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

Abstract

bStandardized 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 for Candida species and Cryptococcus yeasts; 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 pathogen Histoplasma capsulatum, susceptibility to echinocandins differs for the yeast and the filamentous forms, which highlights the need to employ Histoplasma yeasts, not hyphae, in antifungal susceptibility tests. To address this, we developed and optimized methodology for the 96-well microtiter plate-based measurement of Histoplasma yeast growth in 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 of Histoplasma representing different phylogenetic groups. This methodology fulfills a critical need for the ability to monitor the effectiveness of antifungals on Histoplasma yeasts, the morphological form present in mammalian hosts and, thus, the form most relevant to disease.T he increasing incidence of fungal disease necessitates adequate and timely assessment of antifungal susceptibility to guide the selection and implementation of antifungal therapies. Consequently, the Clinical and Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) have established reference test methods for susceptibility to the major classes of antifungals, which include the polyenes, azoles, and the recently developed echinocandins (1, 2). These standards utilize broth macrodilution or microdilution assays for testing yeasts (M27-A3 [3] and E.DEF 7.1 [4]) and filamentous fungi (M38-A2 [5] and E.DEF 9.1 [6]). Procedures, including inoculum preparation, media, assay duration, and endpoint definitions, have now been established for testing some of the more commonly encountered yeast-form pathogenic fungi (i.e., Candida species and Cryptococcus). However, notably missing from the M27-A3 and E.DEF 7.1 standards for testing of yeasts are methods for testing the yeast forms of the dimorphic fungi.The dimorphic fungi that cause systemic disease are characterized by distinct yeast and filamentous morphological states (7,8). Temperature is the principal morphology-determining factor, with yeast forms characterizing infections in mammals (9). Histoplasma capsulatum is the most common clinically encountered dimorphic fungal pathogen in the United States, with an estimated 10,000 to 20,000...

In Vitro Antifungal Susceptibility of Yeast and Mold Phases of Isolates of Dimorphic Fungal Pathogen Emergomycesafricanus (Formerly Emmonsia sp.) from HIV-Infected South African Patients

“…This confirms the findings of an earlier antifungal susceptibility study of six E. africanus yeast phase isolates (5). There are no well-standardized methods for MIC determination for thermally dimorphic fungi (19); nevertheless, in vitro activities of polyenes (i.e., amphotericin B), azoles, and echinocandins have been established for some of these organisms (20). We used Clinical and Laboratory Standards Institute (CLSI)-approved standards as a guide to yeast and mold phase testing but used a larger inoculum for the mold phase and a prolonged incubation period to facilitate growth and endpoint determinations, in line with previous studies (5,19,(21)(22)(23).…”

“…There are no well-standardized methods for MIC determination for thermally dimorphic fungi (19); nevertheless, in vitro activities of polyenes (i.e., amphotericin B), azoles, and echinocandins have been established for some of these organisms (20). We used Clinical and Laboratory Standards Institute (CLSI)-approved standards as a guide to yeast and mold phase testing but used a larger inoculum for the mold phase and a prolonged incubation period to facilitate growth and endpoint determinations, in line with previous studies (5,19,(21)(22)(23). Antifungal susceptibility testing for thermally dimorphic fungi is often limited to the mold phase, results of which may be misleading because the yeast phase is responsible for human disease (20).…”

Antifungal therapeutics for dimorphic fungal pathogens

“…This is not an insignificant question as the yeast and hyphal forms of the dimorphic fungi can have dramatically different susceptibilities. [62][63][64][65][66][67][68][69] For example, early studies with echinocandins showed antifungal effects on the hyphal form of Histoplasma. 67 However, subsequent tests against the yeast form indicated that caspofungin was anywhere from 20-to 1000-fold less effective.…”

Eng1 and Exg8 Are the Major α-Glucanases Secreted by the Fungal Pathogen Histoplasma capsulatum

“…Cell Wall Sensitivity Assays-Yeast cells were grown in 96-well microtiter plates (38) with graded concentrations of antifungal compounds (fluconazole (Glenmark Generics); caspofungin (Merck); and nikkomycin Z (gift from John Galgiani)) and cell wall-destabilizing compounds (Calcofluor White (Sigma); Congo red (MP Biomedicals); SDS; and Uvitex) (39). Growth after 4 days was measured by optical density at 595 nm and compared with the turbidity of wells lacking inhibitors.…”