Aspergillus species have become the leading cause of infection related mortality in immunocompromised patients. At the same time that multiple drugs have become available for treatment of Invasive aspergillosis, antifungal drug resistance of Aspergillus is being recognized. However, the true clinical significance of antifungal resistance remains controversial, largely due to a paucity of studies to identify and characterize resistant isolates, and a lack of standardized susceptibility testing methods for filamentous fungi. We have developed a rapid conidial viability assay that is reproducible and results correlate well with those of the proposed National Committee for Clinical Laboratory Standards (NCCLS) methods. Our method may be more suitable for use in clinical microbiology lab as it is rapid (4-6 hrs) and less subjective than other available techniques. We have also identified a phenotypically and genotypically unique variant of A. fumigatus, which appears to have a high degree of resistance to Itraconazole. The studies proposed herein will facilitate our understanding of the clinical significance of a new Aspergillus variant and define the potential application of a rapid new susceptibility testing method. Aim 1 will define parameters of a fluorochrome based microplate (FM) susceptibility assay and determine if detection of in vitro resistance predicts clinical failure in previously developed animal model. Experiments will be performed to determine how well the FM assay results correlate with NCCLS results for a large number of filamentous fungi and different antifungal drugs. Isolates found to be resistant in vitro will be used to infect mice and therapeutic efficacy will be determined by qualifying fungal burden and death. Aim 2 will determine the clinical significance of the itraconazole-resistant A. fumigatus variants. Specifically, culture collections from multiple institutions will be screened to determine if similar variants of have caused disease and susceptibility of identified isolates will be tested against a battery of antifungal currently used to treat aspergillosis. Clinical significance of in vitro resistance will be assessed in animal models. Also, preliminary studies to evaluate the mechanism of ITZ resistance will be performed. These two aims have been developed to take is closer to the overall goal of decreasing Aspergillus associated mortality by developing more effective, laboratory directed therapeutic strategies.