Determination of dermatophyte and other fungus agents in conventional and PCR methods in skin scraping samples taken from patients with suspected cutanus fungi infection

Dermatophytosis is a disease of global importance caused by pathogenic keratinolytic fungi called dermatophytes. Based on the their morphological features, dermatophytes are divided into three genera as Microsporum, Epidermophyton and Trichophyton, while according to their natural resources as anthropophilic (human origin), zoophilic (animal origin) and geophilic (soil origin) dermatophytes. The easiest, fastest and the most practical examination method in fungal infections of keratinized tissues is the direct microscopic examination of the samples. Since direct microscopic examination depends on the sampling quality, the experience and skill of the microbiologist, its sensitivity varies. Culture aids direct microscopy and should be used in the diagnosis of all superficial fungal infections and dermatophyte infections requiring any systemic treatment. However, mycological cultures and advanced species-level identification methods used in the diagnosis of dermatophytosis delay the diagnosis. In this study, we aimed to detect dermatophytes and other fungal agents by conventional and PCR methods in skin scraping samples taken from patients who applied to the dermatology outpatient clinic with the suspicion of cutaneous fungal infection. Hair, scalp, skin scraping and nail samples taken from 130 patients who applied to the Dermatology Outpatient Clinic of Fırat University Medical Faculty Hospital between May 2020 and July 2020 with a preliminary diagnosis of dermatophytosis were included in the study. Each clinical sample was first prepared with 20% KOH for direct microscopic examination and examined under the light microscope using eyepieces with 10x) and then 40x magnification. In the examination, fungal hyphae/micelle and spores were searched. For mycological culture of clinical samples, the samples taken in sterile Petri dishes were inoculated into SDA, antibiotic SDA and PDA media. Colonies in cultures grown macroscopically in SDA and/or PDA medium at the end of a 4-week period were evaluated in terms of texture, surface appearance, surface and base pigmentation of colonies. PDA medium was evaluated for its ability to produce pigmentation on the base and surface of the colony. The CTAB method was used for DNA isolation from colonies grown in culture. In the study, PCR was applied with primers targeting ITS (ITS1/ITS4) gene regions. Sequence analysis was applied to cultures defined as mold or yeast by conventional methods after PCR analysis. Identification according to the sequences obtained was made by comparing the nucleotide sequences in the gene bank with the nucleotide BLAST program. Fungal hyphae and/or spores were found in direct microscopic examination of 102 (78.4%) of 130 clinical samples taken from patients with prediagnosis of dermatophytosis. Fungal growth was observed in mycological cultures of a total of 106 (81.5%) samples, including samples that had been evaluated as culture -positive (n: 99 ;76%) , and culture-negative (n:7 :25%) under direct microscopic examination. In the distribution of dermatophytes identified using the further identification methods of the cultures, most frequently T. rubrum (49%) species was detected. Results of the identification of the strains grown in cultures by PCR method using primers targeting ITS gene regions were not different from those identified by conventional methods excepting only strain no. 36 defined as T. mentagrophytes by the conventional method was revealed as T. rubrum by PCR. In our study, agents of dermatophytosis isolated from culture could be identified at the species level quickly by using molecular diagnostic methods before applying advanced identification methods. We have concluded that molecular methods can be an appropriate diagnostic tool in the routine diagnosis of dermatophytoses in laboratories with suitable infrastructure. Key words: dermatophytosis, Trichophyton, Epidermophyton, Microsporum, PCR