Prevalence of Candida Species Isolated from Vaginal Discharge of Women Undergoing in vitro Fertilization-Embryo Transfer in Vietnam

Do Ngoc Anh*1, Nguyen Duy Bac2, Le Tran Anh1, Le Bach Quang1, Nguyen Khac Luc1, Trinh The Son3, Hoang Anh Tuan4 and Do Quyet5 1Department of Medical Parasitology, Military Medical University, Vietnam 2The Institute for Medical Research, Military Medical University, Vietnam 3Military Institute of Clinical Embryology and Histology, Military Medical University, Vietnam 4The Centre for Toxicology Research, Military Medical University, Vietnam 5103 Military Hospital, Military, Military Medical University, Vietnam


Introduction
Vulvovaginal candidiasis is estimated to be the second most common cause of vaginitis after bacterial vaginosis [1]. Many studies have shown that approximately 75% of healthy female population at least one symptomatic episode of vulvovaginal candidiasis (VVC) and 40-50% will have recurrent episodes during their lifetime [2,3]. Candida albicans was accounted as the first vulvovaginal candidiasis causative agent [1], followed by non-albicans Candida (NAC) species including Candida glabrata, Candida parapsilosis, Candida tropicalis and Candida krusei [4]. The species distribution of Candida isolates varies between countries, regions, and institutions [5]. NAC species have increasingly been identified as the cause of vulvovaginitis [6]. The data of some reports indicated that NAC species were responsible for 10% to 30% of episodes in certain geographic regions [6]. In clinical practice, the yeast identification is based on morphological and biochemical markers, including the automated methods [7]. However, not all the species are precisely identified by such procedures. Therefore, molecular markers were utilized in the present study to enable identification and detection of various strains [8]. According to the literature, identification of the Candida isolates is important for treatment, prediction of the prognosis, performing infection control, finding epidemiological data and avoid antifungal failure [9,10]. In addition, the species identification of the yeast should be performed with a high accuracy [11]. Previously, very limited data is available on species composition of Candida in Vietnam, especially the women undergoing IVF-ET. This study has been performed to find the prevalence of Candida species isolated from vaginal discharge of women undergoing IVF-ET in Vietnam.

Yeast Identification
The yeasts were first identified according to morphological and physiological characteristics using germ tube test (in human serum), cultured on Brilliance™ Candida Agar (Oxoid, UK), and microscopic morphology on cornmeal agar (Difco, USA) with 1% Tween 80 (Merck, Germany).

DNA Extraction
Genomic DNA were extracted from yeast cultures using QIAamp DNA Mini Kit (QIAGEN, Hilden, Germany), following manufacture recommendation. DNA concentration (ng/μl) was estimated using a NanoDropTM 2000 Spectrophotometer at 260 nm (Thermo Fisher Scientific, USA).

PCR Reactions
To amplify an approximate 500-900 bp region of the ITS1-

Data and Sequence Analyses
IBM SPSS statistics 20.0 software was used for data processing in this study. The sequences generated were compared to available data in the NCBI database using BLAST guidelines.  Table 1). C. albicans was the most commonly isolated species in our study. The second leading strain was C. glabrata. All cases were monomicrobial infections, no mixed infections were detected in the present study (Figure 1a & 1b). DNA sequencing of three D1/D2 were compared to available data in the NCBI database using BLAST guidelines. BLAST results showed that C. tropicalis from China (MG871744.1) possessed the sequence most like those in these 2 isolates with 100% identity for HN-SD20 isolate (MK464263.1) and HN-SD51 isolate (MK464265.1). D1/D2 sequencing of HN-SD42 strain was 100% like C. albicans from China (KY825125.1).

Discussion
There are many techniques that have been proposed to detect and distinguish Candida species in clinical samples [13]. In present study, we used three phenotypic methods and two molecular methods, namely PCR-RFLP and DNA sequencing, to identify Candida species in 83 strains from vaginal discharge of women undergoing IVF-ET. The results of our study showed that C. albicans continues to be the species most commonly isolated from vaginal discharge of women. The result from our study was like that of some previous researches in China [9,14], Iran [3,15] and Brazil [8].
Although C. albicans was still the most frequently isolated species, we describe a very high proportion of non-albicans Candida species (60.24%), with C. glabrata, being almost as common as C. albicans.
C. tropicalis was the third most frequently isolated Candida species (10.84%), followed by C. krusei (8.43%). In this series we found only two C. parapsilosis (2.41%). The results of our study were like that of some previous researches.
Most reports have shown that C. glabrata, C. tropicalis and C.
krusei were accounted as the second, third and fourth common agents of disease, respectively [16,17]. According to the results from our study, C. glabrata was the second most commonly occurring species in Vietnam. Abbasi et al. (2017) reported similar findings in Iran, where C. glabrata was also the second most common species isolated from vulvovaginal Candidiasis [15]. Conversely, a report from India indicated that C. glabrata was the most common species which was isolated from 50.4% patients with vulvovaginal candidiasis [16]. The reasons for such differences in the distribution of Candida spp. are not fully understood. However, the high prevalence of C. albicans and C. glabrata in our study was remarkable results. According to Castrillón-Duque (2018), the presence of C. albicans or C. glabrata affects seminal parameters [18]. The data from this study showed that more studies are needed to determine the effects of vaginal yeast infections on results of IVF-ET.

Conclusion
The most common species in women undergoing IVF-ET were C.
albicans, C. glabrata and C. tropicalis, respectively. The frequency of the non-albicans Candida species isolated from women undergoing IVF-ET is gradually increasing, especially C. glabrata.