Antifungal susceptibility pattern of Candida spp. isolated from vaginal discharge of pregnant women

Vaginal candidiasis is common fungal infections among pregnant women. The aim of this study is to determine antifungal susceptibility pattern of Candida spp. isolated from vaginal discharge and to investigate possible risk factors associated with Candida infection. One hundred high vaginal swabs were collected from pregnant women complaining from abnormal discharge from various clinics in Gaza city, during 2019. Samples were plated on Sabouraud Dextrose Agar supplemented with chloramphenicol. Candida isolates were identified to the Genus level based on cultural and morphological characteristic. The susceptibility to seven antifungals was determined using disc diffusion method. Demographic, environmental, behavioral and other possible risks factors associated with vaginal candidiasis were recorded through an interview with patients before collecting the vaginal swabs and after obtaining a verbal consent. Data analysis was done using the SPSS software. Candida spp. was isolated from 43% of vaginal swabs of pregnant women with discharge. Variations existed in the susceptibilities of the different Candida isolates to antifungals. Amphotericin B the most effective with 0% resistance followed by itraconzaol and nystatin with 9.4% resistance. Resistance to miconazole, ketoconazole, voriconazole and fluconazole was 11.6%. Statistically significant symptoms associated with positive Candida spp. cultures among pregnant women included; redness of vaginal area, secretions, yeast-like smell, urinary urgency, low sexual desire, itching and irritation (p ≤ 0.05). No statistical significance difference with regard to burning urinary sensation and painful intercourse. Candida spp. was isolated from 43% of vaginal swabs from pregnant women. Risks factors should be minimized by patient education and routine testing of pregnant women.


Introduction:
Candida species are ubiquitous, ranging from free saprophytes through endo-symbionts of animals, to pathogens in many animals and in humans. Some of the pathogenic species are of medical importance, especially Candida albicans. However, the prevalence of other non-albicans Candida species as human pathogens is on the rise worldwide (1).
Vulvovaginal candidiasis (VVC) is considered as the second most common cause of vaginal infections, after bacterial vaginosis that is reported at gynaecological clinics (2). An estimated cost of diagnosis and treatment of VVC which take into consideration, loss of productivity and absenteeism in the U.S. is about $1 billion yearly (2). Candida albicans is the most frequent causative agent in about 80 − 90% of VVC infections even though other non albicans Candida species are increasingly reported (2).
VVC is the most common gynecological ruling among women of child-bearing age (3). Among sexually active women, 75% have at least once experienced symptomatic VVC, which usually presents as soreness, burning, itching and abnormal curd-like vaginal discharge (4,5). Pregnancy was the commonest factor (55%) followed by usage of broad-spectrum antibiotics (8%) (6).

Recommendations
for vulvovaginitis diagnosis included speculum examination, and microscopic examination of slide from vaginal exudates to look for protozoan (Trichomonas vaginalis), bacterial (Gardnerella vaginalis) or fungal (Candida spp.) agents, followed by vaginal fluid specimen culture to confirm the diagnosis. Otherwise, the confirmatory diagnosis based on culture is not routinely performed nor generally advised in many regions, because all procedures are expensive and time consuming (7).
Many reports from around the world reports increasing frequency of Candida resistance to common antifungals. The main objective of this study is to determine the prevalence of vaginal candidiasis among pregnant women with abnormal discharge and their resistance profile to antifungals.

2.1
Sample collection and culture A total of 100 high vaginal swab samples were collected from symptomatic and asymptomatic pregnant women aging between (18-49) years attending ante-natal clinics during the period from December 2018 to June 2019. Samples were transport to the Microbiology laboratory at the Islamic University of Gaza within 2 hours and were inoculated onto the surface of Sabouraud Dextrose Agar (SDA) with chloramphenicol, then incubation on 37 0 C for 48 hours.

2.2
Antifungal susceptibility testing Antifungal susceptibility testing was undertaken by the disk diffusion method. Fluconazole disk (10 μg), itraconazole (10 μg), voriconazole (10 μg), amphotericin B (10 μg), nystatin (100 IU), and Ketokonazole antifungal discs were applied on MHA as recommended by the Clinical Laboratory Standard Institute (CLSI). The plates were incubated in ambient air at 35°C and read after 24 hours. Interpretation of all antifungal susceptibility (susceptible S, intermediate I and resistant R) was done according to CLSI standards (8).

Results.
The study sample comprised of swabs from 100 pregnant woman attending Gaza clinics, aged between 18-49 years (Table 1).

Discussion
This present study investigated the prevalence of vaginal candidiasis among pregnant women with symptoms of vaginal infection attending different gynecological clinic over the period of five months in a different area in Gaza city.
In the present study, the isolation rate of Candida spp. from pregnant women was 43%. The highest rate was among 18-28 age group with a prevalence of 46.4%. These results are higher than those observed in previous reports on vaginal Candida spp. (33.8%) in women in the age range of 20-29 years, followed by (24.3%) those in the age range of 30-39 years, and the overall prevalence of VVC was 25% (9). Other researchers reported lower results than ours including 17% obtained from Malaysia (10), 15-20% in Taiwan (11), 14% in Ibadan (12), 30% in Nigeria (13), 37% in Turkey (14) and 38% in Hyderabad (15). While our results were lower than (55.4%) those obtained in Cameroon (16) and even very much lower than (92%) in a report from Libya (17). Feyi (18) in Tanzania and Menza et al (19) in Kenya reported prevalence rates in agreement with our result; they reported that, prevalence of vaginal candidiasis among pregnant women were 42.9% , 42.7% respectively.
The high prevalence of vaginal candidiasis may due to many different reasons include; suppression of the immune system due to the pregnancy as it is among the contributing factors of vaginal candidiasis (20), extended and misuse of antibiotics which leads to the destruction of good and beneficial bacteria resulting to reduction of vaginal immunity could have also contributed to the increase of the prevalence of the infection (21,22).
Virulent factors of Candida spp. like dimorphism, phenotypic switching, protease and phosphatase which enhance its attachment to human epithelium play an importance role in their high occurrence. High incidence rate also may due to increased physiological changes, estrogen and rich glycogen content of the vaginal mucosa thereby providing an adequate supply of utilizable sugar that favor Candida albicans growth during pregnancy (17).
In this study, only 5 (11.6%) of Candida spp. was resistant to fluconazole, this is higher than studies conducted in United States and Brazil were all C. albicans vaginal isolates were susceptible to fluconazole (23), and zero resistance Malaysia (10). A study in Argentina (24) showed a slightly higher result (13.46%).
Age, level of education, income, and residency as possible risk factors for vulvovaginitis were investigated in the present study. We did not find associations between some socio-demographic characteristics and the prevalence of vulvovaginal candidiasis. Among socio-demographic characteristics, age seems to be not an important factor in the overall occurrence of vulvovaginal candidiasis. Out of 43 patients with vulvovaginal candidiasis, 32 (46.4%) patients were in their 18-28 of life. Our result was comparable with previous studies (27,28).
The study of Janković et al. (29), showed that vulvovaginal candidiasis was statistically associated with continual wearing of tight underwear, a finding which is consistent with our result (p<0.01).
In the present study, the presenting complaints in 71.4% of positive patients reported itching and irritations, 75% Redness of the vagina area, 84.4% suffered from thick and white/creamy secretions, 92% Secretions smell Yeast like, 66.7% lower sexual desire, and 54.2% Urinary urgency. These is comparable to the prevalence rate obtained from Pakistan (25). This most likely owes to raised level of hormones during pregnancy, which is an energy source for Candida growth (6). pregnancy resulted significantly associated to asymptomatic infection (30). In other study, 39.5% of pregnant women had symptoms of VV (31).
Discharge was the most symptoms were noticed in pregnant followed by itching while odor and burning are present lowest percentages in all subjects (17). The frequency of vaginal candidiasis during pregnancy was found to be 38%, in which 27% were symptomatic and 11% were asymptomatic group (15).
As this is the first study that investigated Candida among pregnant women, the authors were not able to compare the results of this study with local studies. Other studies should be conducted, to include larger sample and covers the entire Gaza strip. Gynecology clinics are recommended to perform culture and sensitivity to provide an appropriate antifungal. The concerned authorities should work closely to monitor the use of therapeutic antifungals to minimize drug resistance. Education of women on genital hygiene and regular screening for candidiasis in antenatal program is highly recommended.