This study was carried out in the Department of Microbiology in collaboration with Dept of Obstetrics & Gynecology (OG), Aarupadai Veedu Medical College and Hospital, Puducherry, India during the period of August 2010 to September 2012. The study was started after getting the ethical clearance from the scientific research committee of the institution. An informed written consent was obtained from all the subjects. The study group consisted of 180 women between the age group of 15 to 56 years with the complaints of pruritis, pain and vaginal discharge. One hundred & eighty women with no history of vaginal discharge were included as controls. Materials used for this study consisted of high vaginal swabs from patients, with relevant obstetric and gynecological history, attending OG OPD. A questionnaire was completed with information covering complaints, nature of vaginal discharge, personal history, marital history, predisposing factors, per vaginal examination and collection of high vaginal swabs. Data collected included name, age, complaints like vaginal discharge, pain and itching, duration of illness whether acute or chronic, any past history of presenting episode, any systemic diseases such as diabetes/hypertension/tuberculosis. Two sterile, cotton tipped swabs were used to collect specimens from lateral wall of vagina of each woman. One of the two swabs was used to determine the presence of yeast by direct wet-mount microscopy using a drop of 10 per cent potassium hydroxide solution. The other swab was used for culture onto Sabouraud’s dextrose agar (Hi-Media, Mumbai, India) supplemented with 0.06 mg/ml gentamicin, with and without cycloheximide (0.5%) & 5% sheep blood agar incubated at 37⁰C. Species identification was done as per standard procedures [5]. Susceptibility testing to fluconazole was performed using E-test [6]. HiComb MIC test(A-strip- 256-2mcg & B-strip- 2.048-0.016 mcg). Species identification of yeast isolates were was done by standard procedures including morphology, germ tube test, cornmeal agar test (Hi-Media, Mumbai, India), HiChrome agar (Hi-Media, Mumbai, India) and assimilation of various sugars.

Fifty six (31%) patients were having itching as the presenting complaints, 53 (29.4%) presented with vaginal discharge as the initial complaint, 28 (15.6%) patients presented with pain as the initial complaint, 24 patients (13.3%) presented itching, pain & discharge as complaints. The commonest organism isolated was bacterial in origin which constitutes 71 patients (39.4%). Candida species were isolated from 40 patients (22.2%). The other organisms isolated were Trichomonas vaginalis and Gram negative bacilli.

Pregnancy was the commonest risk factor for VVC 22(55%) followed by broad spectrum antibiotic usage 8 (20%). Other risk factors were Diabetes mellitus,usage of oral contraceptive pills and tuberculosis [Table/Fig-1]. Out of 40 isolates, 27 (67.5%) were from pregnant women. Thirty five (87.5%) Candida isolates were sensitive to fluconazole, 3 (7.5%) were moderately sensitive and 2 (2.5%) were resistant. The results were analyzed statistically using Chi-square method at appropriate places.

In given study 93% normal healthy females taken as controls were between 15-45 years of age. Only 7% was in the age group of 46- 55 years [Table/Fig-2]. Various studies have reported the prevalence of asymptomatic vaginal colonization of Candida species as 5% to 30% [7,8]. The vaginal carriage rate of Candida species in asymptomatic controls in our study was 8%, which is similar to the study done by de Oliveira JM et al., [9] who reported the asymptomatic prevalence of VVC as 10%. Vaginal colonization is more frequent in diabetic women [10]. Numerous studies worldwide have shown that C.albicans can convert from being a commensal into a disease-causing pathogen, in response to a change in the host environment causing infections in the oral, gastrointestinal and genital tracts. Moreover as a commensal, C. albicans asymptomatically colonizes epithelial surfaces presumably in the form of blastoconidia. In the present study Gram staining of high vaginal swab in these normal healthy controls revealed Candida only as a blastoconidia not as elongated hyphae or pseudohyphae. In the present study 88.9% of the women were between the age group of 15-45 years. Studies done by various authors say the incidence of reproductive tract infections in women is highest in the age group of 15-45 years and followed by a decline [11]. The reason for the high incidence in this age group includes low levels of protective cervical antibodies, increased sexual activity, and new influence of reproductive hormones that may lead to increased susceptibility to reproductive tract infections [11]. Our result is in consistent with other studies [11]. Postmenopausal women appear to be more resistant to Candida colonization, although the incidence of VVC rises among women using hormone replacement therapy, the prevalence of asymptomatic Candida declines with increasing age. In our study only in 6 symptomatic postmenopausal women Candida species was isolated. This reduction in prevalence of Candida in postmenopausal women may be due to decreased levels of reproductive hormones. High levels of reproductive hormones are generally thought to provide a better source for growth of Candida by inducing higher glycogen contents in the vaginal epithelial cells and also some studies say that estrogens have a direct effect on the growth of Candida and its adherence to the vaginal epithelium [12].

In our study, 31% patients were having itching as the presenting complaints, 29.4% presented with vaginal discharge as the initial complaint,15.6% patients presented with only pain as the initial complaint, 13.3% presented itching, pain & discharge as complaint. Other complaints reported are dysuria, redness, dyspareunia, vaginal and vulvar erythema. Our study is in consistent with other authors [13,14].

In the present study, 74.5% of the women belonged to low socioeconomic status. Despite therapeutic advances, vulvovaginal candidiasis remains a common problem worldwide, affecting all strata of society. In our study women of lower socioeconomic strata, poor genital hygiene & illiteracy showed significantly higher incidence of VVC. The use of synthetic clothes could be contributing to VVC by increasing perineal moisture. Our study is in consistent with work done by Jindal et al.,[15].

In the present study behavioral and host-related risk factors associated with VVC and recurrent episodes were assessed. Statistically highly significant difference in incidence of VVC was observed between pregnant and non pregnant women. In the present study 55% of the females who presented with the complaints were pregnant. This may be probably due to high level of reproductive hormones during pregnancy which provides an excellent carbon source for growth of Candida [12] and also an increased susceptibility to infection by species of Candida, resulting in both a higher prevalence of vaginal colonization and a higher rate of symptomatic vaginitis. Our findings were comparable with other studies [15,16].

Significant influence was observed during use of broad spectrum antibiotics by increasing the incidence of VVC in the present study. Candidal vulvovaginitis is a common occurrence after systemic use of broad spectrum antibiotics [13,14]. Antibiotic agents increase vaginal yeast colonization and are thought to act by eliminating lactobacilli, thereby facilitating Candida to grow, adhere and germinate. The concept of interaction between lactobacilli and Candida includes competition for nutrients and stearic interference of adherence to vaginal epithelial cells [13,14].

In our study diabetes was the third most common risk factor found in females with VVC. Many investigators have suggested that vulvovaginal candidiasis (VVC) occur more frequently in diabetics. Further, they also suggest that chronic recurring VVC may be a marker of diabetes. Several studies report increased rates of incidence of symptomatic infection are seen in diabetic women, but results are inconsistent. Potential risk factors for VVC include type of diabetes, severity, and degree of glucose control [17].

High incidence of VVC is also observed in patients on oral contraceptives, and is similar to the findings of other investigators [13,14].

In the present study, culture for Candida species was positive in 40 (22.2%) of 180 women attending our Obstetrics & Gynaecology OPD. This gave a prevalence of 22.2%. This study is in agreement with the work conducted by Bauters et al., [7] who isolated candida species in 20.1%. Mohanty et al., [14] reported 18.5% prevalence of vulvovaginal candidiasis in a community setting. In the present study the prevalence was found to be higher in the age group of 15-45 years. Out of 40 candida species isolated, C.albicans accounted for 65%, followed by C.glabrata in 22.5%, then C. tropicalis in 7.5%, C. parapsilosis in 5% [Table/Fig-3]. Studies conducted in various countries revealed C. albicans to be the most common species in women with VVC (76 to 89%), followed by C. glabrata (7 to 16%) [18–20]. The percentage of non C. albicans species associated with VVC in these countries ranged from 11% to 24%[18–20]. Some studies have reported an increasing trend in the occurrence of non-C. albicans species over time [20,21]. In the present study C.glabrata (22.5%) was the most common non albicans species isolated which is consistent with a study done by Ahmad et al.,[13] in Aligarh, India.

C.albicans isolated in the present study was lesser than the study done by other authors [22,23]. Vaginal culture is also essential for identification of various candida species. In the present study, although C. albicans (65%) predominated, nonalbicans species were found to be present in 35% of infections. Recently several authors have also reported an increase in the incidence of VVC caused by nonalbicans species of candida [22]. The highest proportion of nonalbicans candida reported is that of C. glabrata, which is similar to the finding of our study [21–23]. These nonalbican yeasts are relatively nonpathogenic but ultimately get selected and start appearing more frequently because of the widespread abuse of over the counter antifungals, use of single dose oral and topical azole regimens, and long term maintenance regimens of oral azoles. Therefore vaginal culture is valuable not only for identifying the species of vaginal candida but also for monitoring the changing trends in the microbiology of vulvovaginal candidiasis which is essential for the complete and prolonged treatment [21,22].

The antifungal susceptibility patterns to fluconazole for the 40 candida isolates were performed by E-test method as per standard protocols. Out of 40 isolates, 87.5% were sensitive to flucanozole, 7.5% were moderately sensitive & only 5% were resistant. Out of the 3 candida isolates that were moderately sensitive, 2 were C.glabrata & one was C.tropicalis. Most non-albicans Candida species have higher azole MICs and infections they cause are often difficult to treat [24,25]. With multiple antifungals and varying susceptibility patterns of Candida, it has now become necessary to perform antifungal susceptibility testing and make reports available to the clinician for effective therapeutic outcome Fluconazole resistance in vaginal C. albicans isolates is an uncommon occurrence. In our study, none of the C.albicans isolates were fluconazole resistant.

VVC caused by C.albicans is prevalent in our region. The high frequency with which C. albicans was recovered in our study and its susceptibility to fluconazole supports the continued use of azole agents for empirical therapy of uncomplicated candidal vulvovaginitis in the community. More prospective studies are needed to determine the optimal therapy for candidal vulvovaginitis caused by non-albicans species.