Indian Journal of Medical Microbiology Medknow Publications on behalf of Indian Association of Medical Microbiology ISSN: 0255-0857 EISSN: 1998-3646 Vol. 24, Num. 3, 2006, pp. 212-215

Indian Journal of Medical Microbiology, Vol. 24, No. 3, July-September, 2006, pp. 212-215

Brief Communications

Prevalence of dermatophytes and other fungal agents isolated from clinical samples

Kannan P, Janaki C, Selvi GS

Department of Dermatology, Madras Medical College and Research Institute, Chennai - 600 003, Tamilnadu
Correspondence Address:Department of Dermatology, Madras Medical College and Research Institute, Chennai - 600 003, Tamilnadu, dr_cjanaki@yahoo.com

Code Number: mb06061

Abstract

The common cause of skin infections are dermatophytes and opportunistic fungi. Aim of this study was to isolate and identify the fungal agents from clinical samples from patients with different mycoses. Clinical samples from 165 patients were subjected to potassium hydroxide (KOH) examination and culture isolation; causative agents were identified macroscopically and microscopically. All the 165 specimens were KOH positive and 110/165 (66.7%) samples were culture positive. Of these, highest isolation rate was obtained in opportunistic mycoses such as candidiasis (29/29, 100%). Dermatophytes were isolated in 53/80 (66.3%) specimens and Trichophyton rubrum was the commonest isolate in skin samples (17/24) among the patients suffering from dermatophytosis. Phaeoannellomyces wernecki was isolated in a patient suffering from tinea nigra. The study signifies the importance of mycological examination in the diagnosis of various mycoses for their effective management.

Keywords: Clinical samples, dermatophytes, opportunistic fungi, prevalence

Although fungi are world wide, only few of them are considered pathogenic. The pathogenic fungi may give rise to infections in animals and human beings. Most of the agents cause infection of the superficial layers of the integument and only very few give rise to systemic involvement. Recently there has been an increase in the incidence of fungal infections. This increase may be a result of frequent usage of antibiotics, immunosuppressive drugs and various conditions like organ transplantations, lymphomas, leukemia and human immunodeficiency virus (HIV) infections.[1]

Skin infection due to dermatophytes has become a significant health problem affecting children, adolescents and adults. Mycetoma caused by filamentous fungi (Eumycotic mycetoma) and filamentous bacteria (Actinomycotic mycetoma) need to be differentiated by culture studies. A correct diagnosis is important to initiate appropriate treatment and also essential for epidemiological purposes. In the background of immunosuppression, detection of these agents becomes mandatory for they effective management of mycoses to prevent further invasions. The present study was undertaken to isolate various fungal agents causing mycoses among the patients attending mycology section of the Department of Dermatology, Madras Medical College attached to Government General Hospital, which has got an average new outpatient turnover of 7000-8000 per year.

Materials and Methods

This study was undertaken for a period of two years from January 2001 to December 2002. All the clinically suspected 165 cases were subjected to mycological work up. The specimens included skin scales, hair, hair roots and pus in cases of superficial mycoses. Biopsy tissue and grain were the specimens in deep mycoses.

Microscopic examination
Direct microscopic examination was undertaken in 10% potassium hydroxide (KOH) wet mount for the specimens of skin scales, pus crust, biopsy tissue and grains, while 40% KOH was employed for hair and nail speicmens.[2] Grains of mycetoma were also subjected to Gram stain and modified Ziehl Neelsen stain (1% H ₂ So ₄ ).

Culture study
The KOH positive cases were subjected to culture study, scraping site was cleaned aseptically with 70% ethanol and the scales were collected in a sterile slide with the help of sterile scalpel. The culture was performed in two different sets of antibiotic incorporated Sabouraud dextrose agar (SDA) media, one with chloramphenicol 50 mg/L and the other with cycloheximide 500 mg/L and in addition to chloramphenicol.[2] The culture tubes were incubated at 30oC and the culture growth was observed and the tubes were discarded only after six weeks in the absence of growth.

The mycological identification was based on macroscopic and microscopic examination of the culture isolates. The macroscopic examination of dermatophytes was characterized by duration of growth, surface morphology and pigment production on the reverse. Commeal agar (CMA) was used to differentiate Trichophyton rubrum from T. mentagrophytes based on pigment production on the media. In addition, hair perforation studies were carried out to distinguish between these two species.[2] The microscopic examination of fungal growth was observed with lactophenol cotton blue stain. Nature of mycelium and conidia formation (macro and micro conidia) helped to differentiate various genera and species.

Budding yeast cells of Candida spp. were identified microscopically. Candida species were classified as albicans and non-albicans group by the production of the chlamydospores on corn meal agar and germ tube formation. Olive oil (2%) was over laid on the media for the isolation of Malassezia spp. in clinically diagnosed cases of pityriasis versicolor. Plain SDA medium was used in cases of pityriasias versicolor. Plain SDA medium was used in cases of mucorales and eumycotic species. Lowenstein-Jensen medium was used for the primary isolation of the agents in actinomycetoma.[2] Eumycotic agents were confirmed based on microscopic observation of morphology and conidilogenesis.

Results

Eighty out of 165 cases (48.5%) were dermatophytoses, 39 (23.6%) were pityriasis versicolor, 29 (17.1%) were condidiasis and 12 (7.1%) were cases of mycetoma. Tinea nigra was the clinical diagnosis in one and nondermatophyte onychomycoses was suspected in 4 (2.4%) patients. Specimens from all these cases were KOH positive [Table - 1]. Those patients with actinomycotic mycetoma showed gram-positive filaments of actinomycetes. On the whole 110 (66.7%) cultures were obtained from the KOH positive specimens. Isolation of opportunistic pathogens was 29/29 (100%) in candidiasis [Table-2].

Among dermatophytoses 24 isolates were obtained from skin scales, 25 from scalp and scalp hair and four from nail clips. The dermatophytes isolated were T. rubrum, T. violaceum , each in 21 specimens, T. mentagrophytes in six, T. simii in three and Epidermophyton floccosum in two specimens [Table-3]. T. Rubrum was isolated from skin scales in 17 (81%) and two (8.3%) each from scalp/scalp hair and nail. Candida spp. were isolated from 29 cases, of which 26 (89.7%) were from HIV patients presenting with oral candidiasis and rest were women with vulvovaginal and intertrigenous candidiasis, Candida spp. (non albicans species) were 17 (58.6%) and C.albicans were 12 (41.4%).

Malassezia species were isolated from 22 patients with pityriasis versicolor with 14 samples from chromic type and 8 from achromic type. Phaeoannellomyces wernecki was isolated from a patient with tinea nigra. Non dermatophytes such as Scopulariopsis brevicaulis and Helminthosporium spp. could be isolated from 2 out of 4 patients suspected with non-dermatophyte onychomycoses.

Of the 12 cases of mycetoma, nine had actinomycotic disease while three had eumycotic disease. Isolates could be obtained only from the cases of eumycotic and all the three isolates were identified as Madurella mycetomatis.

Discussion

Of the total number of 165 KOH positive specimens, only 110 (67.1%) isolates could be obtained. The isolation rate of opportunistic mycoses was more i.e., 29/29 (100%) in candidiasis. The higher load of the organism in the immunocompromized background could be the reason for such higher isolation rate. Among 80 patients with dermatophytosis isolates could be obtained from 53 (66.3%) patients. The isolation rate in this study seemed to be higher when compared to various other studies where it has ranged from 45.3-52.2%.[3],[4],[5] More isolates could be obtained from scalp/scalp hair compared to ski scales and the isolates were least from nail specimens. Among the cases of mycetoma, the isolation rate was much lower (3/12) comprising only of eumycotic agents.

T. rubrum was the chief islate form skin scales (17/24) in the present study similar to many other reports.[6],[7] This is the commonest agent islated form glabrous skin of the body, groin folds and the feet.[8] T. mentagrophytes was the second common isolate from the body site 4/24 (16.7%) as has been observed in other studies.[9] E. floccosum was isolated from two specimens obtained from the skin and this was the third common isolate from the glabrous skin.[10]

T. violaceum seemed to be the chief isolate from the scalp/scalp hair (20/25). This agent is still the commonest isolate from cases of tinea capitis in India.[6] This agent was also isolated from an specimen of skin scales (1/24). T. simii, the zoophilic species, could be isolated from scalp/scalp hair and this formed the second common isolate from the scalp 3/25 (12%). The prevalence of the T. simii among dermatophytes in general has been observed to be 1 %.[11] The prevalence of T. simii in tinea capitits was found to be 1.4% in Tamilnadu, India[12] and as high as 10% has been reported from Sri Lanka.[13] T. rubrum was the least common isolate (2/25) from the scalp and this considered to be the common agent causing glabrous type of tinea capitis which is usually encountered in adults.[8] The isolates from the nail specimens were T. rubrum (2/4) and T. mentagrophytes (2/4) and these agents are the common species infecting the nail.[6]

Malassezia spp. were isolated from 22/39 (56.4%) samples and the isolates were more in chronic type 14/22 (63.6%) than achromic type 8/22 (36.4%). This was probably because of more number of chronic pityriasis veriscolor included in the study group. The isolation rate of this agent in the present study seemed to be much higher compared to a study wherein a low culture positivity (27.8%) has been reported.[5] Among the Candida spp. non albicans group was predominant when compared to C. albicans group because of more number of HIV patients included in the study.

S. brevicaulis and Helminthosporium spp. could be isolated from non dermatophyte onycomycoses and these are rare isolates that infect the nail.[14] A rare and unusual human pathogen of P. werneckii was isolated from a case of tinea nigra. There have been sporadic reports of this condition from South India.[15]

The eumycotic and actinomycotic mycetoma cannot be differentiated on clinical grounds. Histopathological diagnosis is also inconclusive with regard to the agents causing the disease and mycological examination plays an important role in the diagnosis. M. mycetomatis was isolated from all three eumycotic disease and it is considered as the commonest agent involved in eumycotic mycetoma.[16] Isolation is difficult in actinomycotic disease which might involve culture in enriched media and biochemical procedures.

The present study shows that mycological examination of causative agent is necessary to differentiate and treat dermatophyte and non-dermatophyte onychomycosis. Isolation rate of all the fungi has been observed to be much higher in this study except with mycetoma and it can be concluded that isolation rate can be enhanced with aseptic and proper culture techniques.

References

Copyright 2006 - Indian Journal of Medical Microbiology

The following images related to this document are available:

Photo images