Susceptibility testing of Aspergillus flavus:: Inoculum dependence with itraconazole and lack of correlation between susceptibility to amphotericin B in vitro and outcome in vivo

We have attempted to validate in Aspergillus flavus the main in vitro methodologies that have been used to detect resistance in Aspergillus fumigatus. We developed a murine model with two ri. flavus isolates, one that was apparently resistant in vitro to amphotericin B (AFL5) and another that was resistant to itraconazole (AFL8), No correlation was found for amphotericin B in AFL5, since the in vivo response was compatible with a susceptible isolate. Modification of the in vitro susceptibility test methodology for amphotericin B was unsuccessful. Although AFL8 was apparently resistant to itraconazole in vitro, it was found to be susceptible in vivo. Additional in vitro work has detected weaknesses in the in vitro susceptibility methodology validated for A. fumigatus when applied to A. flavus. The principal problems are that changes in the inoculum have a large effect on the MICs of itraconazole for some A. flavus strains and that a trailing end point and spore sediment often appear when an inoculum with a higher colony count is used. We propose a modified method using a final inoculum of 2.5 x 10(4) CFU per ml of RPMI 1640 medium with 2% glucose buffered to pH 7.0 in a microtiter format, incubated for 48 h with no growth end point. Validation of this methodology requires one or more itraconazole-resistant A. flavus isolates, which have yet to be identified.