Rapid quantification of pathogenic fungi by Cellometer image-based cytometry.

Area of Science:

• Microbiology
• Cell Biology
• Biotechnology

Background:

• Traditional colony forming unit (CFU) assays for quantifying pathogenic yeasts lack sensitivity and efficiency.
• Existing flow cytometry methods for live/dead yeast detection are limited by cost and biosafety concerns.
• Accurate quantification of fungal pathogens is crucial for diagnosing and treating infections.

Purpose of the Study:

• To develop a sensitive and efficient image-based cytometric methodology for quantifying viable pathogenic yeasts.
• To establish a cost-effective and safer alternative to traditional CFU assays and flow cytometry.
• To assess the utility of image-based cytometry for evaluating antifungal drug efficacy.

Main Methods:

• Utilized acridine orange/propidium iodide staining with Cellometer image-based cytometry.
• Quantified Histoplasma capsulatum growth within RAW 264.7 macrophages.
• Assessed Candida albicans infection in bone marrow-derived macrophages using a GFP-expressing strain.
• Performed dose-response experiments with amphotericin B and itraconazole to evaluate antifungal drug susceptibility.

Main Results:

• Image-based cytometry accurately recapitulated H. capsulatum growth trends observed with CFU enumeration, but with significantly increased sensitivity.
• Demonstrated direct assessment of C. albicans infection in macrophages.
• Showcased the ability of image-based cytometry to rapidly assess the kinetics of antifungal-induced cytotoxicity.

Conclusions:

• Developed a rapid, accurate, and economical image-based cytometry method for detecting and quantifying human fungal pathogens.
• The methodology is effective for analyzing yeast quantification alone or in association with host cells.
• This approach provides a valuable tool for antifungal drug susceptibility testing and research on fungal pathogenesis.