THE PREVALENCE OF VULVOVAGINAL CANDIDIASIS IN PREGNANT WOMEN ATTENDING SEVERAL HOSPITALS IN SANA’A, YEMEN

Candida species are opportunistic yeasts affecting the genitourinary tract which causes the vulvovaginal candidiasis in the most female especially in developing countries. This study aims to determine the prevalence of vulvovaginal candidiasis caused by Candida species causing among pregnant women in Sana'a City, Yemen. This study was carried out at the department of microbiology of AL-Kuwait University Hospital. 250 vaginal swabs were collected and cultured on Sabouraud dextrose agar. Candida species identification and antifungal susceptibility testing were determined according to standard microbiological methods. The results showed that out of 250 samples, 63(25.2%) were positive for Candida species. It was found that the C. albicans (68.3%) was the most common species isolated followed by non-albicans species that are C. tropicalis (20.6%), C. glabrata (6.3%), and C. kefyr (4.8%). Also, it was recorded that the highest prevalence of Candida species was within group aged of 36-46 year. Susceptibility tests revealed that the most isolated species of C. andida were sensitive to nystatin (95%). miconazole was the next effective drug with 64% sensitive followed by amphotericin B (29%) and clotrimazole (24%). But only 6% of the isolates were sensitive to fluconazole. It can be concluded that the vulvovaginal candidiasis are quite common in Yemen country with a high prevalence. Also, the nystatin remains the effective agent against all isolated of Candida species. In contrast, the increase resistance of Candida species to fluconazole that commonly used antifungal is an alarming increase of vaginal candidiasis caused by antifungal-resistant Candida species.


INTRODUCTION
Candida vaginitis is the infection of vagina by several type of Candida species, also often called vulvovaginal candidiasis/candidosis 1,2 . Vulvovaginal candidiasis (VVC) considered to be the most common manifestation of genital candidiasis 1,3 . It is representing over 25% of infectious vaginitis 4,5 . It is reported that 75% of women are affected by vulvovaginal candidiasis in their lifetimes 3 . Also, it was found that more than 40% of women that affected will have 2 or more vulvovaginal candidiasis episodes 6,7 . Clinical manifestation of vulvovaginal candidiasis is pruritus, vaginal discomfort, burning, and soreness 8 . The distribution of Candida sp. in vulvovaginal candidiasis cases differs widely depending on the geographical location and population studied 9. Some reports have documented that among women with acute vulvovaginal candidiasis were caused by C. albicans that accounts for 80-90% of all vaginal candidiasis cases, whereas other species are less frequently isolated 1,10,11 . However, in last year's, there are different species of Candida non-albicans that are C. parapsilosis, C. glabrata, C. krusei, C. lusitaniae, C. tropicalis, C. dubliniensis, and C. guilliermondii isolated from vaginal samples 12,13,14 . Misuse of antifungal drugs and lack of effective polices that control the use of antifungal especially against the vulvovaginal candidiasis lead to increase resistant of Candida species to several antifungal drugs 15 3,19 .

Examination of Specimen Microscopic examination
The first swab was subjected to wet mount examination. One drop of normal saline was added to each sample and shaking vigorously and examined microscopically under 10x and 40x 20 .

Culture methods
The second swab was cultured on surface of Sabouraud Dextrose Agar (SDA) (Himedia, India) with and without chloramphenicol (250mg/L). The plates were incubated for 48 h at 37°C. The morphological features for colony were studied and confirmed by observing the budding characterization with pseudohyphae by using the Gram tube test. The growth of bacteria on the SDA plates without chloramphenicol was purified on Blood agar and MacConkey agar and identified by biochemical test 21 .

Candida species identification
Candida species were identified depending on morphological features on a culture medium, germ tube formation and carbohydrate assimilation test as the following: Germ tube test A small portion was taken from pure colony of C. albicans by sterile loop and inoculated into sterile tubes containing 0.5ml of human serum. The tubes were mixed and incubated aerobically for 2h at 37°C. One drop of each serum was transferred to a clean slide and examined by microscope under high power (x40) to detect the presence of germ tubes that are short hyphal initials 22 .

Sugar assimilation test
The overnight of culture yeast suspension was added to basal carbohydrate-free medium (II) of molten agar, cooled to 45 °C, and poured to plates and leaved the plates to solidified. Discs saturated with 1% of sugar and placed on the surface of plates and incubated for five day at 37°C. The occurrence of growth around each disc indicates the carbohydrate assimilation of tested sugar. The glucose, D-galactose, maltose, sucrose, lactose, raffinose, xylose and trehalose were used in the sugar assimilation test 23 .

Statistical analysis
The statistical analysis was performed by using program version 20 SPSS (Statistical package for social Science). Percentage and Chi-square test were used to evaluate the degree of the significance with 95% confidence (p<0.05).

RESULTS
The result from the current study revealed that the 250 vaginal swabs were collected from pregnant women who presented with genital manifestations. Only 185 samples (74%) were showed as positive growth in culture media and 65 samples (26%) were reported as negative growth in culture media as shown in Figure 1. This study revealed that an overall isolation rate of candidiasis was 34.06% and 65.94% were bacterial vaginitis from vaginal swabs as listed in Table 1.
From the 63 positive isolates of Candida species, it was found that the highest prevalence of vulvovaginal candidiasis was among women from urban (80.20%) compared to women from rural areas (19.80%) as shown in Figure 2. Also, this study showed that the highest percentage for first time infection was 52/193 (26.9%), while the recurrent infection was 11/57 (19.3%). This study showed that the most clinical symptoms among women with vaginitis and frequency of Candida isolated were burning, discharge, and itching ( Table 4).

DISCUSSION
Vulvovaginal candidiasis is caused by the overgrowth of yeast in the mucosa membrane of the female genital tract and frequently diagnosed as a daily practice of gynecologist 14,25  This study analyzed the predisposing factors of vaginal candidiasis from positive Candida isolated; pregnancy has been the most frequently associated risk factor (33%). This high percentage due to the increased amount of glycogen in the vagina and high levels of estrogen hormones. It provides a good source of carbon, which favors the growth of Candida species 30 , and lowest occur with kidney transplantation (3) and leukemia (2) this may be depended on the type of sample at this study. The result of this study is in agreement with the work of Abu Baker 31 and Babin et al. 32 . In this study, the frequency of isolated Candida among women complained of burning, discharge and itching were (27%) which was the highest percentage. This study has differed with Falahati et al., 33 who showed that the frequency of isolated Candida among women complained of discharge was 55 (82.1%), itching 42 (62.7%), and burning 33 (49.3%). The result of this study was indicated that C. albicans 43(68.3%) are responsible for the greatest number of symptoms associated with the vaginal candidosis. This finding is in agreement with the work of Al-mamari et al. 26 who found that the 65.95% of isolated Candida species was C. albicans. Another study by Omar et al.,34 in Egypt who found that the C. albicans was the highest (78.3%) isolated species from infected women by vaginitis. During the last three decades were noticed that the increase in percentage of vaginitis caused by non-albicans species of Candida. The present study showed the increase in the frequency of non-albicans species as potential causes of vaginal candidiasis. It was found that the C. tropicalis (20.6%), C. glabrata (6.3%), and C. Kefyr (4.8%) were recorded in this study. This finding was supported by Babin et al., 32 in Iran.
In the antifungal susceptibility results, it was reported that the all isolates were highly sensitive to Nystatin and this finding is in agreement with the work of with Al-mamari et al. 26 and Mahmoudabadi et al., 35 . In addition, in this study of 63 positive cases, Candida species were sensitive to Miconazole and this finding in agreement with work of Al-mamari et al. 26 and Abruquah 36 . This study revealed that the most of Candida species showed higher resistance to Fluconazole which may be due to frequent use of these drugs as therapeutic alternatives to Amphotericin B. Azole antifungal agents are easy for administration and are less toxic 9,32,26 .

CONCLUSION
In conclusion, vaginal infections are very common in the region and have a high frequency. It was found that C. albicans (68.3%) was the predominant isolated species followed by C. tropicalis. All isolates were susceptible to nystatin and fluconazole. This is the first report on the types of Candida sp., causing vaginal candidiasis and their antifungal susceptibility patterns in Yemen.

AUTHORS' CONTRIBUTION
The manuscript was carried out, written, and approved in collaboration with all authors.

CONFLICTS OF INTEREST
There are no any conflicts of interest.