Evaluation of Aspergillus fumigatus Metabolic Activity In Vitro: A Comparison Between Resazurin and XTT Methods

Aspergillus fumigatus is a significant opportunistic pathogen that causes a range of diseases collectively known as aspergillosis. Measurement of filamentous fungal growth in molds, such as A. fumigatus, is crucial for the development of antifungal drugs and for clinical and epidemiological monitoring of drug resistance. However, determining their fungal growth is a challenging approach, since these organisms have multinucleate hyphae that make the individual colony-forming unit assessment difficult. Measurement of cellular metabolic activity is, therefore, an alternative method to assess the impact of stressful conditions on fungal growth resulting from drug activity. The current protocol presents a comparative analysis of resazurin and XTT assays as indicators of metabolic viability for A. fumigatus under different experimental approaches. Resazurin, a redox-sensitive compound, is widely used due to its high sensitivity in detecting early-stage metabolic activity, particularly in conidia. In contrast, XTT, a tetrazolium-based assay, is particularly effective for evaluating metabolic activity in mature hyphal forms and biofilms, as it allows for shorter incubation times with the fungus, with the formation of a stable and soluble formazan product. The protocol outlines the respective advantages and limitations of each assay. It provides a guide for researchers in selecting the most appropriate assay for evaluating fungal viability, antimicrobial effectiveness, drug interactions through checkerboard assays, and biofilm-related resistance.