Cervico-Vaginal Candidiasis in Married Women

The present study is aimed to identify the isolated yeasts from vagina and cervix of the pregnant and non-pregnant women. The study included 100 patients (50 pregnant; 50 non-pregnant women), in addition to 50 apparently healthy women. The clinical specimen were collected during the period from December 2013 to May 2014. From each patient high vaginal and endo cervical swabs were collected, in addition to the control group. Three slides were prepared from each swab for direct examination (Slide immersed in normal saline, slide immersed in 20% KOH wet mount and Gram stained slide).The clinical specimens cultured on Sabouraudʼs agar and Brain heart infusion blood agar. Each culture was identified to yeast species by germ tube test, Chrom agar and API 20 C system. The tested women considered infected with Candida spp when the culture from the clinical specimen for each contain > 10 colonies together with positive direct examination and symptoms and signs. The main isolates were C.albicans from pregnant (84.8% from vagina ; 89.7% from cervix) and non-pregnant (66.7% from vagina ; 64.3% from cervix) women. In addition to the control (50% from vagina ; 0% from cervix) group.

were enrolled in the current study as a control group. All of them non-pregnant women. Their age ranged between 16 -50 years.

Sample collection and processing
A total of 200 samples were collected from symptomatic women attending the Outpatient Clinic of Al-Batool and Al-Khansaa Teaching Hospitals for various gynecological and obstetrical problems in Mosul. The samples consisted from 100 high vaginal swabs and 100 endocervical swabs collected aseptically under full illuminated condition using sterile cotton swabs. From the 50 control married females, both vaginal and endocervical swabs were also obtained and processed in same manner as for the patients.
All the swabs from patients and control group were collected in sterile containers, then brought to the laboratory within two hours.

Isolation of the Yeasts
Each swab (vaginal and cervical) was inoculated onto both Sabouraud dextrose agar with chloramphenicol and Brain -Heart Infusion (BHI) blood agar with chloramphenicol. The specimens were streaked on all the surface of the media to obtain separated colonies. The plates were then incubated aerobically at 37Cº for 2 -3 days, then checked for growth of yeasts depending on colony morphology and microscopy in lactophenol mount and considered negative and discarded after a third day of incubation [12]. Pure cultures of the yeasts were obtained by subculture of each isolate on Sabouraud dextrose agar at 37Cº for 2 days then preserved as stock culture at 4Cº for further study [13]. Three slides were prepared from each clinical specimen. The first slide immersed in normal saline, the second slide immersed in 20% KOH solution with parker ink, and the third heat fixed smear was stained by Gram's stain.

Tests for identification of the yeasts
1-Lactophenol mount of small portion of the isolated colonies, to determine the morphological features of different yeasts [14].
2-Germ tube test for the production of short initial hyphae [15].
3-Chrom agar medium was inoculated with a small portion of each yeast colony, then incubated for 2 -3 days at 37Cº, producing colonies of different colors [16].
4-API -20 C system was used for the identification of yeasts according to the analytical profile index.

Statistical analysis
Data will be recorded on a specially designed questionnaire, collected and entered Statistical Package for Social Sciences (SPSS) version 22, and then analyzed statistically by using tables, pie and bar charts according to Dunn and Clark 2009 [17].Chi square and T-test were used to find out the relationship (association) between different variables. Statistical results were considered significant at P level of < 0.05

RESULTS
The 100 pregnant (50%) and non-pregnant (50%) women with genital infection that were included in the present study were of age ranging between 16 -50 years. They were categorized into 4 age groups ( Stained slides with Gram's method and others mounted slides with 20% KOH solution with parker ink for all the clinical specimens showed budding yeast cells with or without pseudohyphae as shown in Table 3. In pregnant women, 64% of the HVS showed the presence of budding yeast cells and in 44% of the ECS with no significant difference between them at P-value of 0.16 . In non-pregnant women, 70% of the vaginal swabs revealed budding yeast cells, while in 40% of the ECS with a significant difference between them at p-value of 0.001. On the other hand, the control group showed the presence of budding yeast cells in 24% of the HVS only with a significant difference between the tested women and control group at P-value of 0.01.

Volume 12, Issue 4, September 2017 ISSN 1992 -0849
Web Site: www.kujss.com Email: kirkukjoursci@yahoo.com, kirkukjoursci@gmail.com No significant difference between HVS and ECS of pregnant women at P-value of 0.16. Chi square test was used. A significant difference between HVS and ECS of non-pregnant women at P-value of 0.001 using proportions and Chi square test. A significant difference between the tested women and the control group at P-value of 0.01 using Chi square test.
Out of the 50 HVS from pregnant women, 33 (66%) of them showed positive culture for yeasts on Sabouraudʼs agar and Brain heart infusion blood agar, while from the 50 ECS of the same group of the patients 29 (58%) showed yeast colonies.    were tested to species level by API-C-20 (Figure 1-B).
The types of yeasts and their number that were isolated from vagina and cervix of pregnant and non-pregnant women present in (  On the other hand, from the control group , C.albicans was isolated from the vagina (50%), in addition to C.glabrata (30%) , and S.cerevisiae (20%) .   Vaginal candidiasis is considered the second most common cause of genital infection in women of reproductive age, although it represents a problem of global importance in public-health, its exact incidence is unknown [18,19]. The main reservoir for Candida is thought to be the rectum, but vaginal colonization is also common [20].
The present study showed that women aged from 21-30 years had highest The clinical presentations in the studied pregnant and non-pregnant women were vaginal discharge followed by itching, odor and oedema. Vaginal discharge is one of most frequent gynecological problems encountered in females especially during their reproductive stage [23]. Other authors mentioned that the signs and symptoms of vaginitis include thick cottage cheese-like vaginal discharge associated with vulvar pruritus, pain, burning, erythema and /or oedema [24].
The pregnancy represents a risk factor in the occurrence of vaginal candidiasis.
Odds, 1988 [25] mentioned that the Documented risk factors of vaginal candidiasis are pregnancy (30-40%), use of high estrogen content oral contraceptives, antibiotics, steroids and chemotherapeutics. The risk factors in the current study were pregnancy (50%), contraception (19%), diabetes mellites (15%), antibiotics (9%) and corticosteroids (7%). The increased secretion of reproductive hormones during pregnancy favors the formation of infection [26]. Other investigators reported that the incidence of infection, as well as the increase in colonization of the mucosa by the yeasts, is also higher in women with diabetes due to their higher glycogen levels and in those with HIV due to immune suppression [27].  [34]. In this study, the women were considered to have Candida infection when swabs were positive for yeasts by both microscopy and culture with number of colonies for each culture more than 10 as presented in Table 5. On the other hand, when the microscopy was negative or showed few budding yeast cells and few numbers of colonies (< 10 colonies) were appeared on culture, this was taken to indicate colonization with Candida rather than infection. Hopwood et al., 1985 [35] reported that when the number of yeast colonies isolated from clinical specimens > 10 considered the causative agent of infection.
In this study, Candida albicans was identified by the production of germ tube and confirmed by growth on Chrom agar with the production of green colored colonies. Babić and Hukić, 2010 [5] mentioned that the germ tube test proves yeast germination, and it is characteristic for the detection Candida albicans. Furthermore, Chrom agar is a selective medium. It can be used for identification of non-albicans species, as well as C.albicans, if germ tube test was not characteristic. During this study, the unidentified yeast species by germ tube formation and color production on Chrom agar mainly these non-albicans Candida, they were identified by API 20 C test. The need for rapid identification of Candida species has led to the development of several media that differentiate yeast species based on colony color [36]. These media contain chromogenic substrates that react with species-specific enzymes secreted by various Candida species producing colonies with various pigmentations [37]. Bhesania and Narayankhedkar et al., 2017 [24] reported that assimilation tests is of importance for identification of yeast isolates, and the method consists of essentially growing yeast on a basal carbohyhdrate-free medium supplemented with test sugar.
In this study, C.albicans was predominant, accounting the higher incidence of isolates in both colonized patients and those with VC. It represents 84.8% of the  [38] reported that C.albicans is considered as the most common causative agent and isolated from 85%-90% of cases of VC. Candida glabrata was the second common pathogen detected in our study followed by C.tropicalis, which was in agreement with previous studies reported by several investigators [39]. In many parts of the world, non-albicans isolates notably C.glabrata affect 10-20% of women [24]. Vaginitis induced by non-albicans species is clinically indistinguishable from that caused by C.albicans but such species are more resistant to treatment [40].

CONCLUSION
The studied cases of pregnant and non-pregnant women have cervico-vaginal candidiasis when both direct examination and culture were positive for the presence of yeasts, in addition to the sign and symptoms. Furthermore, the number of colonies > 10 on culture media for each patient. The main isolates were C.albicans in addition to non-albicans Candida including C.glabrata and C.tropicalis .