Introduction
Oral candidiasis, also called as oral thrush, affects the mucous membrane of the oral cavity. It’s an opportunistic infection caused by Candida species. It is a common problem amongst the immunocompromised patients and can also be a mark of systemic disease such as diabetes mellitus. The most common species of yeast isolated from patients with oral candidiasis is C. albicans.1 Oral candidiasis results from penetration of the oral tissues when the host's physical and immunological defenses have been undermined and yeast overgrowth. Tissue invasion may be assisted by secreted hydrolytic enzymes, hyphal formation, and contact sensing. While these and other phenotypic characteristics may endow certain Candida species or strains with a competitive advantage in the oral cavity, it is the host's immune competence that ultimately determines whether clearance, colonization, or candidiasis occurs.2 Most of the patients having oral candidiasis has yeast strain isolates present in the oral cavity as commensal flora and Oral Candidiasis is the most common HIV related oral lesion.3 Risk of many HIV-related diseases varies with the patient’s degree of immunosuppression. Most commonly used surrogate markers of immune function are CD4 counts and quantitative HIV-1 RNA levels.4 Although the introduction of antiretroviral therapy (ART) has had a major impact on the infectious complications of AIDS,5 Oral thrush or other candida infections remains a common opportunistic infection in HIV-infected patients. The low absolute CD4+ T-lymphocyte count has traditionally been cited as the greatest risk factor for the development of OPC. The risks are higher in patients with a CD4+ cell count of less than 200 cells/mm. During HIV infection, the rate of Candida infection is inversely related to the CD4 counts of the patient which in turn depends on the use of Anti-retroviral treatment.6 Although the introduction of HAART that is Highly Active Antiretroviral Therapy has dramatically reduced the incidence of opportunis tic infections in HIV-positive individuals who have received antifungal drugs, OPC w ith a shift in the spectrum of Candida species remains the most frequent HIV associa ted oral lesion including developing countries.
Materials and Methods
This study was carried out in a period of one year at Department of Microbiology, MGM Medical College & Hospital, Navi Mumbai, India. Two o ropharyngeal swabs each were collected from 50 HIV infected patients who were clinically suspected of having oral candidiasis, after taking a written consent from them. Out of the two swabs collected from each patient, one was used for Gram’s staining and the other was inoculated on SDA slant and incubated at 37°C, it was observed for fungal growth after 48 hours. Isolates were identified using combination of microscopic examination, colony characteristics, germ tube test, morphology on cornmeal agar, species identification on CHROM agar, and sugar assimilation test. All the yeast isolated were subjected to antimicrobial susceptibility testing. The antifungal susceptibility testing was done on Mueller Hinton Agar supplemented with 2% glucose and 0.5µg/ml methylene blue (as per CLSI guidelines M 44-A) using the colonies directly from the CHROM agar plates.
The following antifungals were tested against the isolates:
Fluconazole (Flu), Itraconazole (Itr), Ketoconazole (Keto), Voriconazole (Vori), Amphotericin B (Amp), and Clotrimazole (Ctr)
Results and Discussion
Microbiological analysis of 50 sero-positive patients with clinically suspected oropharyngeal candidiasis was done. The results are as under, the incidence of oral candidiasis is a most common manifestation in HIV infected population. Lower CD4 counts between 200-300µ/ dL, age and gender are considered as predisposing factors for the increasing incidence rate. During the course of HIV infection, the rate of Candida infection is inversely related to the CD4 counts of the patient which in turn depends on the use of antiretroviral treatment.7 Out of the 50 sero-positive patients, Candida was isolated from 22 patients. Hence the incidence of OPC in our study is 44%. Male to female ratio was found to be 1.2:1. The age distribution of the patients having OPC infection ranges from 31-50 years (Figure 1 ) with a mean of 40 years. These results agree with previous study from Inés María Bravo et al.,8 Berhanu Yitayew. et al.9 also studied oral Candida carriage with an age distribution of 19-70 years old; comprising 29.8% males and 70.2% of females. In another study, Okonkwo E. C. et al10 the number of women screened in this study were found to be 176, while men were 64. This is probably because most men rarely go for routine checkup, until the disease has reached symptomatic stage.
Candida albicans was the most frequently isolated spp. (72.7%) which was followed by Candida tropicalis (22%) (Figure 2) in our study. This correlates to the study by Antoine Berberi et al.11 2014 who found that C.albicans (73%) continues to be the most frequently isolated species in OP C. Shobha D Nadagir et al.,12 also showed in their study that the predominantly isolated species was C.albicans (90.6 6%). A study by Francis Kwamin et at., also showed that the majority of Candida species isolated was C.albicans (68.5%) foll owed by C.tropicalis (7.4%).13
Oral Candida colonization and invasive infection occurs more frequently in HIV-positive patients and are significantly more common in patients with CD4+ cell counts <200 cell/mm. Based on this finding it was seen in our study that 59% of sero-positive patients who developed OPC had l
ow CD4 counts <200 cells/mm3 (Figure 3) same as Shiva Kumar K.L et al., 2013,14 who found that patients with lower CD4 counts were more exposed to the incidence of oral candidiasis. During the course of HIV infection, the rate of Candida infection is inversely related to the CD4 counts of the patient. A study carried out by Antoine Berberi et al.,11 showed that among distribution of OPC lesions and CD4+ cell counts, 73% was associated with <200 cells/mm3.
It was also seen that 1(4.5%) patient with CD4+ count > 500 and 3 patients >400 (13.6%) also having oral candidiasis (Figure 3).
Antifungal susceptibility testing is receiving an increased attention with the advent of newer antifungal drugs.7 In-vitro susceptibility testing provides a measure of the activities of two or more drugs and monitors the development of resistance. It also provides a means to co-relate clinically the in-vitro activity with the outcome of therapy. Fluconazole is widely used as a prophylactic drug because of its high oral bio-availability, minimal drug interaction and minim al adverse effects.15 On the contrary, our study showed that 90% of Candida isolates were resistant to Fluconazole (Figure 4). Whereas, minimum resistance was shown by Voriconazole and Amphotericin B (100% sensitivity). A study from Ethiopia16 also reported an 11.9% resistance rate of C.albicans to fluconazole. A study of clinical isolates of Candida from several human sources in KBTH (2008-9) found that the majority of isolates were C.albicans, and its resistance against fluconazole was 62%.
Based on our results, it was observed that Voriconazole and Amphotericin -B(Hi-media laboratories) were most effective against the isolated yeast species.
Fortunately, the vast majority of cases of oral candidiasis are not life-threatening and readily respond to appropriately administered antifungal agents. It must always be remembered, however, that the organism has the capacity to produce fulminating fatal infection by hematogenous dissemination from seemingly innocuous oral infections that serve as a portal of entry to the systemic circulation. Consequently, all oral candidial infections in compromised patients must be treated vigorously and effectively.
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