Comparison of quantification methods of morphological forms and biofilms of Candida albicans submitted to anti-fungal therapies

• Grant number: 21/08890-1
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: November 01, 2021
• End date: August 31, 2022
• Field of knowledge: Health Sciences - Dentistry
• Principal Investigator: Ewerton Garcia de Oliveira Mima
• Grantee: Maria Carolina de Albuquerque
• Host Institution: Faculdade de Odontologia (FOAr). Universidade Estadual Paulista (UNESP). Campus de Araraquara. Araraquara , SP, Brazil

Abstract

Candida albicans is the main fungus pathogen that affects humans and has the ability to develop in different morphologies (yeasts, pseudo-hyphae, and hyphae), a characteristic called polymorphism. This polymorphism and antifungal therapies compromise the quantification methods that demand and accuracy, such as research involving filamentous fungi. The aim of the study will be to compare the methods of quantification of C. Albicans by colony forming units (CFU/mL), by quantitative polymerase chain reaction (qPCR), and by metabolic activity (XTT Assay) with viable cell count and cell nuclei in Confocal Microscope after antifungal therapies. A standard strain of C. Albicans (SC5314) will be cultivated in yeast and filamentous forms and subjected to treatment with fluconazole and antimicrobial Photodynamic Therapy (aPDT) mediated by curcumin and LED light. Treated cultures will be subjected to three methods of quantification: CFU/mL, cell count, and qPCR. For cell counting, the Confocal Microscope will be used with a fungal suspension treated with different fluorescent markers to show viable cells, non-viable cells, and cell nuclei. For qPCR, an intercalating DNA cross-linking agent will be used to exclude DNA from non-viable cells. Biofilms will also be cultivated for 48 hours, treated with fluconazole and aPDT, and submitted to the same tests used for planktonic cultures, in addition to the XTT assay. Data will be submitted to analysis of agreement (Intraclass Correlation Coefficient and Bland-Altman) and correlation (± = 5%) between methods (CFU/mL, cell count, qPCR and XTT). (AU)