Defining conditions for microbroth antifungal susceptibility tests: influence of RPMI and RPMI-2% glucose on the selection of endpoint criteria

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Abstract
We have compared amphotericin B, flucytosine, ketoconazole and fluconazole susceptibilities of 40 clinical isolates of Candida albicans by broth microdilution in two different media: RPMI 1640 (RPMI) and the same medium supplemented with 18 g of glucose per L (RPMI-2% glucose). Preparation of media, drugs and inocula, as well as incubation temperature, followed the recommendations of the National Committee for Clinical Laboratory Standards (Villanova, PA, USA) antifungal agent working group for broth macrodilution tests with antifungal agents. Microtitre plates were agitated for 5 min before spectrophotometric readings were performed with an automatic plate reader. MIC endpoints were defined in three different ways: (i) MIC-100% for amphotericin B and flucytosine; (ii) MIC-80% for azole-drugs and also for flucytosine; (iii) IC½ for azole-drugs. The MIC endpoints were compared between the two different media in order to ascertain which was the best criterion. For amphotericin B, 100% of strains had a maximum difference of a twofold dilution in both media. For flucytosine, MIC values were very similar in both media, irrespective of the MIC endpoint chosen, MIC-100% or MIC-80%. For azole-drugs the (MIC-80%)50 and (MIC-80%)90 in RPMI were higher than those in RPMI-2% glucose. However, (IC½)50 and (IC½)90 were identical in both media as well as (MIC-80%)50 and (MIC-80%)90 in RPMI-2% glucose, The limited growth of yeasts in RPMI precludes the selection of an azole-MIC-80% endpoint, although the MIC determination was performed with an objective turbidimetric method (spectrophotometric reading plus mathematical MIC calculation). The use of RPMI-2% glucose produces the same MIC whatever method was used, IC½ or MIC-80%. However, some minor discrepancies can be expected between IC½ and MIC-80% when strains with higher “trailing effect” are being tested. Therefore, we recommended IC½ in RPMI-2% glucose as the method of choice for MIC calculation, until more studies correlating in-vitro results with clinical outcome have been performed.