An overview of vaginal infections’ etiologies in south-eastern Gabon

Discomfort in women of childbearing age associated with vaginal infections, namely Bacterial Vaginosis (BV), Aerobic Vaginitis (AV), VulVovaginal Candidiasis (VVC), and Trichomoniasis (TV), represent a serious and ongoing gynecological complication throughout the world. This study aimed to investigate the etiologies of vaginal infections among outpatients in south-eastern Gabon. A cross-sectional study was designed using participants referred directly by their treating doctor for a vaginal swab. Socio-demographic data were collected using a structured questionnaire. Microscopic examinations were used for TV and BV diagnostic. All vaginal swabs were cultured for AV and VVC isolates using standard microbiology methods. A total of 573 women of reproductive age participated in the study. The most common identied vaginal infections were BV (62.8%) and AV (51.1%) followed by VVC (34.1%). No signicant difference was observed for each etiology compared to socio-demographic data. Streptococcus B (23.9%), Staphyloccocus aureus (17.7%), Klebsiella spp. (11.6%), and E. coli (5.8%) were the bacteria most associated with AV. A high incidence of Non-C. Albicans Candida (NCAC) strains causing vulvovaginitis were found. The prevalence of TV (2.1%) was low. Mixed infections had been common among participants. No association was found with TV and other vaginal infections, unlike others studies. The present study identied BV 228 (83.5%) and AV 227 (83.2%) as the main cause of mixed infections. The mixed infection AV-BV 113 (41.4%) was the most represented. Also, that simultaneous AV-BV-VVC represented 69 (25.3%) of mixed infections. Molecular analyses would be needed to identify the key species commonly associated with these vaginal infections. etiologies,


Background
In healthy women, the vaginal mucosa is covered by a complex micro ora dominated by lactobacilli (1).
The balance of this ecosystem is essential because it constitutes the main element of defense against genital infections. Some forms of vaginal infections have been well-de ned to date, namely Bacterial Vaginosis (BV); VulVovaginal Candidiasis (VVC), and Trichomoniasis (TV) (2). In the meantime, the term Aerobic Vaginitis (AV) was described for the rst time by Donders and al. (2002) to meet the need to describe another condition of vaginal dysbiosis (3). These infections would be responsible for as many as 50% of all gynecologic visits and represent a major contributor to health care expenses (4). According to some authors, BV and VVC during pregnancy would increase the risk of preterm birth and miscarriage.
While TV and AV can increase the transmission of human immunode ciency virus, VVC, or other sexually transmitted infections (5). Some studies suggest a variety of risk factors. Ethnicity and geographical location would in uence the prevalence of vaginal infections. Several authors reported different inventory of the situation about vaginal's pathobionts, namely in Asia, Europe, Africa, and Latin America (6)(7)(8). As for Sub-Saharan Africa, Kenyon et al. (2013) reported a higher prevalence of BV than other parts (9). Sub-Saharan African women may therefore be less protected from vaginal dysbiosis (10). Given the speci city of women's vaginal microbiota from one region to another, it would be interesting to assess vaginal infection in Gabon, a tropical equatorial country. Also, the semiological approach use by medical practitioners often fails due to the absence or lack of symptoms speci city (11). Few studies reported in Gabon to date are limited either by the limitations of the diagnostic techniques (12) or by the size (13) and the type of sampling (14) (15). In this study, existing data were completed and would allow for a better understanding of microbial dysbiosis in the vagina to help improve the care of women in tropical countries.

Study area
Our study was conducted at CIRMF (Centre Interdisciplinaires de Recherche Médicales de Franceville). This center is a sentinel in terms of public health. The CIRMF is located in Franceville, Haut-Ogooué province's administrative center from southeastern Gabon. This province is the second most populous in Gabon and borders the Republic of Congo.

Design and subject selection
Over one year period, between June 2019 and 2020, a cross-sectional study was designed. Women who agreed to participate in this study had been referred by a general practitioner to the Laboratoire d'Analyses Médicale (LAM) at the CIRMF for vaginal sampling. All participants reported different vaginal abnormalities.
All socio-cultural and demographic information on the patients was collected by following a set of tailored questions. The survey questionnaire was organized in separate sections to obtain information on age, employment status, marital status, symptoms, and contraceptive use. The study led according to inclusion and exclusion criteria. The inclusion criteria were de ned as follows: (1) outpatients with vaginal complaints referred by a local general practitioner (2) outpatients with voluntary consent; (3) outpatients of reproductive age; and (4) outpatients who had been menstruating for at least 2 days and had not had sexual intercourse in the 2 days before sample collection. The exclusion criteria were de ned as follows: (1) outpatients under the legal age and (2) outpatients with clinically apparent herpes simplex infection. These selected variables were based on the literature (4,6,7,(16)(17)(18)(19)(20).

Ethical considerations
Female participants were recruited to our study set, after having read and signed individually the informed consent or from their parents or legal representatives. To ensure con dentiality, numbers were assigned to participants via their survey sheets and then plotted on the corresponding samples. Once the diagnosis is done, the results of all participants were transmitted with strict anonymity.

Laboratory Processing
Once the form was completed, sampling through the vaginal route were performed for each woman. Vaginal swabs were taken using ESwab Collection. Transport System (Copan Diagnostics, Inc., Brescia, Italy) were used to prepare the smear for microscopic examination of the vaginal microbiota and inoculation onto agar plates. The comparison of microscopic observations and cultures was used for the isolates identi cation. Swabs collected were transported and delivered immediately for analysis. Vaginal swabs were used to prepare smears on clean, grease-free slides. The Gram and MGG staining were used for diagnostic. The Gram-stained smear was used to assess the intensity of a possible imbalance in the vaginal microbiota based on Nugent score, gold-standard method diagnostic (21). Slides were read according to Nugent's score as described by Bitew et al. (22). A score in the range 0-3 indicated normal ora, 4-6 indicated mixed vaginal ora, which is not constituted BV, and 7-10 was constituted of BV (23,24) .The smear stained with MGG made it possible to observe the presence of in ammatory cells, to identify Trichomonas vaginalis and the presence of yeasts or pseudomycelium.

Isolation and identi cation of microorganisms
Microbiological analyses were performed according to standard procedures of clinical laboratory to characterize pathogenic microorganisms. Different culture media were used for the microbiological investigations. Brie y, blood and chocolate agar were used as a general non-selective medium, Columbia CNA + 5% Sheep Blood agar (bioMérieux, France) for Gram-positives bacteria, BCP dextrose agar (BIOKAR Diagnostics, France) for bacilles gram-negative and SAB-CHL agar (Sabouraud chloramphenicol, bioMérieux, France) for presumptive Candida spp.
All characteristic colonies isolated were identi ed by the VITECK 2 compact (VITEK-2 automatique système, bioMérieux, France). The VITEK is an automated biochemical system that enabled the fast and accurate identi cation of medically relevant yeasts via uorescence technology (25). It allows the fast and accurate identi cation of medically relevant yeasts to species level via uorescence technology. Brie y, the inocula in the VITEK 2 use were prepared in sterile saline to a turbidity equal respectively to 0.5 and 2.0 McFarland standard for bacteria and yeast according to a densitometer (DensiChek instrument, bioMérieux, France). Each standardized inoculum suspension was placed into a VITEK 2 cassette, namely VITEK 2 GN for Gram-Negative organisms' inoculum, VITEK 2 GP for Gram-Positive organisms' inoculum, and VITEK 2 YST for yeast or yeast-like organisms' inoculum, along with a sterile polystyrene test tube. The cassettes were placed in the VITEK 2 instrument and the respective microbial suspensions were diluted appropriately. Then, the cards were lled, incubated, and read automatically by the VITEK 2.
The time of incubation varied from 10 to 30 h based on the growth rate in the free control well.

Data analysis
Data were entered into Microsoft Excel 2016 and exported into the statistical software R version 4.0.2 for data analyses. Descriptive statistics such as percentages were carried out. Percent prevalence was determined by multiplying the number of the positive sample by 100 and dividing by the total samples number. Comparison of genital infection etiology between age group, employed status, marital status, symptoms, and contraceptive use was determined using the X 2 test. The statistical level of signi cance was considered at p ≤ 0.05. Venn diagrams were generated using InteractiVenn (26). A generalized linear model was made using the function GLM of the package stats version 3.6.2. and the step function of the MuMin package was used to select the best model

Characteristics of study participants
Microscopic examination characteristics of the most common vaginal infections microbiomes in the outpatient cohort from this study have been outlined. A total of 573 participants with vaginitis complaints have been involved in Figure 1.
The socio-demographic characteristics of women screened through this study for the period. These outpatient has been divided into four groups for age. The rst group with an age range of 12- The occurrences of single and mixed infections within the framework of vaginal infections were illustrated in the Venn diagram (Fig.2). According to the GLM model, there is a statistically signi cant association between BV and vaginal infection in south-eastern Gabon outpatients (p 0.0001).

Discussion
The most common causes of vaginal infections among reproductive women are usually associated with infectious diseases, namely BV, VVC, AV, and TV (1-3). The diagnosis is often made based on the symptoms and the physical examination results or the biological tests carried out in the o ce or the laboratory (27). Microbiological analyses of vaginal swabs were systematically carried from women after healthcare providers' examination as described by Barry et al. (28). Once the infection pro le was determined, we looked for close relationships between several socio-demographic elements of the outpatients and the vaginal infections. In the study, the socio-demographic data provide various information including an overview of the context. Franceville is a town in the southeast of Gabon, a semiurban area in a developing country that has the particularity of being located in the heart of the African inter-tropical forest.  (32). Similar results were also found in Yemen and Ethiopia (2, 33). In our study, no statistically signi cant difference was found between different vaginal infections and outpatient age groups, employed status, marital status, symptoms, and contraceptive use.
In some countries (2, 32, 33), religion, age, living in a rural area, and having lower abdominal pain was signi cantly associated with bacterial vaginosis. These Varying prevalence could be due to multiple factors, including socio-demographic characteristics, immune status of patients, treating patients with broad-spectrum antibiotics and immune suppressive drugs, and hormonal in uences, etc. (34). Also, the bad practice in terms of sanitary napkins use with the accumulation of menstrual blood could contribute to the vaginal ecosystem's enhancements (16,35). Another important but obvious point would be the involvement of the women's ethnicity and body hormone level in the diversity of the vaginal microbial community. It has been shown that the decrease in estrogen levels would imply the maintenance of the vaginal pH neutrality which would be at the origin of the vaginal microbiota destabilization (36). Furthermore, current literature suggests that the decrease in estrogen that would be associate with an elevation of the vaginal pH a few hours after coitus would increase the adhesion of Gardnerella vaginalis to the epithelial cells of the mucosa while simultaneously replacing the lactobacilli from their attachment sites on this same mucosa (37). Although Nugent score's techniques are highly sensitive and speci c for evaluating an imbalance of the vaginal microbiota (21), they are not sensitive to characterize the composition of the vaginal microbiota (29). Without losing sight of all these complexities, it would be interesting to characterize the microbiota present in the vaginal epithelium of women in south-eastern Gabon using molecular analysis to better understand these infectious diseases.
In the present study, VVC-like infections among reproductive-age women were 195 (34.1%). Another study, made in the same population for VVC-like infections have revealed a 28.52% occurrence (38 surveillance studies of changes in species distribution should be routinely screened to improve patient care and effective management. In this research, aerobic bacteria play a signi cant role in the etiology of vaginal infections. AV-like infections with an occurrence of 51.1 % were more commonly observed than VVC-like infections. However, the prevalence of AV-like infections was less prevalent than BV-like infections. According to Donders et al. (2002), it is a clinical entity quite distinct from bacterial vaginosis and causes a signi cant host response (3). Despite a late description, on this date, AV is better evaluated. Many recent publications allow a better understanding of it (37). Compared to the nding of the present study, a similar prevalence (51.0%) was reported for AV among examinees women with signs of vaginitis in Bosnia and Herzegovina (4). Recently, Salinas et al. (2020) found among Ecuadorian women that had a vulvovaginitis infection AV as the main diagnosed vaginal infection (51.6%), followed by BV (24.2%) and nally VVC (7.4%) (6). A high AV prevalence has been postponed in China (65.49%) among sexually active patients who underwent routine gynecological examination (18). AV is a common form of vaginal infection that is distinguished from BV. The micro ora in AV comprises commensal aerobic microorganisms of intestinal and mucosa origin, principally Escherichia coli, Staphylococcus aureus, and Staphylococci (3,5,18). Data from our work on outpatients in south-eastern Gabon show that Streptococcus B (23.9%), Staphyloccocus aureus (17.7%), Klebsiella spp. (11.6%), and E. coli (5.8%) as the bacteria most associated with AV. A report in Gabon had shown Streptococcus agalactiae isolates colonizing pregnant women with serotype distribution in line with other reports from Africa (14). In a country close to Gabon, namely Cameroon, the distribution of AV was as follows: Staphylococcus spp. 8.82%; Streptococcus spp. 1.96% and Enterobacteria 11.78% (20). AV-like infections are easily mixed with other pathogens, especially with BV, VVC, or TV (8).
A low prevalence of TV was found in our study. Also notable was that the lack of diagnostic technique sensitivity used in our study may explain these low TV-like infections rates. According to Abdul Aziz et al.
(2019) relying on microscopic examination for the diagnosis of TV could underestimate the prevalence of the infection, and the use of more sensitive techniques is recommended (2). However, we did not nd an association with TV, unlike other studies where the mixed infections between TV, VVC, or BV were found to be 0.1; 0.3%; 25%, and 30% respectively in Cameroun, Yemen, China, and Bosnia and Herzegovina (2,4,8). While mixed infections had been very common in this study among outpatient. AV-like infections and VVC-like infections with respectively 227 (77.5) and 160 (82.1) were most involved in mixed infections. About BV-like infections, single infections (31.9%) were also less frequent than mixed infections (68.1%). The results contradict the Jahic et al. and Zhang et al. studies that found fewer mixed infections than single infection (4,18). The present study identi ed BV 228 (83.5%) as the main cause of mixed infections. Although, AV 227 (83.2%) is also a key component of mixed infections, no statistically signi cant association was found between AV and vaginal infections in our population set according to the GLM model (p 0.05). However, there is a statistically signi cant association between BV and vaginal infections (p 0.0001). This observation con rms that when the vaginal ecosystem is disturbed, the vaginal epithelium is less protected and vaginal infections would set in (6). According to others studies, AV is more commonly associated with BV (3,4,18). Therefore, the absence of lactobacilli would be an AV characteristic (4). More generally, BV would represent a common cause of disturbance in the normal vaginal ora in favor of the agents in vaginal infections (32). It is important to mention that the 69 participants had been simultaneously diagnosed with BV, AV, and VVC in the study. As reported by Salinas et al. and many authors, several sexual partners could represent a risk factor (6). Further analysis would be done in the population set to identify the main key species commonly associated with each mixed infection.

Conclusions
This research indicates a polymicrobial etiology of vaginitis in south-eastern Gabon. Further studies should be conducted in the monitoring of vaginal infections among women. A limitation of this study is the data lack about the antibiotic susceptibility of diverse etiologies while bacterial vaginal infections are one of the major causes of frequent antibiotic use in women of reproductive age. It would undoubtedly be useful the level of antibiotic resistance in vaginal isolates. The use of molecular tools could allow us to assess the colonization status of different microbial taxa and thus identify the species present in the vaginal microbiota with greater reliability among women from south-eastern Gabon. Molecular tools could lead to improved knowledge of the vaginal microbiota of women in south-eastern Gabon, which could result in the development of new context-speci c therapeutic approaches such as the use of probiotics.

Ethical considerations and consent to participate
The study protocol was reviewed and approved by the Ethics Committee of CIRMF. The female participants were recruited to our study set, after having read and signed individually the informed consent or from their parents or legal representatives. To ensure con dentiality, numbers were assigned to participants via their survey sheets and then plotted on the corresponding samples. Once the diagnosis is done, the results of all participants were transmitted with strict anonymity.