Oropharyngeal candidiasis and Candida colonization in HIV positive patients in northern India

Introduction: Oropharyngeal candidiasis (OPC) is the most common opportunistic fungal infection reported in human immunodeficiency virus (HIV) positive patients worldwide. This prospective study was undertaken to investigate OPC and Candida colonization (CC) and their correlation with CD4 cell counts and antiretroviral therapy (ART) in HIV-positive patients. Methodology: In total, 190 HIV-positive patients were enrolled for study in three groups as follows: Group A, 90 patients without ART; Group B, 100 patients undergoing treatment with ART; and Group C, 75 HIV-negative control patients. All HIV patients underwent clinical examination and were subjected to CD4 cell counts. Swabs were collected from the oral cavity of all individuals and plated on Sabouraud’s dextrose agar. Identification of Candida species was performed by conventional methods. Results: Candida species were isolated in 84/190 (44.2%) and 20/75 (26.6%) of the HIV-positive subjects and controls respectively (p<0.01). OPC was noted in 21/190 (11%) of the HIV-positive patients. Candida albicans was the most frequently isolated species. Patients with CD4 cell counts ≤ 200 cells/mm were significantly (p<0.001) more frequently colonized (37/63; 58.7%) and infected (18/21; 85.7 %) with Candida species. Candida species was seen in patients with CC and OPC with CD4cell counts between 201 and 500 (21/63; 33.4% vs 3/21; 14.3%) and > 500 cell/mm (5/63; 7.9% versus 0/21 0%) respectively. Conclusion: OPC and Candida colonization occur more frequently in HIV-positive patients with CD4 cell counts ≤200 cell/mm. ART significantly reduces OPC. C. albicans is the most frequently isolated species in both OPC and colonization, suggesting endogenous infection.


Introduction
The oral cavity is colonized by Candida albicans or other Candida species in 40% to 60% of healthy persons [1].In immunocompetent individuals, Candida species are mostly transient flora.Many factors contribute to the development of oropharyngeal candidiasis (OPC) including malnutrition, poor oral hygiene, dental malocclusion, and immunosuppression [2].The current epidemic of human immunodeficiency virus (HIV) is a major cause of immunosuppression.According to the Joint United Nations Programme (2009) on HIV/AIDS [3] and the National AIDS Control Organization (2008) [4], there are an estimated 2.4 million cases of HIV infection worldwide and 22.7 per 100,000in India respectively.The World Health Organization (WHO) predicts that India will have one of the largest populations of HIV/AIDS patients worldwide in coming decades.OPC has been described as the most frequent opportunistic fungal infection among HIV-positive patients and it has been estimated that more than 90% of HIV-positive patients develop this often debilitating infection at some time during the progression of their disease [5,6].The presence of Candida species in the oral cavity of HIV-positive patients is an indicator of subsequent development of OPC [7].HIV-positive patients experience recurrent episodes of OPC and oesophageal candidiasis.As immunodeficiency progresses, these patients may also receive multiple courses of antifungal drugs contributing to antifungal resistance.In these patients, antifungal agents are also less efficacious and take longer to achieve a clinical response [8].
Until recently, C. albicans was reported as the predominant species in OPC but recent publications suggest that non-albicans Candida species are now emerging [1,9].There is a paucity of microbiological studies on OPC and Candida colonization (CC) in HIV patients from India; therefore, the present study was conducted to determine the profiles of CC and OPC and their correlation with CD4 + cell counts and antiretroviral therapy (ART) in HIV patients.

Study population
This hospital-based prospective observational study was conducted between August 2007 and July 2008.Patients attending the ART Centre and the Integrated Counseling and Testing Centre (ICTC) at Chhatrapati Shahuji Maharaja Medical University (CSMMU), Lucknow, were enrolled for the study.A total of 190 HIV-positive patients were enrolled after they provided informed written consent.Seventy-five HIV-negative subjects in the same age group and socioeconomic status were enrolled as study controls.The study was approved by the institutional ethical clearance committee.Individuals with diabetes mellitus or other systemic diseases, pregnant women, smokers, dentures/orthodontic device users, and those on antibiotics and/or on antifungal treatment in the preceding three months were excluded.
HIV serostatus of the study group was determined using a commercially available enzyme linked immunosorbent assay (ELISA) (HIV-1 Enzaids, Span Diagnostics Ltd, Udhna, Surat, India) and three rapid antibody tests manufactured by CombAids-RS Span Diagnostics Ltd, Surat, India; Retrocheck HIV, QUAL proDiagnostics, Goa, India and Acon Biotech Prolink, San Diego, CA, USA, following NACO recommended algorithms [10].A CD4+ cell count using the Fluorescent Antibody Cell Sorter (FACS, Becton Dickinson, Singapore, BD) Count system was performed in all enrolled HIV-positive patients.

Clinical and microbiological assessment of subjects
An oral examination was performed and suspected lesions were clinically evaluated by the Faculty of Dental Sciences.Candida colonization was defined as isolation of Candida species from the oral cavity without the presence of oral lesions.All patients who had oral lesions and from whom Candida species were isolated were considered to have OPC.Recent CD4 + cell counts and a history of intake of ART were noted for all HIV-positive patients.A single oral swab was collected from each study participant by passing a sterile swab firmly across the buccal mucosa, floor of mouth, dorsal surface of the tongue in cases of asymptomatic patients, and from the base of the oral lesion in cases of symptomatic patients.Swabs were cultured on Sabouraud's dextrose agar (SDA) with chloramphenicol 0.5g/l, then incubated at 37°C and observed daily for seven days.Pure growth of Candida species was considered for analysis.Candida was identified by conventional tests and species identification was performed using the germ tube test, growth on CHROMagar Candida Medium (HiMedia, Mumbai, India), and sugar assimilation tests [11,12].
Statistical analysis was conducted by using the chisquare test.Data were analyzed with SPSS software version 20 (IBM SPSS, Chicago, USA).

Results
In the present study, 190 HIV-positive patients were designated into two groups: group A (n = 90) without ART and group B (n = 100) with ART.The demographic data and clinical profile of these patients is shown in Table 1.The median duration of ART was two years (range, 1.5 months to 6 years).Overall, Candida species was isolated from the oral cavity in 84/190 (44.2%)HIV-positive patients and in 20/75 (26.6%) of HIV-negative patients.The difference was significant (p < 0.01).Of the 84 HIV-positive patients with Candida species, 41 (45.5%) were from Group A and 43 (43.0%) were from the Group B. Twenty-one (11%) HIV-positive patients had oral lesions in the oropharyngeal cavity and Candida species were isolated on culture.

Discussion
Asymptomatic carriage of Candida species in the oral cavity is found irrespective of the immune status of individuals.Many studies have been conducted on oral CC in healthy and immunocompromised individuals.Due to differences in the sample collection techniques used, time and frequency of sampling, yeast assessment methods, and the study population, results from the studies are not comparable.In addition, CC rate can be affected by several factors such as hospitalization, abnormal nutrition, and smoking [2].Oral CC in HIV-positive asymptomatic patients has been reported and is known to be higher than in patients in other risk groups such as diabetes mellitus or other systemic disease [13].
Similarly, in our study, the isolation of Candida was significantly higher in the HIV-positive group compared to that in the controls.The prevalence of OPC (11%) and CC (33%) in HIV-positive patients in the present study was lower compared to that in other studies (62.6% to 81%) [14][15][16][17].The reason for the low isolation rate in the present study could be explained by single sampling performed in this study as compared to multiple sampling in other investigations.
OPC infections are the most common of opportunistic infections in HIV-positive patients, occurring in up to 90% of patients during the course of the disease [13].In India, OPC is reported to be the second most common opportunistic infection in HIVpositive patients [18].Three clinical presentations of OPC have been commonly reported in HIV-positive patients: pseudomembranous, erythematous and angular chelitis [17,19].In our study, acute pseudomembranous candidiasis (exudative) was the most common form of OPC (Table 1), while angular chelitis may be part of vitamin deficiency superimposed with Candida infection seen in 9.5% of the patients.These results are in concordance with those result of several other studies [16,20].C. albicans was also the predominant species isolated from the oral cavities of the patients in the control group.
In HIV-positive patients with OPC, C. albicans is the most frequently identified species; however, nonalbicans Candida has also been reported recently [20,21].C. albicans was the most frequent species isolated from colonized and infected HIV-positive subjects (90.5%) in our study (Table 1).In the present study, a relatively small proportion (9.5%) of isolates were non-albicans Candida.In a study conducted in Italy, Barchiesi et al. described an increase in the frequency of isolation of non-albicans Candida species from 3% to 4% of isolates in 1988/1989 to 16% to 18% of isolates in 1990/1991 [15].Similarly, in another Italian study, Morace et al. (1990) found that 25% of the yeast species isolated from persons with AIDS were non-albicans Candida [22].In Spain, Masia et al. evaluated 153 HIV-positive patients and found that 21% of these patients had non-albicans Candida, the most common being C. glabrata [23].Table 2 shows the distribution of Candida species isolated from HIV-positive patients with OPC from India in the last decade [21,[24][25][26].The findings showed that in India C. albicans continue to be the predominant pathogen and non-albicans Candida has been reported in approximately 14% to 30% of patients.
The reports of an association between CD4 + cell counts and OPC/CC are also contradictory; Costa et al. did not find a significant correlation between CC and CD4 + cell counts, as 16.25%, 61.2% and 22.6% of the patients in their study had CD4 + cell counts ≤ 200, 201 to 500 and more than 500 respectively [27].Little correlation was also observed by Barchiesi and colleagues, with a median CD4 + cell count of397cell/mm 3 and 442 cells/mm 3 in colonized and non-colonized their patients, respectively [28].Schoofs et al., however, reported a significant relationship between CC and CD4 + cell counts less than 200 cell/mm 3 [29].Fong and colleagues also found a strong correlation between asymptomatic CC, the development of thrush, and CD4 + cell counts [30].OPC in HIV-positive persons keeps recurring as the immunity decreases.In our study, CC was significantly higher in the group with CD4 + cell counts ≤ 200 cell/mm 3 (p < 0.001), suggesting that the group is prone to develop OPC.Prevention of opportunistic infections in patients with HIV is important because in all HIV-infected individuals, the risk of infection increases as the absolute CD4 T-lymphocyte count falls.Data from prospective controlled trials indicate that fluconazole prophylaxis can reduce the risk for mucosal (e.g., oropharyngeal, esophageal, and vaginal) candidiasis among patients with advanced HIV disease.However, routine primary prophylaxis is not recommended because mucosal disease is associated with very low attributable mortality, acute therapy is highly effective, prophylaxis can lead to disease caused by drug-resistant species, prophylactic agents can produce drug interactions, and prophylaxis is expensive [31].It is recommended that the HIVinfected asymptomatic cases with CD4 + cell counts ≤ 200 cell/mm 3 should be screened for oral candidiasis to improve the quality of life.
ART has been associated with dramatic decreases in the rate of HIV-related opportunistic infections.Our study suggests that ART does not influence oral CC, but it significantly prevented the development of OPC.In a follow-up study, Yang et al. reported that ART was only marginally effective in eliminating colonization [32].Similarly, Sanchez-Vargas et al. reported that ART did not influence colonization.The authors observed that 36.1% of patients undergoing ART suffered from OPC compared to 45.9% of patients not receiving ART [1].

Conclusion
Oral Candida colonization and invasive infection occur more frequently in HIV-positive patients and is significantly more common in patients with CD4 + cell counts ≤ 200cell/mm 3 .ART significantly reduces OPC. C. albicans continues to be the most frequently isolated species in both OPC and CC, suggesting endogenous infection.

Table 1 .
Profile of HIV patients with Candida colonization and infection and its correlation with ART and CD4 + count

Table 2 .
Studies on OPC in HIV infected patients from India in last decade

Study Group Duration of study Patient size and characteristics Predominant species
OPC: Oropharyngeal candidiasis