A comparative study of the broth micro- and macro-dilution techniques for the determination of the in vitro susceptibility ofAspergillus fumigatus

Abstract

The effects of inoculum size, medium, temperature, and duration of growth on the in vitro susceptibility testing of Aspergillus fumigatus were investigated using broth micro- and macro-dilution techniques. The minimum inhibitory concentrations (MICs) of ketoconazole, miconazole, itraconazole, fluconazole, and amphotericin B were significantly influenced by the inoculum size, regardless of the techniques used. Two- to four-fold higher MIC values were obtained when the inoculum size was increased 100-fold. The use of peptone yeast extract glucose and RPMI 1640 media provided essentially identical MIC values at 30 and 35 °C after incubation for 48 h or longer. A comparison of broth micro- and macro-dilution techniques revealed that, under equivalent conditions, the latter with an inoculum size between 1 × 103and 1 × 104conidia (strain W73355)/mL consistently provided the lowest MICs of fluconazole (256 μg/mL), ketoconazole (8 μg/mL), miconazole (2 μg/mL), itraconazole (0.25 μg/mL), and amphotericin B (0.25 μg/mL). Using the broth macrodilution technique, we screened 24 clinical isolates of A. fumigatus obtained from the Detroit Medical Center in 1994. The MIC values of fluconazole, ketoconazole, miconazole, itraconazole and amphotericin B for all the isolates were 128–256, 8–16, 1–2, 0.25–0.5, and 0.25–1.0 μg/mL, respectively, indicating that none of the clinical isolates that we tested shows acquired resistance to the antifungals used.Key words: Aspergillus fumigatus, susceptibility test, antifungals, drug resistance, broth macrodilution.