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Species distribution & antifungal susceptibility of oral Candida colonising or infecting HIV infected individuals.

Sir,

Oropharyngeal candidiasis (OPC) occurs in up to 90 per cent of HIV infected cases during the course of infection (1) and is considered as an important marker of the disease and its progression. Candida spp. that colonize the oral cavity are the source of yeasts that cause oral candidiasis (2). Although many studies have been conducted on oral carriages of Candida species both in healthy and immuno compromised individuals, but because of different criteria in recruitment and yeast assessment methods, results from these studies are not comparable (3). Though Candida albicans is the most frequently isolated species as coloniser and pathogen of the oral mucosa, other Candida species, such as C. tropicalis, C. krusei, and C. glabrata are recovered increasingly (4). OPC increases morbidity and reduces the quality of life of HIV/AIDS patients and therefore requires prompt diagnosis and adequate therapy (5). The azoles, particularly fluconazole, remain among the most common antifungal drugs, but their intensive clinical use for both therapy and prophylaxis has favoured the emergence of resistant strains. Thus, the present study was carried out to determine the species distribution and antifungal susceptibility profile of oral Candida isolates colonizing or infecting HIV infected individuals.

A total of 210 consecutive HIV positive individuals, attending National AIDS Research Institute (NARI) clinic at Pune, during January 2007--March 2009 were included in the study. During the same period, 125 consecutive HIV negative individuals who came for detection of their HIV serostatus without oral mucosal lesions were included as the control group. The study was approved by the Ethics committee of the institute. All participants were included in the study after obtaining their informed written consent. Individuals with diabetes mellitus or other systemic diseases, pregnant women, smokers, denture/orthodontic device users, and those under treatment with antimicrobials during the preceding three months were excluded.

HIV serostatus of the patients was determined by commercially available ELISA and Rapid antibody tests (Genetic system, Biorad Labs, USA and Tridot, J Mitra & Co., New Delhi), using National AIDS Control Organisation (NACO) recommended algorithm6. CD4 cell counts were measured by using a FACS count system (Becton Dickinson, Singapore BD).

Oral swabs were obtained by firmly swabbing the lesion site with sterile cotton swabs in case of symptomatic individuals and the dorsum of tongue and buccal mucosa in case of asymptomatic ones. The samples were immediately inoculated on Sabouraud dextrose agar (SDA) with chloramphenicol (0.05 g/l) and incubated at 370 [degrees] C for 2 days, and for additional seven days at 300 [degrees] C before being considered as negative. All isolates were identified by standard mycological techniques (7). Identification of all isolates was confirmed by API 20 C AUX (BioMerieux, France). The minimum inhibitory concentrations (MICs) of all Candida isolates from HIV positive individuals to fluconazole, ketoconazole and itraconazole were determined by the E test (AB Biodisk, Sweden) as per manufacturers' instructions. Breakpoint definitions for fluconazole and itraconazole MICs were based on Clinical Laboratory Standards Institute (CLSI) M270A2 (8) standard guidelines. Due to lack of consensual breakpoints for MICs of ketoconazole, arbitrary values based on an earlier study were used (9).

Statistical analysis was performed using SPSS software version 15.0 (SPSS Inc, USA). Chi square test was used to check the association between categorical variables. P<0.05 was considered statistically significant.

Of the 210 HIV positive individuals, 60 had lesions of oral candidiasis (symptomatic) and isolates were obtained from all while, of the 150 asymptomatic individuals, 88 (58.7%) were positive for oral carriage of Candida spp. Twenty eight (22.4%) of the HIV negative individuals showed candidial colonisation.

The Table shows the Candida spp. isolated from HIV positive individuals. C. albicans was the predominant isolate. Higher frequencies of non-albicans species were observed in HIV infected individuals with oral candidiasis (35%) as compared to the asymptomatic HIV positive (19.1%). In HIV positive asymptomatic group mixed candidial colonization was seen in 6 patients (6.81%). The Candida spp. isolated from the HIV negative individuals were C. albicans 21 (75%), C. tropicalis 1 (3.6%), C. krusei and C. glabrata 3 isolates (10.7% each). The range of CD4 in carrier and non-carrier HIV positive patients was 19-987 cells/[mm.sup.3] (median 194.5 cells/[mm.sup.3]) and 61-1105 cells/[mm.sup.3] (median 365 cells/[mm.sup.3]) respectively. All HIV positive individuals with oral candidiasis had CD4 counts less than 250 cells/[mm.sup.3]. There was a statistically significant association between oral candidiasis and low CD4 counts, (i><0.001). Of the 154 isolates obtained from HIV positive individuals, the MIC ranges were 0.032-256 [micro]g/ml for fluconazole, 0.004-32 [micro]g/ml for ketoconazole and 0.016-8 [micro]g/ ml for itraconazole. The Candida spp. showed an overall resistance of 14 per cent to one of the three azoles tested (Table). Of the 210 HIV positive patients, 36 (17.1%) had history of prior exposure to antifungals and 38 isolates were obtained from them (2 patients had mixed infection). Isolates from these patients exhibited greater resistance to azoles (26.3%) compared to isolates from the unexposed group (7.8%) (P< 0.006).

Various studies have shown the isolation rate of Candida ranging from 61-100 per cent in HIV infected individuals with oral candidiasis (10-13), while a wide range of oral carriage rates have been reported for asymptomatic HIV infected (11.0 - 96.0%) as well for HIV uninfected (10.0 - 68.0%) subjects (3). In our study, Candida was isolated from all symptomatic HIV positive individuals, whereas the oral colonization in the asymptomatic HIV infected and uninfected group was 58.7 and 22.4 per cent respectively. This variability in carriage rates reported may be caused by geographic differences, time of sampling or the different methods used for yeast recovery and quantitation. C. albicans was the predominant isolate, while C. tropicalis was the commonest non-albicans spp. in HIV infected, a finding similar to previous studies (4,14). A significant association between oral candidiasis and low CD4 count was noted. Thus, this clinical condition could be useful in estimating immunosuppression in resource limited settings. Overall 14 per cent of Candida species were resistant to at least one of the three azoles tested. Candida non-albicans species were more resistant to azoles compared to albicans, information that can be useful for clinicians dealing with non--responding cases. Thus, eliciting history of exposure to these drugs may be important in choosing appropriate therapy.

Acknowledgment

Authors thank the entire staff of the NARI-Talera Clinic for their help and support in collection of samples and acknowledge the Director, Dr R.S. Paranjape for his valuable guidance.

Arati Mane *, Sapna Panchvalli Shilpa Bembalkar & Arun Risbud

National AIDS Research Institute (ICMR) Pune 411 026, India

* For correspondence:

amane@nariindia.org

arati2478mane@rediffmail.com

References

(1.) Hung CC, Yang YL, Lauderdale TL, McDonald LC, Hsiao CF, Cheng HH, et al. Colonization of human immunodeficiency virus-infected outpatients in Taiwan with Candida species. J Clin Microbiol 2005; 43: 1600-3.

(2.) Costa CR, Cohen AJ, Fernandes OFL, Miranda KC, Passo XS, Souza LKH, et al. Asymptomatic oral carriage of Candida species in HIV infected patients in the highly cative antiretroviral therapy era. Rev Inst Med Trop Sao Paulo 2006; 48: 257-61.

(3.) Campisi G, Pizzo G, Milici M, Mancuso S, Margiotta V. Candidal carriage in the oral cavity of human immunodeficiency virus-infected subjects. Oral Surg Oral Med Oral Path Oral Radiol Oral Endod 2002; 93: 281-6.

(4.) Gugnani HC, Becker K, Fegeler W, Basu S, Chattopadhya D, Baveja U, et al. Oropharyngeal carriage of Candida species in HIV-infected patients in India. Mycoses 2003; 46: 299-306.

(5.) Hamza O, Matee M, Mainen M, Elison S, Ferdinand M, Frans M, et al. Species distribution and in vitro antifungal susceptibility of oral yeast isolates from Tanzanian HIV infected patients with primary and recurrent oropharyngeal candidiasis. BMC Microbiol 2008; 8: 135.

(6.) National AIDS Control Organisation (NACO). Manual on quality standards for HIV testing laboratories. New Delhi: NACO; 2007.

(7.) Naglik JR, Rodgers CA, Shirlaw PJ, Dobbie JL, Fernandes LL, Greenspan D, et al. Differential expression of Candian albicans seceted aspartyl proteinase and phospholipase B genes in humans correlates with active orla and vaginal infections. J Infect Dis 2003; 188: 469-79.

(8.) Clincial and Laboratory Standards Institute (CLSI). Reference method for broth dilution antifungal susceptibility testing of yeasts. Approved standard M27-A2, 2nd ed. Wayne, PA: Clinical Laboratory Standards Institute; 2002.

(9.) Priscilla LSA, Milan EP, Martinez R. Telles FQ, Ferreira MS, Alcantara AL. Multicenter Brazilian study of oral Candida species isolated from AIDS patients. Mem Inst Oswaldo Cruz 2002; 98: 253-7.

(10.) Wabale V, Kagal A, Bharadwaj R. Characterization of Candida spp. from oral thrush in human immunodeficiency virus (HIV) seropositive and seronegative patients. Bombay Hospital J 2008; 50: 212-7.

(11.) Baradkar VP, Kumar S. Species identification of Candida isolates obtained from oral lesions of HIV infected patients. Indian J Dermatol 2009; 54: 385-6.

(12.) Lattif AA, Uma B, Rajendra P, Biswas A, Wig N, Sharma N, et al. Susceptibility pattern and molecular type of species-specific Candida in oropharyngeal lesions of Indian human immunodeficiency virus-positive patients. J Clin Microbiol 2004; 42: 1260-2.

(13.) Chunchanur SK, Nadgir SD, Halesh LH, Patil BS, Kausar Y, Chandrasekhar MR. Detection and antifungal susceptibility testing of oral Candida dubliniensis from human immunodeficiency virus-infected patients. Indian J Pathol Microbiol 2009; 52: 501-4.

(14.) Arora U, Jagdev M, Jindal N. Immunosuppression level in HIV-positive patients with oropharyngeal Candidiasis. Indian J Med Microbiol 2009; 27: 174-5.
Table. Candida species and their in vitro antifungal
susceptibility pattern from HIV positive individuals

 HIV positive HIV positive
 (symptomatic) (asymptomatic)
Candida species N (%) N (%)

Candida albicans 39 (65) 76 (80.9)
Candida non-albicans 21 (35) 18 (19.1)
C. tropicalis 6 (10.0) 5 (5.3)
C. lusitaniae 5 (8.3) --
C. parapsilosis 2 (3.3) 5 (5.3)
C. glabrata 2 (3.3) 4 (4.3)
C. krusei 2 (3.3) 3 (3.2)
C. kefyr 1 (1.7) --
C. boidinii 1 (1.7) --
C. guillermondii 1 (1.7) --
C. norvegensis 1 (1.7) --
C. sphaerica -- 1(1.1)
Total 60 94

 In vitro antifungal
 susceptibility pattern *

 S R
Candida species N (%) N (%)

Candida albicans 93 (83.0) 14 (12.5)
Candida non-albicans 26 (68.4) 7 (18.4)
C. tropicalis 7 (70.0) 1 (10.0)
C. lusitaniae 4 (80) 1 (20)
C. parapsilosis 7 (100) --
C. glabrata 2 (33.3) 3 (50)
C. krusei 1(20) 2 (40)
C. kefyr 1(100) --
C. boidinii 1(100) --
C. guillermondii 1(100) --
C. norvegensis 1(100) --
C. sphaerica 1(100) --
Total 119 (79.3) 21(14.0)

* S, susceptible to all three azoles; R, resistant to one
of the three azoles. Four isolates were susceptible for
one or two of the azoles and susceptible dose dependent
for others, thus data not included
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Author:Mane, Arati; Panchvalli, Sapna; Bembalkar, Shilpa; Risbud, Arun
Publication:Indian Journal of Medical Research
Article Type:Letter to the editor
Date:Jun 1, 2010
Words:1798
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