Prevalence of Candida spp. in cervical-vaginal samples and the in vitro susceptibility of isolates

Keywords

• Vulvovaginal candidiasis;
• Antifungal;
• Susceptibility;
• Resistance

Introduction

Vulvovaginal candidiasis (VVC) is characterized by an infection of the genital mucosa by Candida yeasts, which mainly undertakes the vulva and the vagina. The disease occurs endogenously due to predisposing factors that favor yeast multiplication. Signs and symptoms such as itching, burning, cracking, erythema and vulvar edema, leukorrhea and the presence of whitish plaques on the vaginal mucosa are common. ¹ VVC is considered the second most common cause of genital infection in women of reproductive age, and although it represents a problem of global importance in public health, its exact incidence is unknown. ^2 and 3

Risk factors for vulvovaginitis include pregnancy, use of oral contraceptives, genetic predisposition, and previous antibiotic therapy, among others. The incidence of infection, as well as the increase in colonization of the mucosa by the yeast, is also higher in women with diabetes due to their higher glycogen levels and in those with HIV due to immune suppression.^4 and 5

Studies show that 70–75% of all women of reproductive age develop at least one case of VVC during their lifetime.^6 and 7 The recurrence rate is 40–50%, and approximately 5–8% develop recurrent vulvovaginal candidiasis (RVVC), which is characterized by four or more episodes of disease over a period of twelve months.^{6, 7 and 8}

The main etiologic agent is Candida albicans, accounting for 70–90% of VVC cases. ^7 and 8 Among the non-albicans species C. glabrata is highlighted ^{7, 8 and 9} due to its prevalence and resistance to azoles. ^10 and 11

An increasing number of Candida spp. clinical isolates are resistant to antifungal agents routinely used for the treatment of VVC. In addition, studies in different regions of Brazil suggest that geographical factors interfere in the prevalence of Candida species as well as in the sensitivity of isolates to antifungals. ^{1, 11, 12 and 13} Considering this and the lack of data on this topic in Rio Grande do Sul, this study aimed to identify the prevalence of Candida spp. isolated from the vaginal mucosa of patients with and without vulvovaginitis treated at a university hospital in extreme southern Brazil, as well as the susceptibility of these isolates to four antifungals used in gynecological routines.

Materials and methods

The study included 263 patients attended on from April 2013 to October 2014 at the gynecology clinic of the University Hospital of the Federal University of Rio Grande (FURG) – Rio Grande do Sul – Brazil. The women agreed to participate by signing the Clarified Informed Consent form. The sample size, calculated using Epi Info 6.0 and considering a prevalence of 30%, a confidence level of 99%, maximum permissible error of 20% and losses of 10%, was estimated to be a sample of 249 patients.

During the gynecological exam, a sample of cervical-vaginal mucosa was collected using a sterile brush, which was stored in sterile tubes containing PBS and kept refrigerated until processing. The sample was sent to the Mycology Laboratory of the Medicine Faculty from FURG, where it was processed within 12 h. The cultures were processed in Sabouraud agar with chloramphenicol 0.01% (SCl) and incubated at 37 °C for seven days with daily assessment of growth. The yeasts were identified using phenotypic tests such as micromorphology, chromogenic medium (CHROM Agar Candida^®), germ tube test, and microculture in agar cornmeal and were confirmed by biochemical automated method (VITEK^® 2). Each isolate was maintained on potato dextrose agar (PDA) in the mycology collection at room temperature under freezing at −20 °C in saline with 30% glycerol for subsequent susceptibility testing.

The variables evaluated in the study were obtained by self-administered pre-coded questionnaire and included age, skin color, educational level, marital status, family income, HIV infection status, pregnancy, contraception and vaginal pH at the time of sample collection. Signs and symptoms assessed during the gynecological exam were used to consider women as colonized or presenting VVC, the latter being those that had leukorrhea, pruritus, edema and/or vulvovaginal erythema associated with the presence of whitish plaques in the mucosa and the isolation of Candida spp. in mycological cultivation.

The in vitro susceptibility analysis of clinical isolates was performed by broth microdilution using the M27-A2 standard protocol CLSI (2002). ¹⁴ For the standardization of fungal inoculum, isolates were subcultured on PDA for 24 h at 37 °C, young colonies were homogenized in sterile saline, and the turbidity was adjusted to 0.5 on the McFarland scale by spectrophotometry (530 nm) to obtain a concentration of 1–5 × 10⁶ CFU/mL. Then, a dilution was performed at 1:100 and 1:20 in RPMI 1640, resulting in a concentration of 5 × 10² to 2.5 × 10³ CFU/mL. The inoculum concentration was confirmed using the Pour-plate technique. The susceptibility test was performed at concentrations of 64 μg/mL to 0.125 μg/mL for fluconazole and at concentrations ranging from 16 μg/mL to 0.031 μg/mL for itraconazole, miconazole and nystatin.

The 96-well microplates were filled with 100 μL of the antifungal concentrations and 100 μL of inoculum and were incubated at 37 °C for 48 h along with growth and sterility controls. Each isolate was tested in duplicate, and the results were visually evaluated by turbidity, comparing the fungal growth in the well with the growth control. The minimum inhibitory concentration (MIC) of fluconazole, itraconazole and miconazole was one that inhibited 80% of yeast growth, and the MIC of nystatin inhibited 100% of growth.

According to the MIC result for fluconazole and itraconazole, the isolates were classified as S for sensitive, SDD for sensitive dose-dependent and R for resistant, as established by the cutoff point protocol M27-A2. For miconazole and nystatin, the MIC₅₀ and MIC₉₀ were calculated corresponding to the antifungal concentration capable of inhibiting the growth of 50% and 90%, respectively, of the isolates tested. After reading the results of MIC, 10 μL of the solutions from the wells related to concentrations higher than and equal to the MIC were plated on a Petri dish containing agar SCl and were incubated at 37 °C for 48 h to determine the fungicide minimum concentration (FMC).

The study was conducted according to ethical principles and was approved by the Research Ethics Committee of the Federal University of Rio Grande (65/2012). The results were compiled by conducting descriptive analysis of the data and chi-square tests for categorical variables using SPSS 19.0^® software. p-values below 0.05 were considered statistically significant.