Candida vulvovaginitis or vulvovaginal candidiasis (VVC) is the second-most frequent genital infection after bacterial vaginosis¹ and is characterized by Candida infections of the vulva and vagina². VVC is often called "bacterial vaginosis", and trichomoniasis is the most common cause^3,4. VVC causes significant discomfort and is a focus of concern for clinicians and biologists⁵. It is estimated that 75% of all women will experience at least one episode of VVC during their lifetimes⁶, 40~50% will experience multiple VVC infections, and 6~10% develop recurrent VVC (RVVC). It is defined as at least four proven episodes of VVC in one year^7,8.

Among the species of Candida, C. albicans is the most commonly encountered pathogen in clinical settings, and is, therefore, the most researched^9,10. C. albicans is a complex that includes C. albicans sensu stricto and the related species of C. dubliniensis and C. africana¹¹. As these species are morphologically indistinguishable, molecular amplification of the hyphal wall protein 1 (hwp1) gene is used to discriminate C. dubliniensis and C. africana from C. albicans¹². While C. dubliniensis is predominantly associated with cases of oral candidiasis in patients with human immunodeficiency virus (HIV)¹³, C. africana is mainly responsible for VVC¹⁴. However, it has also been isolated outside the vagina (Odds et al., 2007). This work aimed to update the molecular identification of VVC pathogenesis in Abidjan.

The incidence of VVC has increased over the last decade, as has the frequency of non-albicans Candida (NAC) species, principally associated with RVVC. The latter represents a therapeutic problem, requiring the identification of risk factors and effective treatment^2,16.

The estimated prevalence of VVC in Côte d'Ivoire is ~43, with C. albicans being the most frequently identified fungal species by mycological analysis^18,19. Importantly, all previous studies of VVC were conducted in outpatient referral health- care facilities^17,18,20, potentially omitting data from lower-level facilities. Therefore, this study sought to update what is known about VVC, particularly cases caused by Candida species, and contributing factors to improve our ability to manage these patients. All study patients were seen at one of two different facilities in Abidjan (Côte d'Ivoire).

1. Study design and healthcare facilities

This study was conducted over eight months, from May 2023 to January 2024, on patients who presented with suspected VVC to the gynecology departments of two health- care facilities in Abidjan: the General Hospital of Adjame (GHA) and the Anti-Venereal Dispensary (AVD) of the National Institute of Public Hygiene (NIPH). The GHA, located in Adjame, provides secondary healthcare, while the AVD / NIPH in the Treichville commune specializes in treating sexually trans- mitted infections (STIs). Women were recruited from the gynecology consultation departments from May to June 2023 in the GHA and from September 2023 to January 2024 in the AVD. Mycological analyses were performed at the Parasitology-Mycology Laboratory of the Diagnosis and Research Center on AIDS and Other Infectious Diseases (CeDReS) in the Teaching Hospital of Treichville. Molecular analyses were done at the Malaria Research and Control Center at the National Institute of Public Health.

2. Patient selection

The sample size was determined using the Schwartz formula: N = z2pq/d2, where N = the minimum number of patients, z = 1.96 based on an error risk α of 5%, p = 0.43 (prevalence found in a similar study conducted in Abidjan¹⁷), q = 1 - p, and d = 0.05 (accuracy of our results); thus, at least 376 patients were needed to achieve adequate statistical power.

Non-menstruating women who visited the gynecology departments of GHA and AVD with lesions suggestive of VVC were chosen after providing written informed consent. Before sampling, certain conditions had to be met: (i) no douching for 24~48 hours before the appointment, (ii) no oral or local treatment for the condition for 3~4 days before, and (iii) no sexual intercourse for at least 48~72 hours before.

3. Data collection

Each patient first underwent a complete clinical examin- ation, including an interview and physical examination by a physician. We administered a questionnaire to collect patient-specific sociodemographic (such as age, education level, marital status, and occupation), clinical (burning, itching, and dyspareunia), and behavioral (douching frequency, use of antiseptic douche, condom use, oral contraceptive, and type of underwear) data.

4. Sample collection

The doctor collected vaginal specimens using two sterile cotton swabs moistened with sterile physiological saline. They swabbed the back part of the vagina after placing a speculum. The first swab was used for direct examination, and the second was used to inoculate the culture medium.

5. Mycological analysis

1) Direct examination and isolation

The direct examination used a saline wet mount to identify budding or non-budding yeast with pseudohyphae. Yeast presence and abundance were noted during the examin- ation. Swabs were cultured under strict aseptic conditions on Sabouraud chloramphenicol agar and Sabouraud chloram- phenicol actidione agar for yeast isolation. The incubation conditions were 37℃ for 24~48 hours.

2) Yeast identification

We identified the yeasts after incubation using common macroscopic (appearance and color of clusters) and micro- scopic morphological features and growth parameters. We used chromogenic media (Candida chromatic agar) and sugar assimilation tests (Auxacolor®) to identify the species not recognized by the chromogenic media.

To identify the yeasts using Candida chromatic agar, we inoculated the agar by streaking a drop of yeast suspension obtained from a fresh yeast colony in 1 mL of sterile physio- logical saline, followed by incubation at 37℃ for 24 hours. The ability of the isolates to assimilate carbohydrate sources was determined using an API 20C yeast identification system (BioMérieux, Marcy-l'Étoile, France), according to the manu- facturer's instructions. Lastly, identified clusters of Candida albicans strains were added to 1 mL of a mixture of Brain Heart Infusion Broth and glycerol and stored at -20℃.

6. Hyphal wall protein 1 genotyping

Before analysis, 1~2 drops of stored C. albicans strains were transferred to a fresh Sabouraud chloramphenicol agar under strict aseptic conditions. The agar was then incubated at 37℃ for 24~72 hours, and successful subcultures were extracted.

1) DNA extraction

Parasite DNA was extracted from 1~2 clusters according to the manufacturer's instructions using the Quick-DNATM Fungal/Bacterial Miniprep Kit (Zymo Research, California, USA).

2) DNA amplification

The hyphal wall protein 1 gene was analyzed using a one-step polymerase chain reaction (PCR). The following primer sequences were used for amplification:

Forward : CR-f 5'-GCT ACC ACT TCA GAA TCA TC-3'

Reverse : CR-r 5'-GCA CCT TCA GTC GTA GAG ACG-3'

PCR amplifications were carried out in a final volume of 25 μL, including 7.5 μL of pure water, 0.5 μL of each primer (10 μM), 12.5 μL of GoTaq® G2 Hot Start Green Master (Promega, Madison, Wisconsin, United States), and 4 μL of extracted DNA. All procedures were performed in a class II Biological Safety Cabinet. The amplifications were conducted in SimpliAmpTM Thermal Cycler 96-well plates (Thermo Fischer Scientific, Waltham, MA, USA). The PCR conditions were as follows: 95℃ for 5 min; 30 cycles at 94℃ for 45 s, 58℃ for 40 s, and 72℃ for 55 s; and a final 10-min extension step at 72℃. Following amplification, PCR products were visualized using electrophoresis on a 1.5% agarose gel at 100 mV for 2 hours. The fragments were observed under a UV trans-illuminator (Biocom Biotech, Centurion, South Africa), and their sizes were estimated compared to the 100 bp DNA Ladder BenchTop (Promega, Madison, Wisconsin, United States). The expected sizes were 941 bp for C. albicans, 569 bp for C. dubliniensis, and 700 bp for C. africana.

7. Ethical considerations

The study was approved by the National Committee of Ethics and Life and Health Sciences (In French: Comité National d'Ethique et des Sciences de la Vie et de la Santé) with certificate number N° 059-23/MSHPCMU/CNESVS-km. The study was conducted following the principles of the Helsinki Declaration. Before enrolling, the patients, parents, or legal guardians obtained free and written informed consent.

8. Data analysis

All statistical analyses were conducted using IBM SPSS Statistics version 21 (Armonk, NY, USA). The tests included chi-squared and Fisher's exact tests with an alpha risk of 5%. A p-value less than 0.05 was deemed significant.

The study's participants were 414 women (213 at the GHA and 201 at the AVD). The study's overall prevalence of fungal VVC was estimated to be 53.6%. The prevalence was 62% (132/213) in the GHA and 44.8% (90/201) in the AVD.

1. Mycological analysis

Mycological analysis revealed that the most commonly identified fungal species was C. albicans (70.7%), followed by C. tropicalis (9.9%), as shown in Table 1. No strains of C. krusei were found in the GHA group, and no strains of C. glabrata were found in the AVD group.

2. Molecular analysis

We stored 155 strains identified as C. albicans through mycological analysis. We observed a high subculture suc- cess rate of 97.42% (151 out of 155). The success rate of molecular tests for the hwp1 gene was 96.03% (145 out of 151). All 145 strains identified as C. albicans by mycological analysis were confirmed through molecular analysis. We did not find any C. africana or C. dubliniensis species. The C. albicans isolates provided two genotypes: 941 bp (93.10%) and 941+850 bp (6.90%).

The analysis of sociodemographic, behavioral, personal history, and clinical data was conducted based on mycology results. The average age of the patients was 29.3 years (standard deviation, 8.6 years), with the age group of 25~ 40 years being the most common (50.7%). Regarding socio- demographic and behavioral aspects, it was found that patients under 25 years old (p = 0.018), those living in pre- carious housing (p < 0.0001), and pregnant women (p = 0.006) were significantly more affected than other patients. Additionally, the use of intimate toilets (p = 0.003), products used for intimate toilets (p = 0.003), and the type of under- wear worn (p = 0.037) were associated with the presence of VVC. No significant differences were observed between the two healthcare facilities. The most common clinical sign was vaginal itching (46.4%). The presence of VVC showed significant correlation with vaginal itching (p < 0.0001) and the duration of the disorder (p < 0.0001). Additionally, the appearance of vaginal mucosa (p = 0.030), as well as the appearance of vaginal discharge (p < 0.0001) and its color (p < 0.0001), were found to be significantly associated with VVC. The sociodemographic, behavioral, personal history and clinical data of the patients are presented in Tables 2 and 3.

The purpose of this study was to update the data and improve the management of VVC cases. It also aimed to assess the epidemiological profile of VVC based on the level of care required, as the AVD is a referral center and the GHA is a secondary-level center.

According to this study, the overall prevalence of VVC was 53.6%. The higher prevalence in the GHA than in the AVD can be attributed to differences in the characteristics of women seeking care at the two healthcare facilities. In the GHA group, most participants were pregnant (89.2%), while in the AVD group, only two women were pregnant (1.0%). It is generally observed that vaginal colonization by Candida species occurs in at least 20% of all women and increases to 30% during pregnancy²². The risk of VVC during pregnancy is likely increased by factors such as immune system changes, elevated estrogen levels, and heightened vaginal glycogen production²³. The prevalence of VVC reported in this study was higher than in Abidjan (43; 38.7).

The prevalence of C. albicans as the primary pathogen responsible for vulvovaginal candidiasis (VVC) is approximately 90%²⁴. In our study, C. albicans was the most common species in both healthcare facilities. The C. albicans complex includes C. albicans sensu stricto, C. africana, and C. dubliniensis. This study is the first to explore the genetic diversity of the hwp1 gene within the C. albicans complex in our country. After conducting PCR analysis, no strains of C. dubliniensis or C. africana were identified. The two genetic profiles described in the study have previously been classified as C. albicans²⁵. Notably, C. dubliniensis causes both superficial and invasive infections in both HIV-positive and HIV-negative patients²¹.

Most infected patients in this study were pregnant and visited health facilities for prenatal consultations. We did not consider HIV status in our analysis. C. africana, it is an opportunistic pathogen that causes vaginal infections and, which it can produce germ tubes in serum, C. africana will not produce chlamydospores in nutrient-poor media²⁶, and no tests for chlamydospore production were conducted. C. africana was initially described in 1995 as an atypical strain of C. albicans and later set forth as a new Candida species based on its distinct morphological, biochemical, and physio- logical characteristics compared to C. albicans²⁷. Although C. africana initially appeared to be restricted to Africa and Europe and isolated mainly from genital samples, it is now clear that it has a worldwide distribution and is recoverable from various clinical specimens^15,21,28-31.

The NAC proportion was also high in our study cohort. This center serves as a referral center for managing STIs in the country. Therefore, women attending this center exhibit unusual pathological profiles. NAC species like C. glabrata, C. krusei, C. parapsilosis, and C. tropicalis can cause complications in patients with VVC^32,33. These species have recently gained scientific and epidemiological interest because of their increasing global presence³². These species were detected in the present study, with C. tropicalis dominating. C. tropicalis is genetically similar to C. albicans³⁴. Previous studies have reported the predominance of C. tropicalis among NAC³⁵. A survey of pregnant women identified C. tropicalis as the most prevalent Candida species, with a higher prevalence than that of C. albicans³⁶. C. glabrata was the second-most common species after C. albicans at GHA, and the second-most common species was C. krusei at the AVD. Previous studies in Abidjan found that C. glabrata was the second-most common species after C. albicans^17,18,20. C. krusei is generally less prevalent³⁶. The emergence of NAC species can be attributed to selective pressure due to women's prolonged exposure to RVVC, use of over-the-counter anti- fungal medications, and low-dose azole treatments^37,38. These Candida species—especially C. glabrata—are primarily implicated in cases of RVVC³². The study did not collect follow-up data on women, so the prevalence of RVVC was not determined. However, VVC was the main issue for patients with a personal history in both healthcare facilities.

Common signs and symptoms of VVC include vaginal itching or burning, with or without redness and vulvar swelling, white discharge, and burning or stinging during urination³⁹. In the current study, VVC was significantly correlated with some of these symptoms. Vaginal itching—a leading clinical sign of VVC⁵—is also a predisposing factor and is associated with vaginal discharge. The present study observed white and curdled discharge as a predisposing sign. The duration of the disorder was also noted as a contributing factor in this study, as reported previously¹⁷.

Factors that predispose women to developing VVC include antibiotic use, hormone replacement therapy, pregnancy, diabetes mellitus, and genetic and behavioral factors⁴⁰. In the current study, a typical patient was younger than 25, pregnant, living in a shanty, douching once per week (including with antiseptic products), and wearing synthetic underwear. Anti- biotic use and diabetes did not increase the occurrence of VVC in this study, contradicting many studies' prior findings^16,41,42.

The results from this study emphasize the molecular identification of species within the C. albicans complex. No strains of C. africana and C. dubliniensis were identified. We also observed an increasing prevalence of VVC in Abidjan. There- fore, gaining a better understanding of the risk factors associated with this disease will help improve women's hygiene standards and enable better prevention of its occurrence.