Abstract

Introduction: Oropharyngeal candidiasis (OC) is the most common opportunistic infection occurring in HIV infected individuals. Inappropriate use of antibiotics, antifungal agents and poor infection control practices has led to shift in spectrum towards non albicans candida species.

Materials and Methods: This is a prospective study for 6 months conducted at K.R Hospital, Mysore. HIV seropositive cases with oral candidiasis were included in the study. After informed consent, detailed demographic data was obtained from the study participants and two swabs were collected. One swab was used for gram staining and KOH mount. The second swab was immediately inoculated on two Sabouraud’s Dextrose Agar (SDA) supplemented with antibiotics and incubated at 37ºC and 25ºC. Cream pasty yeast colonies on SDA were subjected to Germ tube test, morphology on Corn meal agar, sugar assimilation test and fermentation tests.

Results: Total of 67 patients were included in the study. Pseudomembranous type of OC was common. All the samples yielded the growth of Candida. Candida albicans was the commonest spp isolated but 68.1% of the isolates were non albicans candida. C. tropicalis, C. guillermondii, C. dublinensis, C. parapsilosis, C. krusei and C. kefyr were the non albicans candida spp isolated.

Conclusion: As some of the non albicans candida are intrinsically resistant to antifungal agents identification up to species level is important. Inappropriate treatment will lead to increased morbidity and mortality among these patients.

Keywords: Oropharyngeal candidiasis, HIV, Non albicans candida, C. tropicalis, AIDS, CD4 count.

Acquired Immunodeficiency Syndrome (AIDS) the modern pandemic has been a public threat since its discovery in 1981. Human Immunodeficiency Virus (HIV) infection prevalence among adults (15 – 49 yrs) of India is estimated to be 0.26% i.e. 21.17 lakh people are living with HIV in India while incidence is around 86 thousand cases per year.^[1] In 2015, 67.6 thousand deaths occurred due to AIDS in India. However, India has successfully achieved the 6^th Millennium development Goal of halting the HIV epidemic.^[1]

CD4+ cells guard the oral cavity from infections. The HIV targets the CD4+ cells and there is progressive decrease in the CD4+ cell count. Oral candidiasis (OC) is first reported when median CD4+ count is around 391 x 10⁶cells/L.^[2] The low absolute CD4+ T lymphocyte count has been cited as the greatest risk factor for the development of oral candidiasis and current guidelines suggest increased risk once CD4 T lymphocyte counts fall below 200 cells/ cumm.^[3] Threshold number of CD4 cells are needed to guard the oral cavity from commensal invasion.^[4]

The yeast like fungi Candida occurs as normal oral commensal in 20-50% of Indian population^[5]. But in HIV infection the colonization rate increases dramatically to 81.3% on an average.^[5] Increased intensity of colonization over time culminates in oral candidiasis. OC is a disease recognized since antiquity but gained significance more recently as an infection frequently seen in AIDS patients.^[4]Use of antibiotics which cause imbalance in the microbial flora of the mouth can also favor fungal infections. The use of antifungal agents, mainly the azoles has increased prevalence of infections by Non albicans candida.^[4]

OC is the first clinical sign in HIV infection and will occur in 50 – 95% of HIV positive persons some time during their progression to full blown AIDS.^[4]Oral candidiasis being painful and recurrent, decreases the compliance for Antiretroviral Therapy (ART) and food intake.^[6] Decreased compliance to ART further increases the viral load and the disease progresses among these patients.

C. albicans has been the most common candida species to cause OC. But among HIV infected individuals Non albicans candida are the common cause of OC. Non albicans candida spp exhibit intrinsic and acquired resistance to antifungals. The non albicans candida spp like C. glabrata and C. krusei are more resistant to Fluconazole and Itraconazole compared to C. albicans^[7]. Some studies have reported resistance even to Amphotericin in 1.61% of candida isolates.^[8] Coexistence of different species poses difficulty in treatment.⁶ The changing susceptibility pattern of C. albicans and emergence of other species of Candida as pathogen have necessitated the identification of candida to species level.^[9]

The present study was undertaken to isolate and identify candida in clinically suspected primary oral candidiasis among HIV seropositive cases.

Materials and Methods

This prospective study was conducted for 6 months after approval from the institutional ethics committee at ART Center, K. R hospital, Mysore.

Patients reactive for HIV 1 or 2 or both by Comb assay, Capillus and Tridot test (NACO recommended algorithm) with clinically suspected oral candidiasis of all age group and both sexes attending to ART Centre of K. R Hospital, Mysore were included in the study. Patients were explained in detail about the study and informed written consent was taken. Detailed history was obtained regarding name, age, gender, socio economic status, occupation, h/o tuberculosis, present complaints, previous similar episodes, antifungal treatment, HAART treatment, smoking and alcoholism that was recorded in a prescribed proforma. After the history the lesion in the oral cavity was examined and sample was collected under sterile conditions. The sample was collected and processed as per standard protocol.^[10],^[11],^[12],^[13]

Collection of sample: Two sterile cotton tipped wooden swabs moistened with saline were used to swab and scrape the lesion without touching any other structure in the oral cavity. Swab was collected after rinsing the oral cavity. Swabs were kept in sterile containers and transported immediately to the laboratory and processed.

Microscopy: One swab was used for direct microscopic examination by Gram stain and 10% KOH mount. Gram stain was examined for pus cells and Gram positive oval budding yeast like cells with/ without pseudohyphae. KOH mount was observed for budding yeast like cells with/ without pseudohyphae.

Culture: Sample from the second swab was inoculated on Sabouraud’s Dextrose Agar containing Gentamicin & Chloramphenicol and 5% blood agar. Culture tubes were incubated at 37°C and room temperature and were observed daily for growth. All the samples yielded creamy white pasty growth by 24 – 48 hrs. Yeast like colony was subjected to Gram stain for confirmation. Other tests for identification and confirmation of candida used are germ tube test, Sugar assimilation and sugar fermentation test.

Germ tube test: Germ tube test was performed on all yeast like cells for presumptive identification of C. albicans and C. dubliniensis.

Inoculation on Cornmeal agar: All the yeast isolated were inoculated onto Cornmeal agar (CMA) by Dalmau plate method and incubated at 25°C for 2 – 5 days. Microscopically the arrangement of pseudohyphae, blastospores and chlamydospore were used to identify various species of candida. Most of the Candida had typical morphological arrangement and were identified.

Carbohydrate fermentation test: Sugar fermentation test was done to detect acid and gas production from 2% Glucose, Maltose, Sucrose, and Lactose with Andrade’s indicator and durhams tube. Final results were obtained after incubation for 5 – 7 days at 25°C.

Table 1: Carbohydrate assimilation test for identification of various candida spp^[11],^[14]

Overnight growth of yeast like colonies were suspended in normal saline with McFarland 5 turbidity. Sterile swab
was soaked and lawn cultured on yeast nitrogen base and incubated at 25°C for 7 days. Presence of growth around the disc indicates the assimilation of the carbohydrates. The pattern of sugar assimilation and fermentation were used for species confirmation as seen in table 1 and 2.

Table 2: Carbohydrate assimilation test for identification of various candida spp^[11],^[14]

The CD 4 cell count were estimated by Flow cytometry using FACS Calibur (Becton Dickinson, San Jose, California).

Results
A total of 67 patients attending to ART centre tested positive for HIV by 3 tests as per NACO guidelines with signs and symptoms of oral candidiasis for the first time were included in the study. The demographic data of the participants are as seen in Table 3.

The mean CD4 count of the study participants was 129cells/µL. The most common presentation was pseudomembranous type of oral candidiasis with painful swallowing.

Table 3: Demographic characteristics of the study population

All C. albicans and C. dublineninsis produced germ tube in fresh human serum at 37°C by 2 hrs.

Most common species of candida isolated was C. albicans (31.9%) followed by C. tropicalis (23.6%), C.
guillermondii (15.3%), C. dublinensis (11.2%). Others isolated were C. parapsilosis, C. krusei and C. kefyr were 6.9%. 6.9% and 4.2% respectively. The alarming observation was 68.1% isolated were Non albicans candida. The species were identified based on their appearance in CMA, sugar assimilation and sugar fermentation test. The appearance of C. albicans (Fig 1), C. dublinensis (Fig 2), C. tropicalis (Fig 3), C. guillermondii (Fig 4), C. parapsilosis (Fig 5), C. krusei (Fig 6) &C. kefyr (Fig 7) are seen below.

Table 4: Distribution of candida species in the study population