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Isolation and Identification of Dermatophytes from Clinical Samples – One Year Study

Dermatophytes are keratinophilic and keratinolytic fungi. They are responsible for dermatophytosis which are superficial mycosis affecting skin (Tinea corporis, Tinea cruris, Tinea pedis), hair (Tinea capitis), beard (Tinea barbae), nails (onychomycosis or Tinea unguium). Dermatophytosis is a nonfatal disease except in extremely rare cases of Hadida and Schousboe’s dermatophytic disease. 1 Infection generally involves skin and restricted to the cornified layers because of the inability of the fungi to penetrate the deeper tissues or organs of immunocompetent hosts. 2 Dermatophytes are a group of closely related organisms that can use keratin as a nitrogen source. There are three genera of Dermatophytes; Trichophyton, Microsporum and Epidermophyton. International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 6 Number 11 (2017) pp. 1276-1281 Journal homepage: http://www.ijcmas.com


Introduction
Dermatophytes are keratinophilic and keratinolytic fungi. They are responsible for dermatophytosis which are superficial mycosis affecting skin (Tinea corporis, Tinea cruris, Tinea pedis), hair (Tinea capitis), beard (Tinea barbae), nails (onychomycosis or Tinea unguium). Dermatophytosis is a nonfatal disease except in extremely rare cases of Hadida and Schousboe's dermatophytic disease. 1 Infection generally involves skin and restricted to the cornified layers because of the inability of the fungi to penetrate the deeper tissues or organs of immunocompetent hosts. 2 Dermatophytes are a group of closely related organisms that can use keratin as a nitrogen source. There are three genera of Dermatophytes; Trichophyton, Microsporum and Epidermophyton.
Because of difficulty in clinically differentiating dermatophytosis from other non-mycoticdermatosis, particularly in the dystrophic nails, it is important to establish an accurate diagnosis. Knowledge of the zoophilic or anthropophilic origin of the dermatophyte may allow setting up prophylactic measures 1 .
Any clinical diagnosis needs to be supported by laboratory diagnosis. Culture is a necessary adjunct to direct microscopic examination for definitive identification of etiological agent and in many instances the choice of therapy depends upon the specific identification of the invasive mould. This is especially important in nail and skin infection, often caused by non-dermatophytic filamentous fungi, which are often resistant to usual dosage of the therapy used for dermatophytic infections. Before starting treatment for dermatophytosis, it is essential to establish the diagnosis of the disease, so that specific therapeutic modalities can be monitored during the course of the treatment. 3 Rapid identification of dermatophyte species and knowledge of their host preference and ecology play an important role in epidemiology, public health and infection control.
The varied clinical presentation of Tinea, which results in delay in diagnosis, poor compliance in follow up of cases, and consequently spread of infection in the community has rekindled interest in rapid identification of species 4 .
The main aim of this study is to isolate and identify the fungi causing dermatophytosis.

Materials and Methods
This study of dermatophytosis was carried out in the Department of Microbiology, KLE'S Dr. Prabhakar Kore Hospital and Medical Research Centre Belgaum, over a Period of one year.

Inclusion criteria
All skin, hair and nail samples from clinically suspected cases of dermatophytosis were included in the study.

Exclusion criteria
Patients who are already on treatment for dermatophytosis were excluded from the study

Statistical Analysis
Was done using percentage and chi-square test.

From the skin
The affected area was first thoroughly swabbed with 70% alcohol to remove surface contaminants. After the alcohol dries, the skin scrapings were collected from the border of the active lesions with a beard parker blade in a sterile black paper envelop.

From the scalp
Hairs from the scalp were epilated with a flame sterilized forceps and the active border area was scraped with a scalpel to collect epidermal scales on a sterile black paper envelop.

From the nail
The affected nail was first cleaned with 70% alcohol. The upper portion of the infected nail was scraped away and material were collected from the deeper part of the distal end of the nail on to sterile black paper envelop.
The samples of skin and hair were subjected to 10% KOH and 20% KOH preparation for nail. After 15-20 minutes, the specimen was examined for the presence of hyphae or arthrospores.

Culture
The specimens collected were inoculated on to Sabourauds Dextrose agar containing Chloramphenicol and Cycloheximide irrespective of demonstration of fungal elements on KOH mount. Each sample was inoculated into two tubes. One tube with antibiotic and other without antibiotic and were incubated at 27 0 C. The cultures were examined daily for a period of 4 weeks. Slopes showing no growth for4 weeks were discarded. If growth was obtained on Sabourauds Dextrose agar, identification was made based on colony morphology, microscopic appearance and other relevant tests. The isolates were inoculated on potato dextrose agar for better conidiation.

Macroscopic examination of culture
The growth on Sabourauds dextrose agar was observed to study the colony morphology, the color of the surface, the reverse of the colony, the texture of the surface, the topography and the rate of growth.
Microscopic Examination of Culture was done using LPCB preparation and slide culture was done in case the morphology was not clear in LPCB preparation.

Urease test
This test is done on Christensen's medium. This is done to differentiate T. mentagrophytes from T. rubrum. T. mentagrophytes, hydrolyse urea and the medium becomes deep red while T. rubrum does not hydrolyse urea. Urea broth may also be used which is more sensitive

Results and Discussion
Out of 100 cases included in our study male preponderance of 73 (73%) cases was seen. 27 (27%) were females. Incidence of Dermatophytosis was high in males and male to female ratio of 2.7:1.
Tinea cruris was the most common clinical type of dermatophytosis with incidence of 32, Tinea capitis was seen in 3 cases, Tinea unguium was seen in 20 cases, Tinea pedis was seen in 12 cases, Tinea manuum was seen in 2 cases and 31 cases presented with Tinea corporis (Table 1).
Tinea cruris was the most common and Tinea manuum was the least common clinical type of dermatophytosis encountered in our study.
Out of 100 clinical samples 58 samples were culture positive and KOH positive. 19 samples were Culture positive and KOH negative. 18 samples were Culture as well as KOH negative. 5 samples were KOH positive and culture negative.
In present study 100 clinically diagnosed cases of Dermatophytosis were studied. Of them 77 were skin scrapings, 20 were nail clippings and 3 were hair stubs. Out of these samples, dermatophytes were isolated in 63 cases.
Present study shows that out of 100 clinically diagnosed cases of dermatophytosis 63 were affected by dermatophytes. Our results are comparable to studies conducted by Abu Elteen et al., (1999) where in isolation rate was 56.8%. In a study conducted by Bindu et al., (2002) and Seemabose et al., (2013) isolation rate was 45% and 60.67% respectively, which is again comparable to results of present study. Extreme variation in isolation rate in few other studies may be due to differences in techniques used for sample collection, culture and other methods of identification.
In present study most common type of dermatophytosis studied was Tinea cruris with incidence of 32 (32%). Study conducted by Sanchita Karmakar et al., (1995) also showed similar results.

33% 20%
In present study T.rubrum was the commonest dermatophyte isolated from 33 clinical samples. Second most common dermatophyte isolated was T. mentagrophyte with the incidence of 20. In various studies conducted by Agarwalla et al., (2001), Asticcioli et al., (2008), Bindu et al., (2002) and Sumana et al., (2002) T. rubrum was the commonest isolate with incidence ranging from 42.3% to 66.2%. The present study was comparable with Seema Bose et al., (2013). Some species of dermatophytes are endemic in certain parts of the world. Various studies done in India have shown T. rubrum as the commonest isolate (Table 2).
Dermatophytosis is the most common type of cutaneous fungal infection. It is very common in our country with several contributing factors like hot humid climate, poor hygiene, increased outdoor activities, occupational trauma and immunosuppression.
The incidence of Dermatophytosis is increasing in India due to widespread and indiscriminate use of corticosteroids and antifungal agents without appropriate microbiological investigations.
The overall isolation rate of dermatophytes was 63%. Tinea cruris accounted for maximum number of cases