Vitro Antifungal Susceptibility Testing And Molecular Identification Of Common Dermatophytes

Objective: Dermatophytosis is a common disease and prevalent among patients indermatological clinic. There are many obstacles, especially the etiological identification,high recurrence, met in epidemiological investigation and therapeutic studies. In thisstudy, we investigated the etiological characteristics of clinical specimens, determined theMinimum Inhibitory Concentration (MIC) of frequently seen dermatophyte, andperformed molecular identification of the common dermatophytes.Method: 1. Etiological analysis of clinical specimens: This study included 120 cases ofonychomycosis with age from 18 to 70 years old (mean age: 38 years old). The sampleswere collected from different districts of Qingdao region (eight. departments ofdermatology in different hospitals). We used the KOH methods under microscopecombined with cultivation on the Mycosel and PDA medium methods to isolate thedermatophytes. And then we performed etiological identification of the isolateddermatophytes using flat plate and microculture methods. 2. Analysis of antifungalsusceptibilities of the common dermatophytes: To determine the Minimum InhibitoryConcentration (MIC) with the National Committee for Clinical Laboratory Standards(NCCLS) approved standard broth microdilution method (M38-A). 3. Molecularidentification of the common dermatophytes: Using the method of random amplificationof polymorphic DNA (RAPD) and a simple repetitive oligonucleotide (GACA)₄ toamplify the DNA of Trichophyton rubrum, Trichophyton mentagrophyte,Epidermophyton floccosum and Microsporum gypseum. The amplified products aredetected by the agarose gel electrophoresis; the dermatophytes can be distinguishedaccording to the banding patterns of their DNA.Results: 1. Etiological analysis of clinical specimens: Four kinds of different strains ofdermatophytes (Trichophyton rubrum, Trichophyton mentagrophyte, Epidermophytonfloccosum and Microsporum gypseum) were isolated from the 120 specimen. 2. Analysis of antifungal susceptibilities of the common dermatophytes: Terbinafine effectivelyinhibited all the dermatophytes tested, and with good reproducibility and feasibility, theM38-A scheme can be used for the antifungal susceptibility test to dermaphytes in thelaboratories. 3. Molecular identification of the common dermatophytes: The randomamplification of polymorphic DNA for the identification of common species ofdermatophytes is a stable, prompt and sensitive method.Conclusion: 1. Etiological analysis of clinical specimens: The KOH methods undermicroscope combined with cultivation on the Mycosel and PDA medium methods canisolate the dermatophytes successfully. We identified four kinds of dermatophytesetiologically using flat plate and on the Mycosel and PDA medium. 2. Analysis ofantifungal susceptibilities of the common dermatophytes: With good reproducibility andfeasibility, the M38-A scheme can be used for the antifungal susceptibility test todermaphytes in the laboratories. And this may instruct the clinical treatment and new drugevaluation. 3. Molecular identification of the common dermatophytes: Our resultsdemonstrate that it's possible for the method of random amplification of polymorphicDNA (RAPD) and a simple repetitive oligonucleotide (GACA)₄ to unambiguouslydiscriminate most strains of common dermatophytes. RAPD technique has a wide rangeof current and potential applications to pathogenic fungi, such as clarifying taxonomicquestion, performing epidemiological studies and improving the diagnosis of superficialmycotic diseases...