Microtiter broth dilution method for yeast susceptibility testing with validation by clinical outcome

Area of Science:

• * Clinical Microbiology
• * Mycology
• * Antimicrobial Susceptibility Testing

Background:

• * Current laboratory methods and culture media for pathogenic yeast susceptibility testing are suboptimal.
• * Screening of six candidate media (RPMI 1640, yeast nitrogen base, Casamino Acids, Mueller-Hinton, Sabouraud dextrose, and minimum essential medium-Eagle salts) was performed.
• * Two media, RPMI 1640 (chemically defined) and Casamino Acids (chemically complex), were selected for further evaluation.

Purpose of the Study:

• * To evaluate and compare different culture media and techniques for antifungal susceptibility testing of pathogenic yeasts.
• * To determine Minimum Inhibitory Concentrations (MICs) of four antifungal agents against Candida species.
• * To correlate MICs with clinical and animal model outcomes and establish reliable susceptibility cut points.

Main Methods:

• * Spectrophotometric absorbance was used to screen media for nucleic acid and protein content.
• * MICs of 5-fluorocytosine, miconazole, ketoconazole, and amphotericin B were determined using agar dilution and microtiter broth dilution methods.
• * Results were correlated with clinical outcomes and validated in a murine model of systemic candidiasis.

Main Results:

• * RPMI 1640 with L-glutamine exhibited the lowest absorbance for nucleic acid and protein, indicating higher purity.
• * The microtiter broth dilution technique proved more efficient and easier to perform than agar dilution.
• * RPMI 1640 medium yielded MICs that correlated better with clinical and animal outcome data compared to Casamino Acids medium.

Conclusions:

• * RPMI 1640 medium with L-glutamine is a superior choice for pathogenic yeast susceptibility testing due to its purity and performance.
• * The microtiter broth dilution system using RPMI 1640 medium is a simple, precise, and economical method for antifungal susceptibility testing.
• * Validated susceptibility cut points enhance the reliability of this microtiter broth system for clinical and research applications.