Abstract: Vaginal candidiasis is a common infectious disease involving genital organ. Candida albicans is the major cause of Candida vaginitis and the other species are less common but an increase of nonalbicans species has been observed. The aim of this study was to identify the etiological agents of vaginal candidiasis in Shiraz, southern Iran. A total of 183 patients suspected of Candida vaginitis were enrolled in our study. The specimens were collected by using vaginal swabs and cultivated on Sabouraud dextrose agar media supplemented with antibiotics. All the cultures were incubated at 35-37°C for 24-48 h. Identification of Candida species was performed based on Germ tube, Chlamidoconidia, Thermo tolerant tests and CHROMagar Candida medium. Eighty (43.71%) cases of Candida vaginitis were diagnosed among 183 patients suffering vaginitis. Candida albicans was the most dominant isolates and nonalbicans species were responsible for 21.25% of vaginal candidiasis. The frequency of Candida isolates were: Candida albicans 63 (78.75%), Candida glabrata 7 (8.75%), Candida krusei 5 (6.25%), Candida tropicalis 2 (2.5%) and Candida sp. 3 (3.75%). No species of Candida dubliniensis was distinguished from Candida albicans by Thermo tolerant test and Chromogenic media. Present study indicated that the rate of Candida vaginitis caused by nonablicans species was high. It seems that inadequate antifungal therapy and incomplete self medication could be a major cause for increasing drug resistance in nonalbicans Candida species.
INTRODUCTION
The most common infection treated by primary care physicians is vaginitis. It has been reported that 30-50% of vaginitis episodes are due to Candida sp. and that two-thirds of all women experience at last one acute episode of vaginal candidiasis during their life time (Ross et al., 1995). Some workers have suggested that a high percentage of women will suffer from it before the age of 25 years and approximately 75% before the age of 40 (Sobel, 1997, 1999). Vaginal candidiasis is an infectious disease involving vaginal ecosystem (Lanchares and Hernandez, 2000). It can occur in mild to severe forms, starting with the typical symptoms of itching, increased thin discharge, which later turns cheesy, marked reddening of the vagina and later stinging. About 5% of those affected suffer from chronic, recurrent vulvovaginal candidiasis, which is defined as episodes reoccurring at least four times within one year (Mendling and Seebacher, 2003). Candida ablicans is causative agents in 80-90% of all cases while C. glabrata is the second followed by C. krusei, C. tropicalis, C. kefyr, C. parapsilosis, C. guilliermondi which is called nonablicans (Mendling, 2003; Boselli and Seebacher, 2004; Ben-Haroush et al., 2004).
In recent years a change in epidemiological trends has been observed. There has been a significant increase in infections caused by non-ablicans species of Candida, particularly in recurrent cases. At present, these species are found in approximately 20-30% of cases of recurrent vaginal candidiasis. Among them, Candida glabrata is the most common type (Lanchares and Hernandez, 2000). Since we have no information about the composition of Candida species causing Candida vaginitis in Shiraz, the aim of this study was to determine the incidence of Candida vaginitis (CV) and the etiological agents in premenopausal women in Shiraz, southern Iran.
MATERIALS AND METHODS
From April 2004 to March 2005, 183 patients suspected of Candida vaginitis which attended to gynecology clinic of Shiraz Medical School with presence of Vaginal Candidiasis symptoms (irritation, pruritis, soreness and altered discharge) were enrolled to our study. Vaginal swabs were collected from vaginal secretions and used for direct smear examination and culture. Microscopic examination of wet mount with KOH (potassium hydroxide 10%) and smear stained with Methylen blue and Giemsa were performed. The swabs were cultivated on Sabouraud dextrose agar supplemented with Penstrep-400 (Penicillin 200,000 IU L-1 and Streptomycin 200 mg L-1), Chloramphenicol (200 mg L-1) and Gentamicin (40 mg L-1). The cultures were incubated at 37°C for 24-48h or until the colonies appear. Identification of Candida species was performed by different methods such as Germ tube test, Chlamidoconidia test (Cultivated on Corn Meal Agar plus Tween 80) and Chromogenic media, using CHROMagar Candida medium (Dr. A. Rambach, France).We used thermotolerant test (growth at 42-45°C) and colony color (CHROMagar Candida medium) for distinguishing C. albicans from C. dubliniensis.
The software package Microsoft Excel 8.0 was used for data processing.
RESULTS
A total of 80 cases from 183 patients suspected of Vaginal Candidiasis (aged 15-45) were diagnosed. The prevalence of CV was 43% and Candida ablicans (78.25%) was the dominant agent in this study. All Candida albicans isolates with positive in germ tube and Chlamidoconidia test had green colony color on CHROMagar medium (Fig. 1). The rate of nonalbicans Candida species was 21.25%. Three species of Candida (with presence of pseudohyphea form on corn meal agar) had no distinct color on CHROMagar medium and could not identify by this method. The frequency of Candida species was: C. albicans 63 (78.75%), C. glabrata 7(8.75%), C. krusei 5 (6.25%), C. parapsilosis 2 (2.5%) and un-known Candida 3 (3.75%) as showed in Table 1.
No species of Candida dubliniensis was identified in this study.
| Fig. 1: | Colony color on CHROMagar Candida medium |
| Table 1: | Distribution of Candida species isolated from vaginal candidiasis |
DISCUSSION
Vaginitis is a common gynecologic condition and accounts for more than 10 million office visits each years (Kent, 1991). Vaginal candidiasis caused 20-25% of infectious vaginitis cases, second only to the 40-45% of cases caused by bacterial vaginosis and marked by pruritis, soreness and a change in discharge, dyspareunia, vulvar erhytema, edema and fissures (Sobel, 1997, 1999). Many physicians believes that these signs and symptoms are almost suitable for diagnosing CV without any laboratory results but our study reveal that at least 53% of all patients with similar symptoms had no Candida vaginitis. Although Grigoriou et al. (2006) reported this rate in 12.1%.
It is quite known that direct microscopic examination of wet mount with potassium hydroxide and vaginal cultures on solid media is the simple and good technique for CV diagnosis (Grigoriou et al., 2006; Trama et al., 2005), so positive examination result is necessary before any therapy.
Candida isolation from the samples of vaginal exudates is very frequent finding and it maybe isolated from approximately one-fourth of symptom-free women (Trama et al., 2005). It has been reported that 30-50% of vaginal episodes are due to Candida sp. and that two-thirds of all women experience at least one acute episode of CV during their life time (Sobel, 1997; Kent, 1991). Among the Candida species causing infections, Candida albicans, Candida glabrata, Candida parapsilosis and Candida tropicalis account for 80-90% of fungal isolates encountered worldwide. Ferror (2000) reported that Candida ablicans is usually the predominant yeast isolate according to 80-90% of yeast Vaginitis. C. glabrata is the second most species, causing approximately 5-15% of cases of CV. Over the last 30 years the incidence of the other species of Candida, termed non-albicans Candida, have steadily increased and C. glabrata being the most common species of this subgroup (Phillips, 2005). In this study, 21.25% of all the isolates were nonalbicans species, although this data was close to Grigoriou et al. (2006) in 19.8% but was not in agree with those Vrabik et al. (2007) and Paulitsch et al. (2006) studies which these rates were 8 and 12.1%, respectively. They did not confirm the higher prevalence of nonalbicans species in Candida vaginitis patients.
We could not identify three species of the isolates, because the chromogenic media usually used to identify clinically important Candida but not all kinds of the species (Odds and Bernaerts, 1994). By this reason, Saunte et al. (2005) could not identify four species of Candida albicans on CHROMagar Candida plates. So we highly recommend to use the other diagnostic methods such as biochemical assimilation patterns and molecular techniques for better identification of Candida species (Trama et al., 2005; Tabrizi et al., 2006).
Candida dubliniensis is another opportunistic pathogen that can cause both superficial and invasive infections. It is found mostly in the immunocompromised and AIDS patients and found all around the world (Sullivan and Coleman, 1998; Jabra-Rizk et al., 1999). Moreover, C. dubliniensis almost isolates from the oral cavity of HIV patients and less common in Candida vaginitis (Mendling and Seebacher, 2003). The most useful test for distinguishing C. dubliniensis from C. albicans is to culture them at 42-45°C which most C. albicans will grow (Mariano et al., 2003; Brito et al., 2006). The colonies with light green color on CHROMagar Candida media were considered typical for C. albicans, where those with a dark green color on primary isolation were initially considered atypical C. albicans and suspected to C. dubliniensis (Kirkpatrick et al., 1998; Momani and Qaddoomi, 2005). Gigoriou et al. (2006) founds that 0.5% of their patients had CV due to C. dubliniensis and in accord with our methods. Us and Cengiz (2007) could identified only one strain of C. dubliniensis from 218 pregnant women. In this study, no species of C. dubliniensis was identified and we had no patients with a history of HIV infection.
Candida vaginitis is usually treated, using topical and systemic azoles (Fluconazole or Ketoconazole) and responds to antifungal treatment uniformly well, but the nonalbicans Candida has variable responses to conventional azoles treatment. The widespread use of azoles antifungal drugs is postulated to have promoted the shifting of vaginal colonization and selection of more naturally resistant species (Ross et al., 1995; Phillips, 2005; Soble et al., 2003). Several investigations have recently reported that the cases of sporadic and recurrent vulvovaginal candidiasis caused by nonalbicans species of Candida are increasing. Only a few of these studies have provided data to support this claim. In specialized clinics, more than 10% and occasionally, less than 20% of patients were infected with nonalbicans Candida organisms (Soble et al., 1998). Short-course therapy, either oral or topical may be eliminating the more sensitive C. albicans and selecting for more azole-resistant nonalbicans Candida species. Ferror (2000) pointed out that the increasing detection of nonalbicans species has been related to the widespread and inappropriate use of antimycotic treatment, self medication, long term maintenance treatments and repeated treatments for Candidosis episodes. Although Poch and Levin (2002) could not documented an association of nonalbicans species with increased use of azole antifungal agents.
This is the first study on vaginal Candidiasis in Shiraz which focused on identification of the etiological agents. This study reveals that the rate of nonalbicans Candida species was high and further study needs for evaluation of their sensitivity to antifungal drugs and using molecular methods for better identification.
ACKNOWLEDGMENT
We would like to express our appreciation in Dr. Kamiar Zomorodian and Sakineh Kemarloei for laboratory and scientific supports.