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JOURNAL OF CULTURE COLLECTIONS

Volume 5, 2006-2007, pp. 85-89

 

 

 

INCIDENCE OF MYCOTIC INFECTIONS IN DIABETIC FOOT TISSUE

 

Seema Nair1*, Sam Peter1, Abhilash Sasidharan1, Sujatha Sistla2 and

Ayalur Kodakara Kochugovindan Unni1

 

1Biotechnology Lab, Research C= oordination Division, AIMS, Kochi, Kerala, India;

2Microbiology Department, Jawa= harlal Nehru Institute of Postgraduate Medical Education

and Research (JIPMER), Pondicherry= , India

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

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Summary

The objective of th= e study was to investigate the incidence of fungal pathogens in diabetic foot infections. A total of 74 Type II diabetic patients with non-healing diabet= ic foot infections were recruited for the study. Among the diabetic patients 65 % (48/74) had yeast and mold infections. Pathogenic yeasts were not= ed in 77 % of the patients of which Candida species was predominant (93 %). The major Candida <= /span>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 Tric= hosporon cutaneum and T. capitatum. Trichophyton spp. was the only dermatophy= tic 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 antifun= gal therapy.

 

Key words: diabetic foot, yeast= s, fungi, infection, pathogenic.

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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. Diabe= tes and foot problems are almost synchronous [4, 8, 20]. Diabetic foot infectio= ns frequently result in morbidity, hospitalization and amputations. The bacteriology of diabetic foot ulcers has been studied by numerous investiga= tors [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 ulcera= ted deep tissue samples.

 

Materials and Methods

Sample collection. The study was carried out over a period of 10 months, from Dec= ember 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 t= he study. Grades were defined as: Grade 2 - deep ulcer often infected but no b= one involvement; Grade 3 - deep ulcer, abscess formation and bone involvement a= nd 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 (ta= king 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 a= nd identification of the cultures. Yeast like gr= owth 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 macro= scopic 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 be-low the spore that actively participates in t= he 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, wool= ly, or cottony in texture. The colonies are initially white and become blue gre= en or gray green. The plate reverse is usually pale to yellowish. At 37 ºC the organism grows as a yeast like colony.

 

Results

Am= ong 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, Aspergill= us spp, Fusarium spp, Penicillium spp, and B. rana= rum. 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 a= dopted 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 isolat= ed were Trichosporon cutaneum and T. capitatum.

 

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

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Yeast species

Germ tube

Urease

Sugar fermentation

Sugar assimilation

Tetrazolium reduction

CMA + Tween 80

Glu

Suc

Lac

Mal

Glu

Suc

Lac

Tre

Raf

Can= dida tropicalis

-=

-=

+=

+=

-=

+=

+

+

-

+

-

Maroon

No arthroconidia. Blastoconidia. Produced randomly along hyphae or pseudohyp= hae. Hyphae or pseudohyphae branched.

Can= dida albicans

+

-

+

-

-

+

+

+

-

+

-

Pale pink

No arthroconidia.
Spherical clusters of blastoconidia at regular intervals on pseudohypae. Chlamydospores present on hyphae.

Can= dida krusei

-=

-=

+=

-=

-=

-

+

-

-

-

-

Pink and dry

No arthroconidia.
Elongated clusters of blastoconidia occur at septa of pseudohyphae. Branc= hed pseudohyphae.

Gandida guillermondi

-

-

+

+

-

-

+

+

-

+

+

Pink and pasty

-

Candida parapsilosis

-

-

+

-

-

-

+

+

-

+

-

Rose pink

No arthroconidia. Blastoconidia present but not characteristic.
Giant hyphae with sage brush appearance.

Tri= chosporon spp=

-=

-=

+=

+=

+=

-=

+

+

+

+

+

 

Numerous arthroconidia. Blastoconidia may be present, septate hyp= hae present.

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

 

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

= Sl. no.

Candida species

= Percentage of samples

= 1.

Candida albicans

= 46

= 2.

= Candida tropicalis<= /o:p>

= 27

= 3.

= Candida parapsilosis

= 17

= 4.

= Candida guillermondi

= 5

= 5.

= Candida krusei

= 5

 

The mold species were identified on the basis of their microsc= opic and macroscopic appearance as described by Chander [5]. The highest inciden= ce was Aspergillus spp (65 %) (Table 3). Among the Aspergillus sp., A. flavus (60 %) and A. fumigatus <= /i>(40 %) were observed. 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 r= are occurrence. Out of the-se 13 % showed only sterile growth. T= richophyton is a dermatophytic fungus which might have appeared in the deep tissue samp= le due to surface contamination

 

Table. 3. Mold spec= ies isolated from diabetic foot tissues.

= Sl. no.

Mold species=

= Percentage occurrence

= 1.

Aspergillus spp=

= 65

= 2.

= Fusarium solani

= 9

= 3.

= Penicillium marneffei

= 9

= 4.

= Basidiobolus ranarum

= 4

= 5.

= Sterile hyphae

= 13

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D= iscussion

Foot infections ar= e 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 antifungal 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 t= hat among the fungal pathogens isolated from deep tissues, 93 % where C= andida spp. The investigators could also identify Candida species like C. parapsilosis, C. albicans, C. tropicalis and C. glab= rata from the infected tissues.

The presence of A. = flavus and F. solani has been reported by some workers on diabetic f= oot 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 rare= ly reported in India. P. marneffei infections have been documented in HIV-infected individ= uals 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 pres= ent study signifies the need of a mycological evaluatuion of non-healing diabet= ic foot and prudent antifungal treatment based on the culture results rather t= han depending on broad spectrum antifungals for cure.

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

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References

1.      Bader, M., A. K. Jafri, T. Krueger, V. Kumar, 2003. Scand. J. Infect. Dis.., 35 (11-12), 895-896.

2.&n= bsp;     Bamberger, D. M., G. P. Daus, D. N. Gerding, 1985. = Amer. J. Medicine, 83, 653-60.

3.      Blazer, K., M. Heidrich, 1999. Chirurg., 70 (7), 831-844.

4.      Chander.J., 1995. A text book of medical mycology, 3<= sup>rd edn, New Delhi, India: Inter print.

5.      Chincholikar, D. A, R. B.= Pal, 2002. Indian J.= Pathol. Microbiol., 45 (1), 15-22.

6.  Cooper, C. R. Jr., M. R. McGinnis, 1997. Arch. Pathol. Lab. = Med., 121, 798-804.

7.  Forbes, B. A., D. F. Sahm= l, A. S. Weissfeld, 2002. Bailey and Scotts diagnostics microbiology, 11th edn, London: Mosby.

8.  Frykberg, R. G, 1998. = J. Foot Ankle Surg., 37 (5), 440-446.

9.&n= bsp; Gerding, D. N, 19= 95. Clin. Infect. Dis., 20, S283-288= .

10.  Heald, A. H, D. J. Ohallo= ran, K. Richards, F. Webb, S. Jenkis, S. Hollis, D. W. Denning, R. J. Young, 200= 1. Diab. Med., 18 (7), 567-57= 2.

11.  Lai, C. S, S. D. Lin, C. = K. Chou, H. J, Lin, 1993. Plast. Reconstr. Surg., 92 (3), 532-536.

12.&= nbsp; Lipsky, B. A, R. E. Pe= coraro, S.A. Larson, 1990. Arch. Intern. Med., 150, 790-797.

13.&= nbsp; Louie,T. J, J. G. Bart= lett, F. P. Tally,1976. Ann. Intern. Med= ., 85, 461-463.

14.  Missoni, E. M., D. RadeNa= deral, S. J. Chromatogr, 2005. Analyt. Technol. Biomed. Life Sci., 822 (1-2), 118-123.

15.  Peterson, L. R, L. M. Lis= sack, K. Canter, 1989. Amer. J. Med., 86, 801-807.

16.  Ranjana, K. H., K. Priyok= umar, T. J. Singh, C. C. Gupta, L. Sharmila, P. N. Singh, 2002. J. Infect., 45, 26= 8-271.

17.  Reyes, C. V., J. W. Rippo= n, 1984. Hum. Pathol., 15 (1), 89-91.

18.  Sapico, F. L, J. L. = Witte, H. N. Canawati, 1984. Rev. Infect. Dis., 6 (1), S171-176.

19.  Sharp, C. S., A. N. Bessm= an, W. Wagner Jr, 1979. Surg. Gynecol. Obstet., 149, 217-219.

20.  Shea, K. W., 1999. Pos= tgrad. Med., 106 (1), 85-94.

21.  Wheat, L. J, S. D. Allen,= M .Henry, 1986. Arch. Intern. Med., 146, 1935-1940

 

 

 

РАЗПРОСТР = 40;НЕНИЕ НА ГЪБНИ ИНФЕКЦИИ В ТЪКАНИ

НА ДИАБЕТНО БОЛНИ

 =

Сиима Наир1*, Сам Петер1, Абхилаш Сасидаран1, Суйата Систла2,

Айалур Кодакара Кошуговиндk= 2;н Уни1

 =

Резю= ;ме

Целта на работата = 077; да се изследва разпростраl= 5;ението на гъбни патогени пр = 80; диабетни инфектиранl= 0;я по краката. В проучванетl= 6; са включени 74 болни от диабет тип II пациенти с н= 077;излекувани диабетни инфекции по краката. От д&#= 1080;абетно болните пациенти 65 % (48/74) имат инфекц = 80;и от дрожди и плесени. Патогенни дрожди са ус= 090;ановени при 77 % от тях, като преобладавk= 2; родът Candida (93 %). Основните видове Candida са C. albicans (49 %), C. tropicalis (23 %), C. parapsilosis (18 %), C. guillermondi (5 %) и C. krusei (5 = ;%). Другите изолирани видове дрожди са Trichosporon cutaneum и T. capitatum. Trichophyton spp. е единственаm= 0;а установена дерматофитl= 5;а гъба. Плесен= 080; са открити при 38 % от заразените пациенти, като преобладавk= 2; род  Aspergillus (72 %). Другите изолирани плесени са Fusarium solani, Penicillium marne= ffei и Basidiobolus ranarum. Резултатитk= 7; от изследванеm= 0;о показват необходимоl= 9;тта от микологичнk= 2; оценка на незаздравяk= 4;ащите тъкани по краката на д= 080;абетно болни и подходяща антигъбна терапия.

 

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