RESUMO
Background: Recurrent vulvovaginal candidiasis (RVVC) is a recalcitrant medical condition that affects many women of reproductive age. The importance of biofilm formation by Candida in RVVC has been recently questioned. This study aimed to elucidate the fundamental growth modes of Candida in the vagina of patients with RVVC or sporadic vulvovaginal candidiasis (VVC) and to assess their roles in the persistence of RVVC. Methods: Vaginal tissues were sampled from twelve patients clinically and microbiologically diagnosed as RVVC or VVC at a post-antifungal-treatment and asymptomatic period. High-resolution scanning electron microscopy, fluorescence in situ hybridization in combination with Candida-specific 18S rRNA probes and viable fungal burden were used to qualitatively and quantitatively evaluate Candida growth in the human vagina. The presence of Candida biofilm extracellular polymeric substances was examined using confocal laser scanning microscopy and biopsy sections pre-stained with Concanavalin A. Histopathological analysis was carried out on infected vaginal tissues stained with hematoxylin and eosin. Lastly, the susceptibility of epithelium-associated Candida biofilms to fluconazole at the peak serum concentration was evaluated. Results: Candida species grew on the vaginal epithelium of RVVC patients as morphologically disparate biofilms including monolayers, microcolonies, and macro-colonies, in addition to sporadic adherent cells. Candida biofilm growth on the vaginal epithelium was associated with mild lymphocytic infiltration of the vaginal mucosa. These epithelium-based Candida biofilms presented an important characteristic contributing to the persistence of RVVC that is the high tolerance to fluconazole. Conclusions: In summary, our study provides direct evidence to support the presence of Candida biofilms in RVVC and an important role of biofilm formation in disease persistence.