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. 2025 Mar 27:16:1566171.
doi: 10.3389/fmicb.2025.1566171. eCollection 2025.

New insights in the role of Candida biofilm in the pathogenesis of recurrent vulvovaginal candidiasis: a prospective clinical study

Marta Díaz-Navarro  1   2 , Álvaro Irigoyen-von-Sierakowski  1   2 , Imani Delcán  3 , Ariadna Monte  3 , María Palomo  1   4 , Pilar Escribano  1   2   4 , Jesús Guinea  1   2   4 , Almudena Burillo  1   4   5 , Alicia Galar  1   2   4 , Patricia Muñoz  1   2   4   5 , María Guembe  1   2
Affiliations

New insights in the role of Candida biofilm in the pathogenesis of recurrent vulvovaginal candidiasis: a prospective clinical study

Marta Díaz-Navarro et al. Front Microbiol. .

Abstract

Background: Despite the pathogenesis of vulvovaginal candidiasis (VVC) is multifactorial, this study aimed to assess whether phenotypic characteristics, such as biofilm production and quality, along with clinical symptoms, are associated with recurrent VVC (RVVC).

Methods: Over 1 year (Oct 2021-Oct 2022), we prospectively included 271 patients ≥18 years who attended our institution, had Candida spp. isolated in vaginal swabs, and provided informed consent. Patients were followed for 1 year. Candida spp. isolates were tested by the following techniques: crystal violet (CV) for biomass quantification, XTT for metabolic activity quantification, and microscopy for biofilm area quantification. Clinical and microbiological data were also collected.

Results: Overall, 55 (20.3%) patients experienced at least one recurrence, with 19 (7.0%) meeting the criteria for RVVC (≥3 episodes/year), with 65 episodes in total. Demographic and clinical characteristics were similar in both study groups. Most isolates were C. albicans (90.0%). Median (interquartile, [IQR]) absorbance values for CV and XTT in 18/19 RVVC and 238/252 non-RVVC isolates were as follows: CV, 1.850 (1.578-2.156) vs. 1.426 (1.081-1.823), p = 0.005; XTT, 0.184 (0.116-0.293) vs. 0.228 (0.147-0.331), p = 0.253. Median (IQR) biofilm occupation area percentage in 16/19 RVVC and 16/252 non-RVVC isolates was, respectively: 13.15 (8.54-16.9) and 10.73 (5.88-17.73), p = 0.710.

Conclusion: RVVC was associated to high biomass production. Additionally, RVVC clinical isolates exhibited a tendency toward lower metabolic activity, which may contribute to treatment failure.

Keywords: biofilm; biomass; metabolic activity; pathogenesis; recurrent vulvovaginal candidiasis.

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Conflict of interest statement

JG reports a relationship with Pfizer Inc., Gilead Sciences Inc., Merck Sharp & Dohme UK Ltd. that includes: speaking and lecture fees. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision.

Figures

Figure 1
Figure 1
Description of clinical isolates in both study groups. (A) Candida spp. distribution according to species. * statistical significance p = 0.029. (B) Median crystal violet absorbance. * Statistical significance p = 0.005. The total available number of clinical isolates to be tested was 256/275 (18/19 in RVVC group and 238/252 in non-RVVC group). (C) Median metabolic activity absorbance (XTT). NS, non-significant. The total available number of clinical isolates to be tested was 256/275 (18/19 in RVVC group and 238/252 in non-RVVC group). (D) Median percentage of biofilm occupation area. NS, non-significant. The total number of selected and available clinical isolates tested were 32 (16 from RVVC group and 16 from non-RVVC).
Figure 2
Figure 2
Biofilm structure according to species. We selected a representative image of each Candida species. Biofilm formation was performed on 12 mm circular glass slides pre-treated with Poly-L-lysine (1:100) for 24 h at 37°C and observed using confocal laser scanning microscopy (CLSM, Leica Geosystems AG, Heerbrugg, Switzerland) and the APO 63X glycerol immersion objective. Biofilm images (280×280 μm) were captured from three different areas of the slide in triplicate and then processed using the FIJI/ImageJ software (National Institute of Health, Bethesda, MD). The percentage of area occupied by yeasts and hyphae was calculated for each sample. (A) Candida albicans; (B) Candida parapsilosis; (C) Candida glabrata; (D) Candida krusei; (E) Candida orthopsilosis.
See this image and copyright information in PMC

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