Antifungal Susceptibility Pattern of Candida Isolates: A Comparison in H.I.V. Positive and Negative Patients from A Tertiary Care Hospital of Northern India

Candidiasis is recognized as a significant cause of morbidity, especially in immunocompromised individuals. An epidemiologic change in Candida species and emergence of resistance can impact the usage of antifungal agents as empirical therapy for Candidiasis in patients with or without AIDS. The present study was done to find out: i) The species of Candida isolated from H.I.V. and Non-HIV infected patients. ii) The resistance pattern of these Candida isolates to antifungal agents. A total of 160 Candida species isolates (80 isolates each from H.I.V. and Non-HIV infected patients) were characterized. Identification of yeast isolates was made by standard procedures including morphology (Staib agar, cornmeal agar, CHROMagar), germ tube test, fermentation, and assimilation of sugars and growth at 42°C. In addition, sensitivity testing was done using the broth microdilution method (M27-A2) as per the C.L.S.I. guidelines against amphotericin B, nystatin, voriconazole, fluconazole, ketoconazole, and itraconazole. In both the groups, i.e., H.I.V. and Non-HIV infected patients, Candida albicans was the most common species (61.2 % and 85 % respectively), followed by Candida guilliermondi (16.2 % and 5 %), Candida tropicalis (5 % and 3.7 %), Candida krusei (5% and 2.5 %), Candida dubliniensis 1(5 % and 1.2 %) and others. Among HIV infected patients fluconazole resistance was 16.25%, ketoconazole 13.5%, clotrimazole 12.5%, itraconazole 6.25 %. In the non-HIV infected group, fluconazole resistance was 8.75% and itraconazole 1.25%. For the appropriate treatment of Candida infections, antifungal susceptibility has become an essential tool, especially in the present scenario of increasing resistance.


INTRODuCTION
Candidiasis has been recognized as a significant cause of morbidity, especially in immunocompromised individuals. The use of highly active antiretroviral therapy (HAART) causes inhibition of viral replication, which eventually causes the recovery of CD4 T+ lymphocytes in HIV-positive patients. The prolonged course of H.I.V. infection further puts at risk these patients to repeated episodes of various opportunistic infections like Candidiasis that can increase in severity with the progression of H.I.V. disease. In addition, candidiasis' management causes the development of antifungal resistance amid the course of treatment [1][2][3] . Infection by resistant Candida strains does not respond to antifungal therapy even with standard doses for an appropriate time duration 4,5 .
Another scenario is that multiple exposures to antifungal agents can cause a shift to Candida species other than albicans, which will eventually lead to hard to treat, refractory, and recurrent infections 6,7 . Thus, an epidemiologic change in Candida species and the emergence of resistance could cause a significant impact on the use of antifungal agents as empirical therapy for Candidiasis in patients with or without AIDS. Opportunistic infections, including Candidiasis, aggravate morbidity and decrease the span and quality of life of HIV-infected patients and thus require accurate diagnosis and adequate treatment. In-vitro sensitivity testing is clinically helpful in foreseeing whether the patients are going to improve with treatment or not. However, in India, in the same way as other developing nations, in-vitro antifungal testing is not performed as routine testing. Accordingly, not much is known about the in-vitro antifungal sensitivity of Candida species cultured from HIV-infected patients with Candidiasis. This study was done to find out the species of Candida isolated from H.I.V. Negative and H.I.V. Positive patients and their resistance pattern to antifungal agents.

MATeRIALS AND MeTHODS Patients and setting
One hundred and sixty patients with

Fungal culture and species identification
One Hundred and sixty Candida isolates (80 isolates each from H.I.V. and Non-HIV infected patients) were characterized. Identification of yeast isolates was made by standard procedures including morphology on CHROMagar, Cornmeal agar, and Staib agar (HiMedia® Laboratories Pvt. Ltd, India), germ tube formation, fermentation and assimilation of sugars and growth at 42°C 8 .

In vitro antifungal susceptibility testing
The determination of the M.I.C. (minimum inhibitory concentration) was done using a broth microdilution test as per the C.L.S.I. guidelines (Clinical and Laboratory Standard Institute) M27-A3 and M27-S4). The following antifungal agents were used-amphotericin B, nystatin, ketoconazole, fluconazole, itraconazole, and voriconazole (HiMedia® Laboratories Pvt. Ltd, India) 9,10 . Aliquots of 100 microliters of each antifungal drug at a double concentration of the final concentration were put in the wells of 96-well microtiter plates. Before testing, fungal strains were subcultured and incubated at 30°C for 24 hours. Next, 100 μl of the inoculum was added to each microdilution well containing 100 μl of the serial dilution of the antifungal agents to reach the final concentration; the plates were then incubated at 35°C for 48 hours. The minimum inhibitory concentrations (M.I.C.s) were calculated after 48 hours. American Type Culture Collection (A.T.C.C.) strains recommended by C.L.S.I., C. albicans (ATCC 64550), C. parapsilosis (ATCC 22019), and C. krusei (ATCC 6258) were used as controls.

ReSuLTS
Demographically, both the groups, ie. H.I.V. Negative and Positive patients presenting with Candidiasis were comparable in terms of age and sex. The HIV-positive patients with Candidiasis constituted 47 (59%) males and 33 (41%) females with a mean age of 32 years. The HIV-negative patients with Candidiasis constituted 41 (51%) males and 39 (49%) females with a mean age of 29 years (Table-1). Heterosexual contact was the most common mode of the transmission of H.I.V. infection ( Table-2) Most of the patients with Candidiasis in the H.I.V. positive group presented with oral Candidiasis (51 patients), followed by vulvovaginal Candidiasis, Cutaneous Candidiasis, oesophageal Candidiasis, candidal diarrhea, and candidemia. In the HIV-negative group, 31 patients presented with oral Candidiasis, and 28 patients were diagnosed with vulvovaginal Candidiasis. In addition, five patients were confirmed with candidemia and were of the pediatric age group (Table 3).

DISCuSSION
In this study, oropharyngeal Candidiasis was the most common presentation in HIV-positive  15,16 .
In 80 cases of Candidiasis who were H.I.V. positive, Candida albicans were isolated from 49 (61.2%) while non-candida albicans, including C. guilliermondi, C. tropicalis, C. parapsilosis, C. dubliniensis, were isolated from 31 (38.7%) patients. Khedri et al. 1 reported C. albicans in 52.9% HIV-positive cases with Oropharyngeal Candidiasis. Even Though Candida albicans persists as a significant causative species, the frequency of non-albicans species of Candida has modestly risen. Oral Candidiasis in HIV/AIDS patients caused by non-albicans Candida is well documented 14,15,17 . A study by Ismail H Sahand et al. 17 on the presence of Candida in oral swabs of HIV-infected individuals reports the isolation rate of candida albicans from 52% of patients and non-albicans Candida from the 48% 14,17 reported that 40% of all Candida isolates to be Non-albicans 14 .
Sixteen Percent of the candida species isolated showed resistance to fluconazole, similar to a study that reported fluconazole resistance in 21% isolates 18 . Fluconazole resistance is mainly due to past fluconazole (azoles), specifically multiple and long-term use 19 . Extended-term and repeated antifungals are sometimes required in AIDS patients and are at greater risk of developing an infection with resistant strains. Additionally, resistance in C. albicans is associated with a steady increase in non-albicans species as a causative agent of resistant mucosal Candidiasis, especially in patients with high immunosuppression 12 . Fluconazole resistance was relatively lower (8.7%) in the patients without H.I.V. infection, and maybe this is because of Non-recurrent Candidiasis and minimal prior exposure to antifungal drugs. The development of resistance to antifungal agents, especially to azoles in the candida albicans and non-albicans species, is an area of concern. This species can expand its resistance repertoire with the development of stable resistance to Azoles, including fluconazole. It is well documented that cross-resistance happens between the antifungal drugs 2 , and when these resistant strains infect a majority of patients, this will limit our choices for treating Candidiasis in such patients.
Although enough literature has accumulated in the last years regarding the prevalence of vaginal colonization and vulvovaginal Candidiasis in HIV-Negative and HIV-Positive women, the deficit in our knowledge remains, mainly related to the pathophysiology of the disease, as the frequency of vulvovaginal Candidiasis does not increase with the H.I.V. As the number of patients with H.I.V. infection continues to increase in number due to the increase in survival rates, the problem of opportunistic infection also grows in parallel. A few years back, C. albicans was the significant species implicated in Candidiasis in immunocompromised conditions; although it still is, the incidence of Nonalbicans Candida and its rise is a matter of concern. Therefore, for the appropriate management of Candida infections, antifungal susceptibility has become an essential tool, especially in the present scenario of increasing resistance.