Initial use of a broth microdilution method suitable for in vitro testing of fungal isolates in a clinical microbiology laboratory

Abstract

Antifungal susceptibility testing methods currently lack a standardized procedure. Many factors, such as inoculum preparation, inoculum density, medium selection, pH, incubation time and temperature, and endpoint determination, affect results. We developed a workable procedure for fungal susceptibility testing, with a microtiter method based upon modifications of the proposed guidelines from the National Committee for Clinical Laboratory Standards, using two different growth media. For this procedure, the microtiter tray is prepared as a panel of 6 drugs (amphotericin B, flucytosine, fluconazole, ketoconazole, miconazole, and itraconazole) alone and in combination with amphotericin B. Eagle's minimal essential medium and RPMI 1640 are the two growth media. Two separate susceptibility trays are inoculated for each sensitivity test, with one tray incubated at 30 degrees C and the other incubated at 35 degrees C. After 48 h of growth, results for both temperatures and both media are recorded and interpreted. The four test environments (two media each at two temperatures) provided growth for 100 of the first 104 organisms that were submitted for testing. This approach provides a workable methodology for routine antifungal susceptibility testing in a clinical microbiology laboratory setting.