Quantitative Microplate-Based Growth Assay for Determination of Antifungal Susceptibility of Histoplasma capsulatum Yeasts

Abstract

Standardized methodologies for determining the antifungal susceptibility of fungal pathogens is central to the clinical management of invasive fungal disease. Yeast-form fungi can be tested using broth macrodilution and microdilution assays. Reference procedures exist forCandidaspecies andCryptococcusyeasts; however, no standardized methods have been developed for testing the antifungal susceptibility of yeast forms of the dimorphic systemic fungal pathogens. For the dimorphic fungal pathogenHistoplasma capsulatum, susceptibility to echinocandins differs for the yeast and the filamentous forms, which highlights the need to employHistoplasmayeasts, not hyphae, in antifungal susceptibility tests. To address this, we developed and optimized methodology for the 96-well microtiter plate-based measurement ofHistoplasmayeast growthin vitro. Using optical density, the assay is quantitative for fungal growth with a dynamic range greater than 30-fold. Concentration and assay reaction time parameters were also optimized for colorimetric (MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] reduction) and fluorescent (resazurin reduction) indicators of fungal vitality. We employed this microtiter-based assay to determine the antifungal susceptibility patterns of multiple clinical isolates ofHistoplasmarepresenting different phylogenetic groups. This methodology fulfills a critical need for the ability to monitor the effectiveness of antifungals onHistoplasmayeasts, the morphological form present in mammalian hosts and, thus, the form most relevant to disease.