A study on Vulvovaginal candidiasis among non-pregnant women

Introduction: Candida species are opportunistic pathogens which cause a wide variety of infections in humans ranging from trivial intertriginous infection to fatal candidemia. Vulvo vaginal candidiasis (VVC) is an acute inﬂammatory disease and a frequent reason for gynaecological consultations. The present study aimed at determining prevalence of Candida species from VVC and to speciate isolated Candida with antifungal susceptibility profile among non pregnant women. Materials and Methods: This was a cross sectional study conducted over a period of six monthsin the department of microbiology and Obstetrics and Gynecology. Swabs collected from patients were subjected to microscopy (Gram s stain) and culture on Sabouraud’s dextrose agar. Species level was identified by using chrome agar. Antifungal susceptibility testing: Antifungal susceptibility testing was performed by NCCLS M44-A Disc diffusion method. Results: A total of 209 high vaginal swabs were collected from non pregnant women. 71 patients high vaginal swabs yielded growth of Candida species and accounted for 33.97%. Most commonly isolated species were Candida albicans 31 (43.66%) followed by Candida tropicalis 27(38.03%) and Candida glabrata 13 (18.31%). All species were found to be susceptible to Amphotericin B. Among commonly used antifungals, clotrimazole was found to be most susceptible. But, C.glabrata species was least susceptible to frequently used antifungals. Conclusion: Candida albicans was the most commonly isolated species. CHRO Magar is rapid, technically simple and cost effective compared to time consuming technically demanding expensive conventional methods. Performing antifungal susceptibility is useful in choosing appropriate antifungal in treating vulvo vaginal candidiasis.


Introduction
Vulvo vaginal candidiasis (VVC) is an acute inflammatory disease and a frequent reason for gynecological consultation as it can affect up to 75% of women of child-bearing age [1]. Clinical signs and symptoms include intense pruritus, vaginal discharge, anerythematous vulva and dyspareunia [2]. Until recently, the problem of vaginal candidiasis was often ignored, or treated as an insignificant problem forthe female population. It received more focus onlyafter Herman Gardner said: "Vaginitis can cause morein convenience than any other gynaecological disease. In addition, many mental and emotional problems are associated with vaginitis " [3]. There are many risk factors for development of vulvo vaginal candidiasis, like vaginal ecosystem, pregnancy, hormonal contraception, diabetes, stress recent antibioticuse, dietary practices, gastrointestinal colonization by theorganism, clothing and weaken immune-compromised system [4].
VVC is most often caused byCandida albicans, however, other species of Candidasuch as Candida glabrata, Candida parapsilosis, and Candida tropicalis are emerging [5]. Azole antifungals are being frequently being used to treat infections caused by Candida species Recently studies reported that intrinsic azole resistance in some Candida species as well as development of high-level azole resistance is a problem of critical importance in the clinical setting [6]. Mostof the studies done previously focused on immunocompromised

Material and Methods
This was a cross sectional study conducted over a period of six months in the departmentof microbiology in collaboration with the department of Obstetrics and Gynaecology of Vinayaka mission's medical college and hospital. Study population consisted of non pregnant women attending OPD of Obstetrics and Gynaecology. Verbal consent was obtained from the women before sample collection. Clinical examination was performed of each participant and recorded signs of vaginal abnormalities. A pair of highvaginal swabs were obtained from the posterior vaginal fornix of the subjects aseptically with the help of a vaginal speculum. Swabs collected from patients were subjected to microscopy (Gram s stain) and culture on Sabouraud's dextrose agar.
Species identification: CHRO Magar was used for preliminary identification of yeast and to detect mixed infections. Candida species were differentiated by colonial morphology and colors, which were generated by a chromogenic in the agar, asdescribed by Wortman et al [7].
Antifungal susceptibility testing: Antifungal susceptibility testing was performed by NCCLS M44 -A Disc diffusion method.
Inoculum was prepared by picking five distinct colonies of approximately 1 mm in diameter from 24 h old culture of Candida species. Colonies were suspended in 5 ml of sterile saline and its turbidity was adjusted visually with the transmittance to that produced by a 0.5 McFarland standard. Inoculation of test plates were done with a sterile cotton swab dipped into the suspension. The dried surface of a sterile Mueller-Hinton + GMB (glucose and methylene blue) agar plate was inoculated by evenly streaking the swab over the entire agar surface. Anti fungal disks were dispensed onto the surface of inoculated agar plate. Plates were incubated at 35 0 C and examined after 20-24 hours. The zone of inhibition was measured and the results were recorded as susceptible/resistant [8]. Patients aged between 15-45 years and non-pregnant were included in the study. Immunocompromised patients, menstruating women and patients who were taken antifungal therapy in past two weeks (before sample collection) were excluded. Statistical analysis was done by simple percentage method.

Results
A total of 209 high vaginal swabs were collected from non pregnant women aged between 15-45 years attending with any vulvovaginal candidiasis symptoms (itching, soreness, vaginal secretions, inflammation, rashes etc.). Of the 209 patients included in the study, 71 patients high vaginal swabs yielded growth of Candida species and accounted for 33.97%. Majority of the patients yielded growth were ranged between 26-35 years and accounted for 54% followed by 16-25 years which showed 30%. Least growth was seen in the patients between the age group of 36-45 years which accounted for 14% (Table.1).

Discussion
In the present study infection rate of vulvovaginal candidiasis was found to be 33.97%. But according to the study conducted by Enweani et al [9] V VC in nonpregnant women was 40.6% which is high compared to the present study. Eckert et al [10] found 25% of VVC among non pregnant women. The prevalence of vaginal candidiasis reported by different studies was 16.5%, 21.31%, and 19% [11,12,13]. This relatively low prevalence of vaginal candidiasis among women in other studies probably due to to adequate knowledge, good personal hygiene, and normal levels of estrogens and corticoids [14].
In the present study, Candida species was predominantly isolated in the age group of 26-35 years which accounted for 54%. This may be due to high sexual activity, poor personal hygiene, the use of contraceptives, and drug abuse among this age-group. Alo et al [15] reported a higher prevalence of C. albicans (33.33%) within the age bracket of 36-40 years, while those between 20 and 25 years had the lowest prevalence (20.42%).
In the present study, low Candida infection was found in age group 36-45 years which accounted for 14%. This finding is in line with a previous report by Okungbowa et al [16] who reported prevalence of 10% and 2% within the age-groups of 36-45 and over 46 years, respectively. They reported that this was probably due to the possible increase in vaginal immunity with age as they have decreased levels of estrogen and corticoids, and thus are resistant to Candida infections.
In the present study, Candida albicans 31 (43.66%) was the most predominant species isolated followed by Candida tropicalis 27(38.03%) and Candida glabrata 13(18.31%), similar to other study by Grigoriou et al, the most commonly isolated species from the patients was C. albicans.
C. albicans adheres to vaginal epithelial cells in significantly higher numbers than do other Candida species [20]. Vaginitis induced by non-albicans species is clinically indistinguishable from that caused by C. albicans [21]. The reason for the increase in incidence of VVC caused by non-albicans species is thought to be single-dose antifungal treatment, low-dosage azolemaintenance regimens, and the use of over-the-counter antimycotics [22].
In the present study, all three species of Candida were least susceptible to fluconazole and no Candida species showed resistance to amphotericin B. Few strains of Candida species exhibited multi drug resistance to routinely used antifungals. Relatively low levels of fluconazole resistance were observed in all species of Candida isolated.Resistance to fluconazole is of great concern as it is the most common azole used for the treatment of candidiasis including VVC. Fluconazole is available in both intravenous and oral formulations with high bioavailability and is more cost-effective than other antifungal agents.
As per study by Mishra et al [23] all C. glabrata, 50% C. tropicalis and 12% C. albicans isolates were found to be resistant to fluconazole. But, another study by Khan M et al observed, C. krusei showed 100% resistance to fluconazole [24]. It is worth mentioning that the non-albicans species had the highest levels of resistance compared to C. albicans. C. glabrata was the least susceptible to the tested antifungals. Although amphotericin B iseffective against most strains of Candida spp., its usage is limited due to the nephro toxicity associated with it.

Conclusion
Candida albicans was the most commonly isolated species followed by C.tropicalis. Among commonly used antifungals, clotrimazole was found to be most susceptible. But, C.glabrata species was least susceptible to frequently used antifungals. Hence, species level identification is required to choose appropriate antifungal and chrom agar medium will helpful to mycology laboratories for rapid identification of clinically important candida spp.