Susceptibility of Candida species isolated from recurrent vulvovaginal candidiasis to antifungal agents among women at Institut Pasteur of Côte d ’ Ivoire

Susceptibility of Candida species isolated from recurrent vulvovaginal candidiasis to antifungal agents among women at Institut Pasteur of Côte d’Ivoire Kpongbo Etienne Angora, Vincent Djohan, Valérie A. Ira-Bonouman, Henriette A. VangaBosson, Kondo Fulgence Kassi, Abibatou Konaté, Valérie A. Bedia-Tanoh, Sébastien A. Miezan, Christiane P. Kiki-Barro, Hervé Menan, André Offianan Touré and William Yavo


INTRODUCTION
Vulvovaginal candidiasis is a frequent mycotic infection in women caused by a commensal yeast called Candida lying in the vaginal mucous membrane.Candida to produce either superficial or systemic infections depends on the host immune system, risk factors (Arfiputri et al., 2018), sexual behaviour (Grinceviciene et al., 2018) and a loss of the vaginal equilibrium (Arendrup, 2013).Of sexually active women, 75% have at least once experienced symptomatic of vulvovaginal candidiasis linked to vaginal discharge (Brandolt et al., 2017).Moreover, the recurrent vulvovaginal candidiasis is characterized by the fact that it occurs four times at least in a year (Sobel, 2015).Among the Candida species, Candida albicans is considered the most prevalent and 25 to 34 years age group has the highest prevalence at 9% (Denning et al., 2018).Hence, an efficient treatment generally requires a local and oral route administration of antifungal agents (Fan et al., 2015).Recurrent vulvovaginal candidiasis is a common cause of morbidity in women because of the fact that it is a source of health discomfort.It is also due to the difficulties in the treatment according to the clinical practitioners and fluconazole remains the most effective treatment option (Crouss et al., 2018).
Studies about candidiasis in Côte d"Ivoire showed the antifungal susceptibility pattern of the Candida spp.from vulvovaginal candidiasis (Djohan et al., 2012;Konaté et al., 2014).But there is no data available about recurrent vulvovaginal candidiasis, hence the importance of this study which aims to determine the antifungal susceptibility of Candida spp.isolated from recurrent vulvovaginal candidiasis in Côte d"Ivoire.

Patient selection
This cross-sectional study was carried out at Institut Pasteur of Côte d"Ivoire from May to July 2014 among 400 women.Vaginal swabs were collected from women in ""Centre Hospitalolier Universitaire (CHU) Cocody"" standing for Teaching Hospital of Cocody, and in "Unité d"Accueil et de Réception des Prélèvements" (URAP) standing for reception and sampling unit, Institut Pasteur de Côte d"Ivoire.Candida species identification and antifungal susceptibility testing were performed in the Mycological Laboratory in Institut Pasteur de Côte d"Ivoire.Target patients were women with vaginal discharge associated or not to other symptoms.The recurrent of vulvovaginitis was defined by the fact that it occurs four times at least in a year from a woman (Ringdahl, 2000).An informed consent form was obtained from the participants after the contents of the form were clearly explained.

Questionnaire administration
Upon a clear consent, a questionnaire was submitted to patients for information about each individual"s sociodemographic and clinical living factor.The questionnaire listed among others, points the age, the medico-surgical history, the factors that lead to the infection, the symptoms of the disease, some knowledge about the treatment used and about the recurrence factor.

Sample collection and processing
Before the insertion of the swab stick, the vulva was cleansed.
Then, the swab was obtained from the cul-de-sac with the aid of a speculum.An immediate analysis was performed with each sample, which was streaked on Sabouraud-chloramphenicol medium and then on Sabouraud-chloramphenicol-Actidione medium.Yeasts were identified via chromogenic method (CandiSelect ® 4 Biorad) which shows C. albicans (purple colonies), Candida tropicalis (dark green colonies), Candida glabrata (light green colonies), and Candida krusei (blue-turquoise colonies).The Auxacolor® gallery 2 (Biorad) was used according to manufacturer's procedures in case of diagnosis difficulty.
The isolated Candida spp.were tested to determine their antifungal susceptibility, the micro-dilution method according to the National Committee for Clinical Laboratory Standards (NCCLS) norms using the ATB ® Fungus 3 gallery (Biomérieux, France).Five antifungal agents were tested in accordance with the manufacturing recommendation; there are amphotericin B, 5-fluorocytosin, fluconazole, itraconazole and voriconazole.Briefly, fungal suspension with turbidity of McFarland 2 was obtained with young colonies in an API NaCl 0.85% Medium tube.Then 20 mm of this suspension was poured into ATB F2 Medium with aid of a micropipette to homogenize.After that, 135 mm of the ATB F2 Medium content were divided up into cupules and incubated for 24 h at 37°C.The growth and the quantification factors of the yeasts were identified through visual reading by setting the gallery on a dark background to help along.A growth score for each antifungal agent was determined and noted in each cupule in comparison with the model cupules, beginning by the weakest growth.

Data statistical analysis
Data analysis was performed using SPSS 21 software (Statical Package for Social Science, IBM SPSS Statistics).All variables were classified in a group and the Fisher exact test was used to compare proportions.P-value < 0.05 was considered significant.

Sociodemographic and clinical data
In total, 400 women with a recurrent vaginal discharge were included in the study.The average age of the admitted patients was 29.2 years (standard deviation = 7.2 years) with extremes between 14 and 69 years.The patients between 14 and 19 years old were the most affected by the recurrent vulvovaginal candidiasis in a proportion of 31.6%.However, no statistically significant ratio between age and outbreak of recurrent vulvovaginal candidiasis was noticed.As far as marital status is concerned, women that are single or concubine were more exposed to recurrent vulvovaginal candidiasis (p=0.004).The women with primary school education level represented the most vulnerable.
Clinically, apart from vaginal discharge, vaginal pruritus was the most common clinical manifestation.

Mycological data
From 400 included women with a recurrent vaginal discharge, 94 were positive to Candida spp., a prevalence rate of 23.5% (95% CI: 19.49-28.02).Five Candida spp.were isolated and C. albicans was the most prevalent at 59.6%.No case of mixed infection was seen among women.Figure 1 shows the distribution Candida spp.isolated from women with recurrent vulvovaginal candidiasis infection.

Antifungal susceptibility
The analysis on antifungal susceptibility focused on the three major species observed in the survey, which were C. albicans (56 strains), C. glabrata (18 strains) and C. tropicalis (15 strains).C. albicans did not show resistance to amphotericin B and voriconazole.Susceptibility rate varied from 89.3 to 96.4% with the other forms of antifungal agents.However, only a half of C. glabrata was found susceptible to itraconazole.This species was relatively susceptible to fluconazole (77.8%) and to voriconazole (83.3%).For C. tropicalis, the susceptibility rate to antifungal agents varied from 86.7 to 100% (Table 2).

DISCUSSION
Vulvovaginal candidiasis represents one the most frequent causes of vaginal discharge and infection, hospital admission for women that are admitted for gynecological visits.The cause of a recurrent form is linked to the exposure to some risk factors and to the chronicity of a first episode for vulvovaginal candidiasis (Fidel et al., 2004;Auler et al., 2010;Sobel, 2007).Different clinical symptoms take place in the development of recurrent vulvovaginal candidiasis.Thus, the symptoms documented in our work earlier stated by other authors (Bernstein and Seidu, 2015;Sobel, 2015).
In the survey, recurrent vulvovaginal candidiasis was found in 23.5% of the patients.Because recurrent vulvovaginal candidiasis varied from a country to another different prevalence rates from 8 to 23% were recorded in some other surveys (Corsello et al., 2003;Rylander et al., 2004;Grigoriou et al., 2006).
The frequent appearance of C. albicans in recurrent vulvovaginal candidiasis demonstrates its commensal nature from women genital mucous membrane (Sobel et al., 2004;Amouri et al., 2010;Hu et al., 2015).The present study also sheds light on the role of non albicans in the outbreak of recurrent vulvovaginal candidiasis with C. glabrata predominance.
In fact, recurrent vulvovaginal candidiasis is caused mostly by Candida spp., especially by C. glabrata (Sobel, 2015;Denning et al., 2018).The occurrence of these fungal agents in vulvovaginal candidiasis etiology was previously mentioned in Côte d"Ivoire with prevalence rates ranging from 10 to 32,7% (Bonouman-Ira et al., 2011;Djohan et al., 2012;Konaté et al., 2014) where C. glabrata was also considered predominant.The other species observed were C. tropicalis, C. krusei, Candida parapsilosis and seldom Candida famata (Richter et al., 2005) Candida inconspicua   Ekpenyong et al., 2012;Brandolt et al., 2017).This situation could be linked to the self-medication with antifungal agents in ambulatory treatment or to frequent risk factor exposures.The difference observed in susceptibility rates vary according to the Candida spp.tested and to the antifungal agent used.Other reports about C. albicans susceptibility rates to fluconazole, to amphotericin B, voriconazole and clotrimazole gave similar results (Panizo et al., 2009;Khan et al., 2018).No case of resistance to fluconazole was seen in this study, similar results about susceptibility of C. albicans from recurrent vulvovaginal candidiasis were reported (Shahid and Sobel, 2009).C. albicans spp.isolated from recurrent vulvovaginal candidiasis remain still susceptible to antifungal agents.But recently, fluconazole resistant C. albicans was found from vulvovaginal candidiasis (Sobel and Sobel, 2018).Because of this, they deserve an accepted usage in order to avoid development of resistance.
Non albicans spp.identified in this study, a good susceptibility to in vitro tested antifungals, except for C. glabrata with resistance rates of 50% to itraconazole, 22.2% to fluconazole and 16.7% to voriconazole.Non albicans spp.was particularly reported in the recurrence pathogenicity with a predominance of C. glabrata.Cases of C. glabrata in vitro resistance to antifungals especially to fluconazole were also observed in some surveys (Sobel et al., 2004;Amouri et al., 2010;Sobel, 2015).Fluconazole is an azotic antifungal frequently used through oral route associated to local forms of other antifungals in case of recurrent vulvovaginal candidiasis outbreak (Amouri et al., 2010;Khan et al., 2018).Associating an attacking treatment with a simple health care measures over a period is an advisable management strategy.Suspension of treatment can lead to a relapse in 30 to 50% of patients after three months (Sobel, 2007(Sobel, , 2015)).Thus, adequate treatment and compliance is essential for a reduction of recurrence and in an outbreak of resistance appearance of non Albicans spp. to antifungal agents.

Conclusion
Recurrent vulvovaginal candidiasis is frequent and C. albicans is the most prevalent.The treatment requires a precise diagnosis based on a mycological analysis and on the determination of the species susceptibility to the antifungal agents.Thus, appropriate tests to detect the presence of Candida spp.and assessment of the risk factors linked to the outbreak of vulvovaginal candidiasis from women should help in infection prevention.

Figure 1 .
Figure 1.Distribution of Candida species isolated from the infected patients.

Table 1 .
Table 1 shows the socio-demographic and clinical characteristics of the population studied.Corresponding author.E-mail: angorakpongbo2005@yahoo.fr.Tel: +22507393801.Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License Sociodemographic and clinical characteristics of the patient.

Table 2 .
In vitro antifungal susceptibility patterns of Candida spp.isolated from patients.

strains and antifungal agents Sensitive Resistant n % n % Candida albicans (n=56)
n: Total of Candida strains; %: percentage of Candida strains.