬小孢子菌致病情况。2.探讨使用分子生物学技术鉴定皮肤癣菌的特异性和敏感性,以便在犬小孢子菌所致的真菌性疾病的鉴定中推广应用。3.观察5种常用抗菌药物抗菌效果,进而为临床犬小孢子菌所致疾病(头癣、体癣)的用药提供理论依据。
方法:2007年7月～2007年12月,对兰州大学第二医院皮肤科门诊拟诊为犬小孢子菌(头癣及有可疑动物接触史的体癣)感染的患者,分别取其断发、头屑或皮屑做15%KOH直接涂片镜检。采用调查问卷的方式,详细登记培养结果为犬小孢子菌患者的相关临床资料。对阳性标本行沙堡葡萄糖琼脂及米饭培养基分离培养、鉴定菌种。采用真菌通用引物ITS1(5'-TCCGTAGGTGAACCTGCGG-3')、ITS4(5'-TCCTCCGCTTATTGATATGC-3')对犬小孢子菌rDNA的ITS区进行PCR扩增,从而从分子水平鉴定到犬小孢子菌种的水平。对基因组DNA采用限制性片段长度多态性(RFLP)分析,用HinfⅠ、TaqⅠ两种限制性内切酶对ITS1、ITS4的扩增产物进行酶切并对酶切结果进行分析。采用美国临床和实验室标准化研究所(clinical and laboratorystandardinstitute,CLSI,即原NCCLS)于2003年发表的针对产孢丰富丝状真菌的微量稀释法(M-38A)测定犬小孢子菌对临床5种抗真菌药物的MIC值。
结果:1.55例犬小孢子菌感染患者,表现为头癣者47例(85.45%),体癣者8例(14.55%)。年龄1～47岁,其中10岁以下者52例(94.54%),男40例(72.72%),女15例(27.27%)。家住城市者42例(76.36%),地县及农村者13例(23.63%),49例(84.09%)有2周到1年半不等的动物(猫狗马兔)接触史,8例(14.55%)例有与头癣患者接触史,亲兄弟,表兄妹同时感染头癣各1例。2.引物ITS1、ITS4在犬小孢子菌均扩增出一约760bp特异性条带。3.HinfI、TaqⅠ各将犬小孢子菌切出两条固定条带。4.抗真菌药物的MIC范围如下,伊曲康唑0.25-1μg/ml,益康唑0.25-2μg/ml,特比萘芬0.01-0.04μg/ml,灰黄霉素0.25-1μg/ml,氟康唑1-32μg/ml。
结论:1.调查的犬小孢子菌感染以10岁以下儿童为主,主要表现为头癣中的白癣,并有家族内传染的情况。2.引物ITS1、ITS4可以把犬小孢子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.内切酶HinfⅠ和TaqⅠ可以特异地鉴定犬小孢子菌。4.犬小孢子菌对5种药物敏感,各种药物的最小抑菌浓(minimal inhibitory concentration MIC)之间有显著性差异,并为临床用药提供了有益参考。
Background:Microsporum canis is a kind of dermatophytosis which belongs to Deuteromycotina-HyPhomycetes-Moniliales-Moniliaceae-Microsporum which trends to invade skin and fur of animals.It is found on the skin of some animals like cats, dogs and rabbits and can transfer from animal to animal as well as from animal to people.Previous studies show that M.canis can result in diseases such as tinea capitis, corpus capitis and few onychomycosis,especially tinea capitis.It has always being known as one of the most important superficial mycosis all over the world.With a popular phenomena of keeping pets,the rate of tinea capitis rises sharply during those years.M.canis is the main pathogen of tinea alba and kerion,which plays a crucial role in the prevalence of tinea capitis.Besides,M.canis can cause outbreak of tinea capitis to certain people in some situations.In conclusion,it is significant to farther research for this pathogenic fungus.
Objective:1.To investigate the etiology,clinical features of M.canis in Lanzhou district.2.To inquire into the application of rapid and precise identification for the superficial mycosis by using molecular methods.3.Observe the broth microdilution antifungal susceptibility about M.canis of Lanzhou district and prove the reproducibility and feasibility of M38-A procedure which can be used for the antifungal susceptibility test to dermaphytes in the laboratories,especially M.canis.
Method:The specimens are from suspected patients who suffered from tinea capitis and corpus capitis and came to No.2 Attached Hospital of Lanzhou University during 2007.6-2007.12.The patients were examined microscopically with positive results are questioned about history and related clinical information is detailed.The specimens of these cases were cultured on Sabouraud - chlorlamphenicol glucose agar (SDA)and Rice grain medium(RGM),the species being identified with growth rate of isolates,production and direct microscopic examination.After that,we approached the broth microdilution antifungal susceptibility testing condition which was recommended by CLSI in 2003 tested the minimal inhibitory concentration(MIC)of 5 antifungal drugs to 55 M.canis.We also extracted the DNA of the 55 M.canis by mini-preparation.The internal transcribed spacer(ITS)region of ribosomal DNA were amplified by the fungi-universal primers ITS1(5'-TCCGTAGGTGAACCTGCGG -3')and ITS4(5'-TCCTCCGCTTATTGATATGC-3').Following restriction fragment length polymorphisms(RFLP)was performed by two restriction enzymes(HinfⅠ, TaqⅠ)digesting those production of polymerase chain reaction(PCR)amplification.
Results:1.Among 55 cases,47 patients suffered from tinea capitis(85.45%)and 8 patients suffered from corpus captitis(14.55%).The ages were from 1-47.Patients under 10-year-old were 52 cases which occupied 94.54%in the whole specimens. Male were 40(72.72%)and female were 15(27.27%).Patients living in Lanzhou city are 42(76.36%)and these living in town and countryside were 13(23.63%).The 49 patients(84.09%)admitted to contact animals especially pets during 2 weeks and 1.5 years,while 8 patients once played with children with tinea capitis.We also found 2 cases of more children in the same families got the tinea capitis one by one.2.The patterns of 55 strains of M.canis were isologous after the primer-pair ITS1 and ITS4 being amplified,and about 760 bps were obtained in M.canis.3.The patterns of two restriction enzymes HinfⅠand TaqⅠwere respectively identical with two bands.4. The range of the drugs are:Itraconazole,0.25-1ug/ml,Econazole,0.25-2μg/ml, Terbinafine,0.01-0.04μg/ml,Griseofulvine,0.25-1μg/ml.Fluconazole,1-32μg/ml respectively.
Conelusion:1.Majority of patients' are less than 10-year-old.The main clinical type of tinea capitis is tinea alba.2.PCR of ribosomal DNA intergenic spacer regions is a useful method for species identification of common dermatophyte fungi.3. Restriction enzymes HinfⅠand TaqⅠdigestion of PCR amplified ITS regions produce unique and easily identifiable fragment patterns for M.canis species.4.M. canis are sensitive to the five drugs.There are significant differences of MIC among these 5 drugs,which provide a reference to us to use these drugs properly.
目的:1.分析本地犬小孢子菌致病情况。2.探讨使用分子生物学技术鉴定皮肤癣菌的特异性和敏感性,以便在犬小孢子菌所致的真菌性疾病的鉴定中推广应用。3.观察5种常用抗菌药物抗菌效果,进而为临床犬小孢子菌所致疾病(头癣、体癣)的用药提供理论依据。
方法:2007年7月～2007年12月,对兰州大学第二医院皮肤科门诊拟诊为犬小孢子菌(头癣及有可疑动物接触史的体癣)感染的患者,分别取其断发、头屑或皮屑做15%KOH直接涂片镜检。采用调查问卷的方式,详细登记培养结果为犬小孢子菌患者的相关临床资料。对阳性标本行沙堡葡萄糖琼脂及米饭培养基分离培养、鉴定菌种。采用真菌通用引物ITS1(5'-TCCGTAGGTGAACCTGCGG-3')、ITS4(5'-TCCTCCGCTTATTGATATGC-3')对犬小孢子菌rDNA的ITS区进行PCR扩增,从而从分子水平鉴定到犬小孢子菌种的水平。对基因组DNA采用限制性片段长度多态性(RFLP)分析,用HinfⅠ、TaqⅠ两种限制性内切酶对ITS1、ITS4的扩增产物进行酶切并对酶切结果进行分析。采用美国临床和实验室标准化研究所(clinical and laboratorystandardinstitute,CLSI,即原NCCLS)于2003年发表的针对产孢丰富丝状真菌的微量稀释法(M-38A)测定犬小孢子菌对临床5种抗真菌药物的MIC值。
结果:1.55例犬小孢子菌感染患者,表现为头癣者47例(85.45%),体癣者8例(14.55%)。年龄1～47岁,其中10岁以下者52例(94.54%),男40例(72.72%),女15例(27.27%)。家住城市者42例(76.36%),地县及农村者13例(23.63%),49例(84.09%)有2周到1年半不等的动物(猫狗马兔)接触史,8例(14.55%)例有与头癣患者接触史,亲兄弟,表兄妹同时感染头癣各1例。2.引物ITS1、ITS4在犬小孢子菌均扩增出一约760bp特异性条带。3.HinfI、TaqⅠ各将犬小孢子菌切出两条固定条带。4.抗真菌药物的MIC范围如下,伊曲康唑0.25-1μg/ml,益康唑0.25-2μg/ml,特比萘芬0.01-0.04μg/ml,灰黄霉素0.25-1μg/ml,氟康唑1-32μg/ml。
结论:1.调查的犬小孢子菌感染以10岁以下儿童为主,主要表现为头癣中的白癣,并有家族内传染的情况。2.引物ITS1、ITS4可以把犬小孢子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.内切酶HinfⅠ和TaqⅠ可以特异地鉴定犬小孢子菌。4.犬小孢子菌对5种药物敏感,各种药物的最小抑菌浓(minimal inhibitory concentration MIC)之间有显著性差异,并为临床用药提供了有益参考。
Background:Microsporum canis is a kind of dermatophytosis which belongs to Deuteromycotina-HyPhomycetes-Moniliales-Moniliaceae-Microsporum which trends to invade skin and fur of animals.It is found on the skin of some animals like cats, dogs and rabbits and can transfer from animal to animal as well as from animal to people.Previous studies show that M.canis can result in diseases such as tinea capitis, corpus capitis and few onychomycosis,especially tinea capitis.It has always being known as one of the most important superficial mycosis all over the world.With a popular phenomena of keeping pets,the rate of tinea capitis rises sharply during those years.M.canis is the main pathogen of tinea alba and kerion,which plays a crucial role in the prevalence of tinea capitis.Besides,M.canis can cause outbreak of tinea capitis to certain people in some situations.In conclusion,it is significant to farther research for this pathogenic fungus.
Objective:1.To investigate the etiology,clinical features of M.canis in Lanzhou district.2.To inquire into the application of rapid and precise identification for the superficial mycosis by using molecular methods.3.Observe the broth microdilution antifungal susceptibility about M.canis of Lanzhou district and prove the reproducibility and feasibility of M38-A procedure which can be used for the antifungal susceptibility test to dermaphytes in the laboratories,especially M.canis.
Method:The specimens are from suspected patients who suffered from tinea capitis and corpus capitis and came to No.2 Attached Hospital of Lanzhou University during 2007.6-2007.12.The patients were examined microscopically with positive results are questioned about history and related clinical information is detailed.The specimens of these cases were cultured on Sabouraud - chlorlamphenicol glucose agar (SDA)and Rice grain medium(RGM),the species being identified with growth rate of isolates,production and direct microscopic examination.After that,we approached the broth microdilution antifungal susceptibility testing condition which was recommended by CLSI in 2003 tested the minimal inhibitory concentration(MIC)of 5 antifungal drugs to 55 M.canis.We also extracted the DNA of the 55 M.canis by mini-preparation.The internal transcribed spacer(ITS)region of ribosomal DNA were amplified by the fungi-universal primers ITS1(5'-TCCGTAGGTGAACCTGCGG -3')and ITS4(5'-TCCTCCGCTTATTGATATGC-3').Following restriction fragment length polymorphisms(RFLP)was performed by two restriction enzymes(HinfⅠ, TaqⅠ)digesting those production of polymerase chain reaction(PCR)amplification.
Results:1.Among 55 cases,47 patients suffered from tinea capitis(85.45%)and 8 patients suffered from corpus captitis(14.55%).The ages were from 1-47.Patients under 10-year-old were 52 cases which occupied 94.54%in the whole specimens. Male were 40(72.72%)and female were 15(27.27%).Patients living in Lanzhou city are 42(76.36%)and these living in town and countryside were 13(23.63%).The 49 patients(84.09%)admitted to contact animals especially pets during 2 weeks and 1.5 years,while 8 patients once played with children with tinea capitis.We also found 2 cases of more children in the same families got the tinea capitis one by one.2.The patterns of 55 strains of M.canis were isologous after the primer-pair ITS1 and ITS4 being amplified,and about 760 bps were obtained in M.canis.3.The patterns of two restriction enzymes HinfⅠand TaqⅠwere respectively identical with two bands.4. The range of the drugs are:Itraconazole,0.25-1ug/ml,Econazole,0.25-2μg/ml, Terbinafine,0.01-0.04μg/ml,Griseofulvine,0.25-1μg/ml.Fluconazole,1-32μg/ml respectively.
Conelusion:1.Majority of patients' are less than 10-year-old.The main clinical type of tinea capitis is tinea alba.2.PCR of ribosomal DNA intergenic spacer regions is a useful method for species identification of common dermatophyte fungi.3. Restriction enzymes HinfⅠand TaqⅠdigestion of PCR amplified ITS regions produce unique and easily identifiable fragment patterns for M.canis species.4.M. canis are sensitive to the five drugs.There are significant differences of MIC among these 5 drugs,which provide a reference to us to use these drugs properly.
引文
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