Introduction

Vaginitis is the most prevalent disease among females, which is responsible for 10 million clinical visits every year (1-3). This disease is the second common genital tract infection among females and is usually caused by C. albicans. Nonetheless, the prevalence and causes of vaginitis are not known yet. Vaginitis is normally asymptomatic and has more than one etiology. Most specialists believe that 90% of cases of vaginitis are secondary to bacterial vaginosis, vulvovaginalcandidiasis, and trichomoniasis (4). In addition, vaginitis may occur as the result of non-infectious causes, such as vaginal atrophy, allergies, and chemical irritation (5).

Sometimes, vaginitis is accompanied by pelvic inflammatory disease, post-operative infections, abnormal cervical cytology, chronic cervicitis, and urinary tract infection (6). In England, Fai-Katty (1993) and Gardner (1998) reported the prevalence of Candida vaginitis to be 25% and 27%, respectively (7, 8). Besides, in a study conducted in Georgia University, Freeze et al. (1995) demonstrated that out of 499 cases with vaginitis 20%, 7.4%, 25%, and 14% were diagnosed with candidiasis, trichomoniasis, bacterial vaginosis, and combined infection, respectively (9).

Similarly, Bomsil et al. (2004) conducted a study on vaginal wet mount of 749 females. According to the microbiology results, 50.6%, 25.6%, 17.4%, 5.3%, and 0.3% of the cases had cytolytic vaginosis, Gardnerella vaginalis, Candida vaginitis, trichomoniasis vaginosis, and gonorrhea, respectively (6).

C. albicans is the major cause of Candida infection in patients with vulvovaginitis, yet there are other species that are responsible for this kind of disease (10-13).Given the high prevalence of vaginitis, using serological techniques such as double diffusion method for diagnosis is helpful (14). Pirotta and colleagues' (2006) study showed that C. albicans was responsible for 75% of the infections, whereas other Candida species accounted for 25% of the cases (15).

A retrospective survey of 93,775 samples testing positive in Candida species-specific PCR tests demonstrated consistent yearly distributions of Candida albicans (89%), C. glabrata (7.9%), C. parapsilosis (1.7%), and C. tropicalis (1.4%) (5). Likewise, in some other studies, vulvovaginal candidiasis was demonstrated to have the highest frequency, compared to other vaginitis. (16-20).

According to the literature, the prevalence of recurrent Candida vaginitis and treatment-resistant cases have increased. Since most of these cases are induced by C. non-albicans species (15), it is essential to determine the prevalence rate of the infection and accurately identify Candida species responsible for Candida vaginitis. The inappropriate use of anti-fungal medications can lead to more inflammation, distress, irritation, excoriation, rubbing, maceration, and even secondary infection (21). Regarding this, it is necessary to make an accurate and early diagnosis based on para clinical tests and clinical trials data. Therefore, the aim of the current study was to investigate the prevalence of vulvovaginal infections and characterize the Candida species isolated from patients with vulvovaginitis using three different tests.

Materials and Methods

The sample size of this study was determined using randomized block design; as a result, 105 samples were examined. First, 1100 patients who had referred to gynecology clinics for various problems were evaluated for 7 months. Among these patients, 450 ones had complaints about genital infections. Based on the patients’ history and vaginal discharge characteristics, 280 patients were entered into the study and 105 females were diagnosed with Candida infection using serological diagnosis.

This descriptive study was conducted on 105 females referring to the Gynecology Clinics of Shahid Faghihi, Zeynabieh Hospitals, and Hor-e-Riahi Health Center affiliated to Shiraz University of Medical Sciences, Iran during March to September of 2009. Health centers were selected based on their geographical location in the Northern, centeral, and Southern regions of the city.

 The inclusion criteria of the current study were as follows: 1) being married; 2) being at reproductive age, 3) not having vaginal bleeding; 4) not suffering from any known chronic diseases such as those weakening the immune system; 5) not having used herbal or chemical drugs for treatment of genital infection in the recent 2 weeks; 6) being diagnosed with Candida vaginitis based on direct test and fungal culture; 7) not having undergone vaginal douching within the past 48 h; 8) not having used broad-spectrum antibiotics in the recent 2 weeks; and 9) not having used vaginal creams or suppositories within the past 48 h. On the other hand, the exclusion criteria were negative fungal culture on Sabouraud dextrose agar medium.

 In the first stage, 1100 patients with gynecological problems were visited and 450 females were diagnosed to have genital infections. In the second stage, the patients with Candida vaginitis symptoms (itching, discharge, color, ...) were selected through purposive sampling and were entered into the study after signing written informed consents (N=280). In the last stage, 105 patients were diagnosed with candidiasis using three different tests. It should be mentioned that all the samples were collected from the posterior fornix region which contains the highest amount of discharge.

The data were collected using a form including the following sections. The first part consisted of the patients’ demographic charac-teristics including age, occupation, marriage age, and education level. The second part of the questionnaire inquired the history of infection, drug consumption, recurrence, and the third part investigated the features of the recent infection, including the intensity of itching, burn, color of discharges, etc.

All stages of research were taken by the researchers and demographic data were collected from patient's medical history.

In order to collect the samples, first, the patient lay in lithotomy position and the smell, color, volume, and other features of vaginal discharges were examined. Then, the samples were collected using speculum and sterile swab. Overall, two samples were obtained from each patient. The first sample was directly put on the lam for preparation of smear and staining by methylene blue method. On the other hand, the second sample was used for culturing on Sabouraud dextrose agar medium in sterile conditions. After sampling, the samples were daily transferred to the laboratory in order to determine the type of fungus.

In order to determine the intensity of infection (the number of fungal colonies) and differentiate Candida species, the sterile swabs were dragged on the culture media in a zigzag pattern. Afterwards, the characteristics of the samples, such as number and date, were recorded on the plates containing the culture media. These plates were transferred to the mycology laboratory of School of Medicine at the end of the work shift and were kept in the incubator at 30ºC for 24-48 h. Thereafter, in case of colony growth, the colonies were counted, purified by passages, and kept at sterile distilled water at -20ºC. In this study, fungal species were identified using germ tube test, CHROMagar medium, and chlamydospore formation by an expert in the School of Medicine laboratory.

 This test is the gold standard method for C. albicans diagnosis. In this method, the intended yeast is inoculated in human or rabbit serum diluted by normal saline (1:2) and is kept in the incubator at 37ºC for 2-3 h. In case of Germ tube formation, Candida albicans was considered. (22).

This test aims to differentiate Candida species based on the color produced on the media. In this study, fungal species were purified, cultured, and incubated at 30ºC for 48 h, using a chromogenic medium. Thereafter, the types of fungi were determined based on the colonies’ colors (HiMedia, India). In this study, the following 8 colors were observed: green, light green, green with a blue border, bluish green, purple, blue, white, and white with blue spots. The first 4 colors were all categorized as green and were interpreted as C. albicans. Furthermore, blue and white with blue spots were considered as C. tropicalis, while pink, purple, and violet were identified as C. krusei. In case yeast was observed in chlamydospore test, white was considered as C. glabrata. On the other hand, if pseudo-hyphae was detected in this test, this color represented other C. non-albicans species (23, 24).

 This test is used for identification of Candida species. In general, more than 90% of isolated C. albicans form chlamydospore on corn meal agar medium. Similar to germ tube formation, this feature is also specific to C. albicans. In this study, the yeasts were cultured through a - corn meal agar medium and were kept at 25ºC for a week. In addition to chlamydospore, C. albicans also form pseudohyphae on this medium. In case pseudohyphae and blastospore are detected, C. non-albicans species are identified. On the other hand, growth of yeast cells is likely to be suggestive different yeast species, most probably C. glabrata (23, 24).

 In this study, data analysis was performed using SPSS 16 (Released 2007, SPSS for Windows, Version 16.0., Chicago, SPSS Inc.). The study variables including age, education status, distribution of different types of organisms, Candida species, and color of vaginal discharges were presented as frequency and percentage. The data were checked for normality of distribution and their mean and standard