Prevalence and Antifungal Susceptibility of Candida albicans Causing Vulvovaginitis Among Pregnant Women in Lebanon

Background: Vaginal candidiasis is frequently prevalent in pregnant women and is associated with sepsis and adverse neonatal outcomes. This study determined the presence of Candida species in symptomatic pregnant women and evaluated the antifungal susceptibility profile of the isolated strains. It also aimed to explore whether Candida species predicts gestational complications and adverse neonatal outcomes. Methods: A total of 258 pregnant women at 35 to 37 week of gestation participated in this study. Vaginal swabs from these patients were collected at various obstetrics and gynecology clinics in Lebanon for a period of 12 months. Candida isolates were identified at species level and antifungal susceptibility of Candida albicans to fluconazole (FCZ), amphotericin B (AMB), itraconazole (ICZ) and voriconazole (VCZ) was determined by the agar-based E-test method. Results: Among 258 women tested, 100 (39%) were positive for Candida species. C. albicans, C. glabrata and C. krusei were isolated from 42%, 41% and 17% of the women, respectively. C. albicans had significant positive associations with gestational diabetes while C. kreusi or C. glabrata had significant positive associations with gestational complications and vaginal discharge. The antifungal susceptibility tests of C. albicans isolates revealed 97.5%, 90%, 87.5% and 97.5% susceptibility to AMB, FCZ, ICZ and VCZ, respectively. . Conclusion: The current study revealed high incidence of both C. albicans and non-C. albicans strains causing vulvovaginitis among pregnant women in Beirut, Lebanon. Whereas the susceptibility rates of C. albicans against AMB and VCZ were high, FCZ and ICZ proved comparatively less efficacious. The resistance profile of circulating C. albicans among pregnant women can predict the best outcome of appropriate prophylaxis or treatment of neonatal of determination; CI: Confidence interval; GBS: Group B streptococcus.

candidiasis. Vaginal candida colonization might lead to adverse neonatal outcome or gestational complications thus Candida screening as antennal follow up is advised.

Background
Candida species, which are part of the normal flora in the vulvovagina, may cause opportunistic infections under various circumstances that compromise host immunity. Candida spp. subsist in symbiotic relationship with vaginal microbiota, therefore asymptomatic colonization is common and may persist for years. The rate of genital Candida colonization ranges from 20% in asymptomatic young women to up to 30% in pregnant women [1][2][3][4][5][6]. The risk factors associated with increased rate of vulvovaginal candidiasis (VVC) in pregnant women are immunologic alterations, increased estrogen levels, and increased vaginal glycogen production mechanism [3].
VVC is the result of Candida albicans in 85-95% of cases whereas incidence rate of Non-C. albicans Candida (NCAC) in pregnant women and non-pregnant women is less than 10% as described in previous studies [7]. Treatment of VVC is recommended only in the presence of symptoms since over 20% of women may have yeast as part of their natural vaginal microbiome and are asymptomatic [8]. However, pregnant women may have severe and prolonged symptoms of VVC requiring longer courses of therapy [9]. Recent studies have shown an increase in the development of drug-resistance among C. albicans, less is known about the burden and effects of drug resistant fungal infections.
Candidiasis in newborns has been associated with increased risk of pregnancy complications, such as premature rupture of membranes, preterm labor, chorioamnionitis, and congenital cutaneous candidiasis. Colonization with Candida spp. in neonates may occur by vertical transmission from the mother during the perinatal period or by horizontal transmission in the nursery or the neonatal intensive care unit (NICU) [10][11][12]. It has been shown that 5% to 30% of all colonized preterm neonates will develop invasive Candida infection (ICI) during their stay in the NICU [13][14][15]. C. albicans was shown to play a major role in neonatal colonization in the first days of life and were also documented in a group of premature infants [16].
The epidemiology of antifungal resistance among C. albicans in pregnant women in Lebanon remains poorly reported. Therefore, the objective of this study was to determine the prevalence of Candida species in symptomatic pregnant women with vaginal discharge at 35 to 37 weeks of gestation and to evaluate the antifungal susceptibility profile of the isolated strains of C. albicans. In addition, the study evaluated the association between the presence of Candida species and gestational complications and outcomes.

Study sample and procedure of collection
In this study, a cross-sectional design was adopted for determining the prevalence

Data collection
Socio-demographic data, clinical status and gestational history of 165 (64%) patients were collected through a questionnaire that included information about mothers' risk factors for adverse neonatal outcomes such as gestational diabetes, previous miscarriage, anemia and recurrent urinary tract infections (UTI). The 165 participants were followed up after delivery to gather information about delivery time, delivery type, induced labor, gestational complications (intrahepatic cholestasis, mitral valve prolapse, asthma, hypothyroidism, oligohydramnios and gestational thrombocytopenia) and neonatal outcomes (newborn height, weight and apgar score).

Literature search
To compare the distribution of Candida species isolated from women genital tract in different countries, a Pubmed search was performed that included articles published in the last ten years [21][22][23][24][25][26][27]. Fifteen articles were selected and summarized in Figure 1.

Culture and identification:
Vaginal swabs were cultured on both Sabouraud dextrose agar (SDA) and Chromatic Candida medium (Liofilchem, Italy). The latter allows the selective isolation and differentiation of Candida spp. based on colony color and morphology; it has been well documented in previous studies as for its high sensitivity and specificity for the identification of the most commonly encountered Candida spp. [17][18][19]. Both plates were incubated at 37°C for 48 to 72 hrs. The chromatic characteristics of the colonies were the following: green colonies were identified as C. albicans, creamy colored colonies as C. glabrata, and pink with a whitish border colonies as C. krusei. All isolates were confirmed by API 20 C AUX strip (BioMerieux, Marcy l'Etoile, France). Further phenotypic testing was done to differentiate between C. dubliniensis and C. albicans by growing the germ tube positive yeast isolates at 45°C on SDA for up to 10 days. C. albicans isolates were identified by their ability to grow at 45°C.

Statistical analysis
The presence of Candida species was correlated with the newborn height, weight and Apgar score (overall assessment of new born well-being used immediately following the delivery of the baby) as dependent variables using linear multiple regression analysis. The models took into consideration to control other possible confounding effect of various independent variables including mother's age, mother's education, delivery type, delivery week, induced labor, recurrent urinary tract infection (UTI), gestational diabetes, anemia and other gestational complications. Statistical significance was calculated using p-value and confidence

Results
The socio-demographic characteristics of 165 respondents are summarized in Table   1; 49% of women who participated were between the ages of 36 to 40 Table 3) .
Results of the three multiple regression models with neonatal outcomes (weight, height and Apgar score) as dependent variables are displayed in Table 4. Results  tropicalis, C. glabrata, and C. parapsilosis [30]. Recent emergence of NCAC, such as C. glabrata and C. krusei has been seen in the post FCZ era and in settings with azole selection pressure [31]. Worldwide, there is a variation in the distribution of Candida spp. identified from vaginal swabs and depends largely on the location as well as the population studied ( Figure 1).
Treatment of vaginal candidiasis is successfully achieved by use of azoles [32].
NCAC related disease is less likely to respond to azole therapy, alternative treatment with AMB suppositories with or without topical azole is recommended. In the current study, isolates showed high susceptibility to AMB (97.5%) and this observation has been corroborated by studies done in various other countries including Lebanon [30,[33][34][35]. Resistance rates of C. albicans to VCZ, FCZ, and ICZ and in this study were 2.5%, 10%, and 12.5%, respectively, which are in contrast to earlier data from Lebanon reporting 3 to 6%, 0 to 6%, and 38% resistance, respectively [30]. However, despite high susceptibility rates against FCZ and VCZ in the previous study, their MIC 90 showed an elevated trend over 10 year of study period [30]. The increase in azole resistance in our study can be attributed to the It is reported that vaginitis in pregnancy is related to adverse perinatal outcome [38]. In the current study, we aimed to find correlation between the presence of candidiasis and pregnancy outcome. Our results showed that height decreased with the presence of Candida species. This reduction was statistically significant in the presence of C. krusei or C. glabrata. However no effect was observed on the weight of the baby. This finding was consistent with a study done previously in Iran where they found no association between vaginal Candida colonization and low birth weight [39]. The current study has also shown that Candida species cause gestational complications which is also in agreement with a previous study done in China [40] Among the different studied variables which may be affected by Candida, such as

Ethics approval and consent to participate
This study was approved by the institutional review board of Beirut Arab University under IRB# 0041-S-P-0336. A written informed consent was obtained from all eligible women before entering the study.

Consent for publication
Not applicable

Availability of data and material
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
Competing interest: All authors declare that they have no competing interests  Distribution of Candida species isolated from vaginal swabs of women from various countries