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

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Abstract
There is no ideal laboratory procedure or culture medium in current use for susceptibility testing of pathogenic yeasts. Six candidate growth media (RPMI 1640 with L-glutamine, yeast nitrogen base, Casamino Acids medium, Mueller-Hinton broth, Sabouraud dextrose broth, and minimum essential medium-Eagle salts) were screened by spectromphotometric absorbance for nucleic acid and protein. From these, two media were selected: a chemically defined growth medium (RPMI 1640 with L-glutamine) and a chemically complex medium (Casamino Acids). MICs of four antifungal agents (5-fluorocytosine, micronazole, ketoconazole, and amphotericin B) for 84 clinical isolates of various Candida species were then determined with both media in agar dilution and microtiter broth dilution systems. The resultant MICs were correlated with clinical outcome for those isolates obtained from patients treated with single antifungal agents, and susceptiblity cut points were calculated. Derived MIC cut points for susceptibility were validated in a murine model of systemic candidiasis. RPMI 1640 with L-glutamine was found to have the lowest absorbance values for both nucleic acid and protein, while Casamino Acids medium was highest in both categories. We found that RPMI 1640 with L-glutamine was superior to Casamino Acids medium in the yield of MICs which correlated with actual clinical and animal outcome data. While there were no significant differences in MICs when RPMI 1640 medium was used, the microtiter broth dilution technique was superior to agar dilution in efficiency and ease of performance. We conclude that a microtiter broth system containing RPMI 1640 medium with L-glutamine is a simple, precise, and economical technique for susceptibility testing of pathogenic Candida species. We also suggest that the validation of susceptibility cut points with patient and animal outcome data make this microtiter broth system a preferential method for yeast susceptibility testing.