Molecular Biological Indetification And Examination Of Hair Perforation In Vitro In Dermatophytes

Objective: The dermatophytes are the common pathogenicfungi of superficial mycoses that have the capacity to invadekeratinized tissues (skin, hair, and nail) of humans. Now it hasbeen reported forty-five species. some of them only invadeanimals and about twenty species infect humans. The commondermatophytes include Trichophyton metagrophytes,Trichophyton rubrum, Microsporum canis, Microsporumferrugineum and Epidermophyton floccosum .To study the significance of AP-PCR in indetification andsubtyping, five species were amplified by AP-PCR. And wecompared the differences between Microsporum canis andTrichophyton mentagrophytes in the damage to the hair ofdifferent age groups by light microscope and scanning electronmicroscope (SEM).Methods: All the strains of dermatophytes were isolated fromthe clinical patients and were identified by cultivation,micromorphology,morphology and biochemical test. We isolatedgenomic DNAs from fungi using benzyl chloride. And theDNAs of Trichophyton metagrophytes, Trichophyton rubrum,Microsporum canis, Microsporum ferrugineum andEpidermophyton floccosum were amplified using the randomprimers(AP3, ATG, TCA, and TCT) by AP-PCR. We analyzedvariation among dermatophytes. In vitio hair perforation test,healthy hair of four different age groups (less than 2 yearsold,2～13 years old,13～19 years old and older than 19 yearsold) were collected. The isolated strains suspensions ofmicrosporum Canis and Trichophyton mentagrophytes incubatedrespectively on the surface of the hair on Sabourd agar at 27℃and incubated hairs were observed every day after three daysunder light microscope (LM) and electronic microscope (SEM).Results: The result of indentifying dermatophyte species:Distinct DNA band patterns were observed in different specieswith four different primers. The more distinct patterns wereobserved among these species, especially using primers AP3 andATG, We amplified the same template many times, and theuniform DNA band patterns were obtained. We amplified thetemplates isolated from different strains of same species, andobtained the uniform DNA band patterns.The result of subtyping: The phenotypes of 46 Trichophytonmentagrophytes strains were powdery type and the dictinctpatterns of DNA bands were observed. Two genotypes wereidentified among 46 strains. 25 strains belonged to typeⅠand21 strains belonged to typeⅡ. 11 of 20 strains isolated fromtinea of feet and hands were typeⅠ, 9 strains were typeⅡ. 14 of26 strains isolated from tinea of cruris and corporis were typeⅠ,12 strains were typeⅡ. There was no statistical diffentence ingenotypy between tinea of feet and hands and tinea of cruris andcorporis. Uniform DNA band patterns with primer ATG wereobserved in 46 Trichophyton mentagrophytes strains. UniformDNA band patterns with primer AP3 and ATG were observed inMicrosporum canis isolated from different infection sites.We compared the differences between Microsporum canisand Trichophyton mentagrophytes in the damage to the hairsof different age groups. The criteria was that the hyphas andspores attached the damaged hairs. Microsporum canis andTrichophyton mentagrophytes could lead to damage the hairs.The time of Microsporum canis leading to damage the hairs wassignificantly shorter than that of Trichophyton mentagrophytesin different age groups (p<0.01). The time to damage the hairswas delayed along with the ages growing both in Microsporumcanis and Trichophyton mentagrophytes (p<0.01). Except thediversity between groups (less than 2 years old and 2～13 yearsold) had no statistical significance, the diversity among the othergroups had statistical significance. The time and degreedamaged by Trichophyton mentagrophytes were earlier andseverer than Microsporum canis.Conclusion: (1) AP-PCR may be applied in the indetificationand subtyping of dermatophytes using AP3, ATG, TCA, andTCT as primers. AP-PCR is a rapid, simple and dependablemethod for species identification of common dermatophytes. (2)Trichophyton mentagrophytes had two genotypes. There was norelationship between genotypes and infection sites. (3) Uniform...