Journal of Culture Collections National Bank for Industrial Microorganisms and Cell Cultures ISSN: 1310-8360 Vol. 5, Num. 1, 2006, pp. 85-89

Journal of Culture Collections, Volume 5, 2006-2007, pp. 85-89

INCIDENCE OF MYCOTIC INFECTIONS IN DIABETIC FOOT TISSUE

Seema Nair^1*, Sam Peter¹, Abhilash Sasidharan¹, Sujatha Sistla² and Ayalur Kodakara Kochugovindan Unni¹

¹Biotechnology Lab, Research Coordination Division, AIMS, Kochi, Kerala, India;
²Microbiology Department, Jawaharlal Nehru Institute of Postgraduate Medical Education and Research (JIPMER), Pondicherry, India

*Corresponding author, e-mail: seemanair@aims.amrita.edu

Code Number:cc06012

Summary

The objective of the study was to investigate the incidence of fungal pathogens in diabetic foot infections. A total of 74 Type II diabetic patients with non-healing diabetic foot infections were recruited for the study. Among the diabetic patients 65 % (48/74) had yeast and mold infections. Pathogenic yeasts were noted in 77 % of the patients of which Candida species was predominant (93 %). The major Candida species isolated were C. albicans (49 %), C. tropicalis (23 %), C. parapsilosis (18 %), C. guillermondi (5 %) and C. krusei (5 %). The other yeast species isolated were Trichosporon cutaneum and T. capitatum. Trichophyton spp. was the only dermatophytic fungus found. Molds were isolated from 38 % of the infected patients of which Aspergillus species predominated (72 %). The other molds isolated were Fusarium solani, Penicillium marneffei and Basidiobolus ranarum. The results of the study indicate the need for mycological evaluation of the non-healing diabetic foot tissues and appropriate antifungal therapy.

Key words: diabetic foot, yeasts, fungi, infection, pathogenic.

Introduction

Patients with diabetes represent a unique group of individuals who appear more prone to develop infections than others. Several mechanisms have been proposed to explain the association between diabetes and infections. However, few conclusive studies exist and a considerable debate is going on regarding the evidence for this predisposition. Diabetes mellitus is a chronic disorder that affects a large segment of the human population and is a major public health problem. Diabetes and foot problems are almost synchronous [4, 8, 20]. Diabetic foot infections frequently result in morbidity, hospitalization and amputations. The bacteriology of diabetic foot ulcers has been studied by numerous investigators [2, 9, 12, 15, 19, 21]. However, there is a paucity of re-ports on the incidence of fungal pathogens in deep tissue samples.

The present study was undertaken in order to evaluate the incidence of pathogenic fungi in ulcerated deep tissue samples.

Materials and Methods

Sample collection. The study was carried out over a period of 10 months, from December 2004 to October 2005. Seventy four people with diabetes and a foot ulcer of grade 2 – 4, attending the Podiatry surgery clinic were enrolled in the study. Grades were defined as: Grade 2 - deep ulcer often infected but no bone involvement; Grade 3 - deep ulcer, abscess formation and bone involvement and Grade 4 - localized gangrene. Patients with superficial ulcer or me-re abrasions were excluded from the study.

Tissue specimens were obtained from the depth of the wound (taking aseptic precautions) after debridement. Samples were transferred to the laboratory within an hour in sterile containers. The necrotic areas of the tissues were mounted on KOH and also inoculated to Sabouraud Chloramphenicol Agar (SCA) and Sabouraud's Dextrose Agar (SDA) (Himedia Ltd, Mumbai). The samples were incubated both at room temperature (28 ± 2 ºC) and 37 °C for one month and evaluated daily for growth of fungal cultures.

Characterization and identification of the cultures. Yeast like growth on SCA was evaluated for germ tube formation, urease production, sugar fermentation (dextrose, maltose, sucrose, lactose), assimilation of sugars (dextrose, lactose, raffinose, sucrose and trechalose), Tetrazolium reduction, and microscopic and macroscopic appearance in slide culture and corn meal agar with Tween 80 [4, 7].

Fungal cultures were identified by microscopic (LCB) and macroscopic appearance [3, 5]. Velvety colonies on SDA with red pigment on reverse, tear drop microconidia and long pencil shaped macroconidia were identified as belonging to Trichophyton species. Basidiobolus ranarum shows a flat, yellowish-grey, glabrous, radially folded colonies covered by a fine, powdery, white surface mycelium on SDA after 17 days incubation at 26 °C. They have globose, one-celled conidia from a sporophore. The sporophore has a distinct swollen area just below the spore that actively participates in the discharge of the spore. Penicillium marneffei (thermally dimorphic) shows hyaline, smooth-walled conidiophores bearing terminal verticils of 3 to 5 metulae, each bearing 3 to 7 phialides. Conidia are globose to subglobose, 2 to 3 µm in diameter, smooth-walled and are produced in basipetal succession from the phialides. The colonies of P. marneffei are rapid growing, flat, filamentous, with wrinkled, folded surface and velvety, woolly, or cottony in texture. The colonies are initially white and become blue green or gray green. The plate reverse is usually pale to yellowish. At 37 ºC the organism grows as a yeast like colony.

Results

Among the 74 patients with diabetic foot infections from surgical units, 49 were male and 25 female patients, aged between 48 to 69 years. The fungal species isolated from diabetic foot ulcers were Candida spp, Trichosporon spp, Trichophyton spp, Aspergillus spp, Fusarium spp, Penicillium spp, and B. ranarum. Fungal pathogens were not detected in 26 patients. The incidence of yeast and/or mold infections was 65 % out of the 74 patients studied. Among the fungal cultures identified, 66 % were yeast isolates and 34 % mold like cultures.

Table 1 depicts the identification scheme for yeast cultures adopted in this study. Candida was the major isolated species (82%) (Table 2). Among the Candida species, C. albicans (46 %), C. tropicalis (27 %), C. parapsilosis (17 %), C. guillermondi (5 %) and C. krusei (5 %) were observed. The other yeast species isolated were Trichosporon cutaneum and T. capitatum.

Table 1. Identification scheme for yeast cultures [4, 7].

Legend: glucose (Glu), sucrose (Suc), lactose (Lac), maltose (Mal), trehalose (Tre), raffinose (Raf).

Table. 2. Candida species isolated from diabetic foot tissues.

The mold species were identified on the basis of their microscopic and macroscopic appearance as described by Chander [5]. The highest incidence was Aspergillus spp (65 %) (Table 3). Among the Aspergillus sp., A. flavus (60 %) and A. fumigatus (40 %) were ob-served. The other molds isolated were Fusarium solani (9 %), P. marneffei (9 %), and B. ranarum (4 %). P. marneffei was unique due to its dimorphic nature at different temperatures and also its rare occurrence. Out of these 13 % showed only sterile growth. Trichophyton is a dermatophytic fungus which might have appeared in the deep tissue sample due to surface contamination

Table. 3. Mold species isolated from diabetic foot tissues.

Discussion

Foot infections are a major cause of morbidity in people with diabetes. Devitalised tissue is the site where the microorganisms responsible for the non-healing ulcers inflict damage. Numerous investigations have been carried out on the bacteriology of diabetic foot ulcers [2, 9, 12, 15, 19, 21]. Most diabetic foot lesions are known to have a polymicrobial aetiology [13, 18, 21].

Though there are a few reports on the incidence of fungal pathogens in diabetic foot infections [1, 6, 11, 14], there is a paucity of published work on the incidence of fungal pathogens in deep tissue samples. The study conducted by Chincholikar and Pal [5] showed the presence of various fungal pathogens in diabetic foot ulcer tissues, among which Candida species preponderated. Heald et al. [10] has also reported the association of Candida spp with protracted ulceration in diabetic feet which improved the following systemic anti-fungal therapy.

The presence of various species of Candida (C. parapsilosis, C. albicans, C. tropicalis, C. famata and C. glabrata) was reported in diabetic patients with ulcer and in interdigital spaces of the same and/or the other foot by Misoni et al. [14]. This is in agreement with the findings of the present study showing that among the fungal pathogens isolated from deep tissues, 93 % where Candida spp. The investigators could also identify Candida species like C. parapsilosis, C. albicans, C. tropicalis and C. glabrata from the infected tissues.

The presence of A. flavus and F. solani has been reported by some workers on diabetic foot ulcer [1, 11]. In our study, among other mold species we also isolated A. flavus and F. solani from the infected tissues. Reyes and Rippon have reported a case of simultaneous aspergillosis and mucormycosis complicating diabetic foot gangrene [17]. P. marneffei has been rarely reported in India. P. marneffei infections have been documented in HIV-infected individuals from the northeastern part of India [16]. This infection has been predominantly reported from Southeast Asia [7] where it has been found to be the third most common illness that specifies the Acquired Immuno Deficiency Syndrome (AIDS). P. marneffei is pathogennic particularly in patients with AIDS and its isolation from blood is considered an HIV marker in endemic areas. The present study signifies the need of a mycological evaluatuion of non-healing diabetic foot and prudent antifungal treatment based on the culture results rather than depending on broad spectrum antifungals for cure.

Acknowledgement. We are grateful to the Podiatry Surgery Division, Endocrinology Department, AIMS, Kochi for providing the deep tissue samples for analysis and part of the funding for the successful completion of the work..

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