Background: Candida is the most common agent causing leucorrhea affecting the women of all strata. It is becoming difficult to completely eradicate the infection mainly due to recurrence caused by non-albican species of Candida. Most of the non-albican species of Candida are resistant to commonly used antifungal agent – azole. Therefore, studying the prevalence of Candida species in vaginal secretion is of great significance. Objective: To study the prevalence of different species of Candida and the efficiency of different Candida detection methods in women from low socio-economic setup of Miraj and Sangli, Maharashtra, India. Materials and Methods: The study was conducted on 150 patients with specific complaints of leucorrhea. In the control group, 50 asymptomatic women were included for comparison. Results: In 33% of the women the leucorrhea was due to Candida infection with highest incidence in women of sexually active age (20-40 years). Sabouraud’s culture was the most efficient method (100% efficiency) to detect the Candida compared to wet mount, KOH and gram stain method. Candida albicans was the most common strain identified and Candida krusei was the least common one. Conclusion: Candida infection is the commonest reason for leucorrhea and non-albican candida species significantly contribute to candidiasis in women of Miraj and Sangli.
Keywords: Candida albicans , candidiasis, leucorrhea, Trichomonas vaginalis
Vulvovaginitis is an infectious condition of the female genital tract characterized by whitish or yellowish discharge from the vagina. Candida is the most common microorganism responsible for vulvovaginitis in adolescent women. The survey conducted all over the world indicates that 75% of women will have at least one episode of candidal vaginitis during their life time, especially during their child-bearing age and, 40-50% of women may experience a recurrence. Effective anti-candida defense mechanisms in the vagina allow long-term persistence of candida organisms as vaginal commensals in an avirulent phase.
Majority of the yeast strains (85-95%) isolated from vagina are Candida albicans.  However, incidence of vulvovaginal candidiasis caused by non-albican species is reported to be increasing worldwide.  Most of the non-albican species of candida are responsible for recurrence and are difficult to treat due to their resistance to the commonly used azole-based antifungal agents. To achieve effective infection control, it is necessary to detect the candida species prior to the treatment. The present investigation was carried out to study the prevalence of different Candida species in women of Miraj and Sangli, Maharashtra, who presented to the clinic with complaints of leucorrhea.
The study was conducted on 150 women with specific complaints of leucorrhea attending the Obstetric and Gynecology Department, General Hospital, Sangli, and Government Medical College Hospital, Miraj, from January 2001 to January 2002. In the control group, 50 asymptomatic women were included. During the entire study, recording of the patient history, local examination, collection of the vaginal discharge and the microbiological assessment was performed by a single person.
Duration, color, type, amount and odor/foul smell of the vaginal discharge were noted. Associated complaints such as pruritus, burning micturition, dysuria, backache, abdominal pain, menstrual history, use of contraceptives and obstetric histories were also recorded.
Local examination and sample collection
The patients were examined for vulvitis, vaginitis, cervicitis and cervical erosion. Sims posterior vaginal speculum was introduced and the vaginal discharge was collected from posterior vaginal fornix using sterile cotton swabs. The swabs were placed in sterile aliquots containing 0.5 ml of physiological saline. The acidity of the vaginal discharge was assessed by direct pH measurement using a pH indicator strip.
Rapid microscopic examination of the vaginal discharge for Candida species was done by wet mount technique after treating the sample with a drop of 10% KOH.
For culture, the swab was inoculated in two separate tubes containing Sabouraud’s dextrose agar with Chloramphenicol incubated at 37°C and room temperature. The Candida species were identified by standard procedures based on their morphology,  germ tube test, cornmeal agar test and assimilation of various sugars. ,
The study was conducted on 200 women (150 with leucorrhea and 50 asymptomatic women) belonging to low socioeconomic group residing in Sangli and Miraj of Maharashtra. Among 50 asymptomatic women, 25 were pregnant. The incidence of leucorrhea was highest in women of age group 20-29 years (42.66%) and minimum in women aged above 50 years (3.33%) [Table 1]. The incidence was 34% in women of age group 30-39 years. Only 8% of women were below the age group of 20 years. Similarly, the incidence of candidiasis was highest in the women of age group 20-29 years (44.89%) followed by women of age group 30-39 years (36.73%) [Table 1]. The incidence was three-fold lower (12.24%) in women of age group 40-49 years compared to women of above age groups. The incidence was 4.08% and 2.04% in women of age group <20 years and >50 years, respectively.
When we studied the causative agents in women with leucorrhea, 49 women (32.66%) were harboring Candida out of which 3 (2%) were positive to both Candida and Trichomonas vaginalis [Table 2]. In ~17% of the women, vaginitis was of bacterial origin. T. vaginalis was detected in 14 women (9.3%) among which 3 were positive to both Candida and T. vaginalis. However, in 65 women (43%) the etiology was of unknown origin. Among 50 asymptomatic women only 13 were harboring Candida and all were in 20-29 year age group.
The predisposing factors associated with candidiasis were analyzed in women with leucorrhea and asymptomatic women [Table 3]. Pregnancy was the major predisposing factor among the Candida positive women contributing to ~49%. Even in asymptomatic women, pregnancy was the major predisposing factor (69.23%). Candidiasis was associated with regular intake of oral contraceptives and antibiotic therapy in ~15% and ~13%, respectively, in women with leucorrhea. Other factors such as diabetes mellitus and steroid intake had minor contribution to candidiasis (~7% and 2%, respectively). However, no predisposing factors were identified in 8 Candida positive women (16.32%) with leucorrhea and 4 asymptomatic women (30.77%).
When we analyzed the sensitivity of various assay systems used in the present investigation to detect the Candida, Sabouraud’s culture was found to be the most sensitive method, which efficiently detected all Candida positive cases (100% efficiency) [Figure 1]. KOH preparation and Gram stain technique was found to have an equal sensitivity (48.39%), which was two times below the efficiency of Sabouraud’s culture method. The least sensitive method was wet mount technique, which had an efficiency of 16.12%.
When the vaginal swabs of women with leucorrhea were studied for different species of Candida, 49% of the Candida positive cases tested positive for C. albicans species [Figure 2]. C. glabrata and C. tropicalis were detected in ~22 and 14% of candida positive samples, respectively. Candidiasis due to C. stellatoidea and C. krusei was found to have the lowest incidence among these women (~8 and 6%, respectively). Even in the asymptomatic women, in 70% of the cases the infection was due to C. albicans and rest 30% was equally due to C. tropicalis and C. krusei. Among asymptomatic women, 9 (69.23%), 2 (15.38%) and 2 (15.38%) of women were harboring C. albicans, C. tropicalis and C. krusei, respectively.
In the present investigation, 42.66% of women with leucorrhea were of the age 20-29 and 34% were in the age group 30-39 years, comprising more than 75% of the entire study group. Ovarian activity as well as sexual activity is maximum in women of 20-30 years age. During this period, the ovary produces adequate amount of estrogen, which favors the Candida growth by maintaining the acidic pH and enhancing the yeast adherence to vaginal epithelial cells.  Sexual transmission of Candida also depends upon frequency and periodicity of sexual intercourse and poor sexual hygiene.  The present study was carried out in women of rural area and of poor socioeconomic status who have a poor general health condition, poor sexual hygiene. Studies have shown that the incidence of leucorrhea is significantly higher in women of low socioeconomic group and rural area. 
Candida was found to be the major etiological agent causing leucorrhea in the women of present study group with an incidence of 32.66%. The results are in parallel with an earlier report conducted on Indian women.  Several techniques are available to diagnose Candida from vaginal secretions. Since the symptoms and signs of vulvovaginal candidosis are not specific to the infection, diagnosis cannot be made solely on the basis of history and physical examination. Most patients with symptomatic vaginitis can be readily diagnosed by microscopic examination of vaginal secretions. In our study we were able to detect 8.36% of the Candida positive cases by saline wet mount technique, which agrees with some earlier reports. , A 10% potassium hydroxide (KOH) preparation is more sensitive than a saline preparation in identifying yeast or hyphae. We observed that the efficacy of KOH preparation and gram staining technique is same in detecting candida (48.97%). However, the Sabouraud’s technique had the maximum efficiency (100%), which could detect all the Candida positive cases.
The most common predisposing factors associated with candidiasis are pregnancy, use of contraceptives, diabetes mellitus, antimicrobial therapy, sexual activity and sexual hygiene. The result of the present investigation suggests that pregnancy is one of the major predisposing factors for vaginal candidiasis, which is well supported by some earlier reports.  This was true even in case of asymptomatic women where 9 out of 25 pregnant women harbored Candida. During pregnancy, elevated level of reproductive hormones like estrogen increases the vaginal glycogen content that acts as a carbon source for the growth of Candida species. 
Effect of contraceptives on predisposition to vulvovaginal candidosis is poorly understood and has conflicting supporting evidence. , In our study we found that approximately 15% of the women having candidiasis were taking oral contraceptives. High estrogen content in oral contraceptives may be one of the predisposing factors as estrogen facilitates the adherence of yeast to vaginal epithelium. The use of vaginal or systemic antibiotics is found to increase the vaginal colonization rate by 10-30%.  In our study subjects, ~12% of the Candida-positive women were under antibiotic therapy. Vaginal colonization of Candida is more frequently observed in diabetic women than in non-diabetic women  and women undergoing steroid therapy.  Poor immune status and high glycogen deposition due to hormonal imbalance in diabetes mellitus and lowering of immune defense mechanism by corticosteroids increases the risk of candidiasis. In our study we observed that only 3 (6.12%) and 1 (2.04%) women were diabetic and under steroid therapy, respectively. Candidiasis was of unknown origin in 8 out of 49 candida positive women. Genetic factor and exposure of vaginal milieu to allergens and chemicals may act as predisposing factors. Increased prevalence of vulvovaginal candidosis in African-American women  and people with blood group ABO-Lewis non-secretor phenotype suggests that there could be genetic factors that predispose individuals to colonization or vaginitis.  Chemical contact, atopy, local allergy, or hypersensitivity reactions could alter the vaginal milieu and facilitate transformation from asymptomatic colonization to symptomatic vaginitis. 
In the present investigation, ~50% of women with leucorrhea were harboring C. albicans. The C. albicans have high adherence capacity to vaginal epithelium compared to non-albicans species.  However, we also observed a concomitant increase in the prevalence of non-albicans species in our study group. Among the non-albicans species, C. glabrata was the most common type (22%) and C. krusei was the least common type (6.12%). Studies have shown that C. glabrata is one of the major causes for recurrent vulvovaginal candidosis. More than 20% of the women in the present study were infected with C. glabrata which agrees with the reports from other parts of the world. , Vaginitis induced by non-albicans species is clinically indistinguishable from that caused by C. albicans. The explanation for increase in the incidence of vulvovaginal candidosis caused by non-albicans strains is thought to be because of single-dose treatment, low-dosage azole maintenance regimens and the use of over-the-counter antimycotics.  Therefore for effective control of candidiasis, it is advisable to identify the Candida species before planning the treatment.
In conclusion, the candidiasis is the most common condition associated with leucorrhoea in women of Miraj and Sangli, Maharashtra. More than 50% of women with candidiasis were harboring non-albican species of Candida suggesting the increased prevalence of these species in rural India. For effective treatment of the infection, to prescribe the correct medication and to overcome the recurrence, it may be advisable to identify the Candida species routinely from vaginal swabs of infected women.
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]