In vitro determination of optimal antifungal combinations against Cryptococcus neoformans and Candida albicans.

There is currently no rapid, reliable, and reproducible in vitro technique to describe the growth-inhibitory interactions of antifungal drug combinations over a wide range of drug concentrations. We have developed a microdilution plate assay that was used to determine optimal drug combinations and concentrations of one-, two-, and three-drug regimens of amphotericin B (AmphB), fluconazole (FLU), and 5-fluorocytosine (5FC) for growth inhibition of three isolates each of Cryptococcus neoformans and Candida albicans. These growth inhibition data were then used in a multifactorial design technique to (i) generate contour and surface response plots to aid visual interpretation and (ii) develop mathematical equations describing the growth responses of the fungi to a wide range of antifungal concentrations and ratios. Our data indicated that (i) antifungal drug-drug interactions affecting yeast growth are complex functions of the drugs used in combination, their absolute concentrations, and also their relative (proportional) concentrations; (ii) AmphB-FLU combinations had additive effects against C. albicans over wide concentration ranges for each agent but were indifferent (i.e., were less than additive) in their inhibitory effect on C. neoformans; (iii) other two-drug combinations (FLU-5FC or AmphB-5FC) had indifferent effects on the growth of both fungi; and (iv) three-drug combinations (AmphB-FLU-5FC) showed an additive inhibitory effect on the growth of both C. albicans and C. neoformans. The finding that no antagonism was observed in combinations employing AmphB and FLU in this in vitro model is of critical importance since it argues against the current theoretical concept, based on the individual drug's mode of action, of antagonism between these two drugs. These microdilution techniques provide a method to determine rational regimens of antifungal agents in multidrug combinations for future testing to correlate in vitro activity with in vivo response. The use of this approach has made the evaluation of complex antifungal drug-drug interactions possible and provided important new information to the evolving field of antifungal drug combination.

Susceptibility testing of Cryptococcus neoformans: a microdilution technique.

We studied a series of test conditions in a microtiter system to define the optimal method for determining the susceptibility of Cryptococcus neoformans to antifungal agents. Twenty-one isolates of C. neoformans were grown for 24 or 48 h in four chemically defined media: yeast nitrogen base (BYNB 7); RPMI 1640; synthetic amino acid medium--fungal (SAAMF), buffered at pH 7.0 to select the medium that best supported growth of this fastidious yeast; and yeast nitrogen base, pH 5.4 (YNB 5.4). Maximum growth of C. neoformans, at 35 degrees C, was obtained in YNB 5.4, with the next highest growth levels in BYNB 7, SAAMF, and RPMI. Growth at 24 h was uniformly poor in all media and lacked reproducibility. In contrast, incubation for 48 h gave adequate growth with low standard deviations, and 48 h was selected as the optimal incubation period for this study. Comparison of the relationship between growth kinetics and initial inoculum size for eight cryptococcal isolates showed that 10(4) cells per ml yielded optimal growth in BYNB 7 and YNB 5.4, whereas 10(5) cells per ml was optimal in RPMI and SAAMF. Furthermore, variation of inocula from 10(3) to 10(5) cells per ml showed small but significant inoculum effects in determining MICs of fluconazole, amphotericin B, and flucytosine for C. neoformans. Therefore, 10(4) cells per ml was chosen as the optimal inoculum for susceptibility testing in this study. Mean MICs of fluconazole, amphotericin B, and flucytosine for 21 crytococcal isolates in RPMI and BYNB 7 were low (for example, fluconazole had mean MICs of 1.2 and 1.3 micrograms/ml in RPMI and BYNB 7, respectively) and differed significantly from medium to medium. In contrast, the MICs obtained in SAAMF were significantly higher (e.g., fluconazole had a mean MIC of 2.2 micrograms/ml). Variance in MICs was large with fluconazole and flucytosine but small with amphotericin B, irrespective of the medium used. A microtiter system employing BYNB 7 as the medium, 48 h as the incubation period, and 10(4) cells per ml as the final inoculum is a simple, accurate, and reproducible method for the testing of C. neoformans susceptibility to fluconazole, amphotericin B, and flucytosine.