We sought to determine if substantial differences in susceptibility profiles to common antifungal agents existed among clinical isolates of C. neoformans from three geographically diverse areas. Sixty-five clinical isolates of C. neoformans from Malawi, Thailand, and the United States were available for study. The 16 isolates from Malawi and 29 isolates from Thailand were recovered from the bloodstream of febrile, adult inpatients during previous bloodstream infection studies in these regions (9,10). The 20 isolates from the United States were recovered from the bloodstream, lung tissue, cerebrospinal fluid, and other sterile sites in routine clinical practice in the clinical microbiology laboratories of the Cleveland Clinic Foundation and Duke University Medical Center. The yeast isolates from all of the countries were shipped to Duke University Medical Center for testing and maintained in frozen stock vials at –70°C. Sixty-five yeast isolates were recovered from the frozen stock vials on potato dextrose agar and incubated at 30°C for 48 hours. The antifungal susceptibilities of the isolates were determined by using the Sensititre YeastOne system (Trek Diagnostic Systems Ltd., West Sussex, England), which includes amphotericin B, 5-flucytosine, fluconazole, itraconazole, and ketoconazole. All isolates were incubated for 72 hours, according to the manufacturer’s instructions. Inoculum assessments were performed on all trays and were within acceptable limits. The trays were visually inspected, and the MICs were determined according to the manufacturer’s guidelines. Interpretive guidelines and breakpoints for susceptibility testing of C. neoformans are not yet available from the National Committee for Clinical Laboratory Standards (NCCLS); therefore, only MIC comparisons were performed (11).

For isolates from each country, we recorded the MIC at which 50% of the isolates were inhibited (MIC₅₀) and the MIC at which 90% of the isolates were inhibited (MIC₉₀) and determined the MIC geometric mean for each therapeutic agent. We compared the MIC geometric means for the three countries with a one-way analysis of variance (ANOVA) to determine if significant differences existed. Additional comparisons between the MIC₅₀ and MIC₉₀ were not undertaken, since these were within one dilution of one another.

The C. neoformans isolates from the United States, Thailand, and Malawi demonstrated similar susceptibility profiles to the common antifungal agents against which they were tested (Table 1). The percentage of isolates inhibited at each concentration of antifungal agent over the full dilution series is summarized in Table 2. The isolates from the three countries did not differ significantly in their susceptibility to fluconazole (p = 0.198), itraconazole (p = 0.163), 5-flucytosine (p = 0.713), or ketoconazole (p = 0.531). The geometric mean of the MIC values for amphotericin B in Thailand, the United States, and Malawi was 1.2 μg/mL, 1.4 μg/mL, and 1.6 μg/mL, respectively. These mean values were significantly (p = 0.019) different.

Resistance to antifungal drugs is rare among clinical isolates of C. neoformans but has been reported (4,12). The use of antifungal agents, particularly in long-term suppressive regimens, has raised concern about the development of drug resistance in C. neoformans. However, an extensive survey of the susceptibility profiles of clinical isolates of C. neoformans at a university hospital during 1987 to 1994 helped to allay these fears by indicating no emergence of resistance (13).

This study also demonstrates no evidence of resistance among clinical isolates of C. neoformans from Thailand, Malawi, and the United States. For each country, the MIC₅₀ and MIC₉₀ of isolates to commonly used antifungal agents were within one dilution from each other. In addition, the MIC ranges were similar. Statistical comparison of the MIC geometric means confirmed that no significant differences existed between the three regions for fluconazole, itraconazole, 5-flucytosine, or ketoconazole. The only statistically significant differences were observed for amphotericin B susceptibilities; however, this difference was believed to be clinically irrelevant since the MIC geometric means for amphotericin B were 1 - 2 μg/mL, or within one dilution. Our documentation of the absence of resistance among C. neoformans isolates from the United States is consistent with data published by the Centers for Disease Control and Prevention, which showed in vitro resistance to antifungal agents to be uncommon and unchanged among C. neoformans isolates from 1992 to 1998 (14).

The similarity between the MICs of C. neoformans isolates from Malawi and the United States concurs with data from a previous study of 164 African and 402 North American clinical isolates of C. neoformans isolates that were tested and found to be susceptible to fluconazole and other triazoles, with over 99% inhibited by concentrations of fluconazole <32 μg/mL (5). The MIC₅₀ and MIC₉₀ in that study were lower than those in this study, although the YeastOne trays have been found to agree well with the NCCLS reference method for itraconazole and the other azoles (15). Also, the MICs of fluconazole documented in our study are similar to those previously reported for isolates of C. neoformans from the United Kingdom and Uganda (3,4); the MICs of 5-flucytosine in our study also were similar to those previously reported for C. neoformans isolates from Uganda (4). The itraconazole MICs documented in our study were lower than those reported for isolates from the United Kingdom, Africa, and the United States (4,5). The differences between the susceptibility profiles of C. neoformans to itraconazole reported in our study and those reported previously may be due in part to the poor solubility of this antimicrobial agent in an aqueous solution.

Using a standardized testing method, we found no significant or clinically meaningful differences between the antifungal susceptibility profiles of clinical isolates of C. neoformans from the United States, Thailand, and Malawi. Although rare strains of C. neoformans with elevated MICs to some antifungal agents may exist, they were not detected in this sampling of clinically significant C. neoformans isolates and, therefore, do not appear to be prominent in Cleveland, Ohio; Durham, North Carolina; Bangkok, Thailand; or Lilongwe, Malawi.