Project Summary/Abstract:Invasive aspergillosis (IA) is a leading cause of morbidity and death in immunocompromised patients, with over 200,000 cases worldwide per year. While advances in diagnostic testing for IA and the development of potent, less toxic triazole antifungal drugs have reduced IA-associated mortality from 68% to 25%, an alarming rise in the incidence and geographic distribution of azole-resistant Aspergillus fumigatus (ARAF) over the past decade is steadily offsetting these gains and compromising the use of triazole antifungal drugs as first-line therapy for IA. The major culprit in the rapid explosion of ARAF in the environment and in patient lungs appears to be the increasing global use of azole fungicides similar in structure to medical triazoles in agriculture worldwide, especially in Europe and Asia ? these fungicides persist in the soil and water for months, applying widespread, systematic selection pressure favoring the emergence of ARAF, which in turn spreads quickly through vegetative growth and abundant dissemination of airborne conidia. ARAF is now responsible for up to 30% of IA cases in Europe with associated mortality of 70-100%, with steadily rising incidence elsewhere in the world. Managing patients with ARAF IA is incredibly challenging due to the lack of clinical risk factors, lack of diagnostic methods that can differentiate ARAF IA from azole-susceptible IA in a timely manner, and the need to employ antifungals with substantially lower efficacy and greater toxicity than the azole drugs.We have developed methods of identifying patients with IA via detection of Aspergillus volatile sesquiterpene secondary metabolites in breath, and of examining in vivo responses to antifungal treatment via serial assessment of these fungal breath metabolites over the course of antifungal therapy. In contrast to the attenuation of secondary metabolite release in azole- susceptible A. fumigatus with azole antifungal treatment, these metabolites instead increase in ARAF with azole exposure. We will test the hypothesis that the sesquiterpene secondary metabolite response to triazole antifungal therapy is distinct in azole-susceptible and azole-resistant A. fumigatus by (1) comparing the in vitro response of ARAF (including the most common cyp51A mutations TR34/L98H, TR46/Y121F/T289A, M220, and G54, and ARAF with phenotypic multi-azole resistance despite wild-type cyp51A) and azole-susceptible A. fumigatus strains to azole antifungal therapy and (2) comparing the in vivo response of ARAF and azole-susceptible A. fumigatus to azole antifungal therapy in breath using a neutropenic murine IA model. Ultimately, we expect to delineate marked differences in the dynamics of volatile sesquiterpene secondary metabolite release in ARAF vs. azole- susceptible A. fumigatus, both in vitro and in vivo. Successful completion of these aims would set the groundwork for a novel, rapid breath assay that can distinguish patients with ARAF IA from azole-susceptible IA, providing an in vivo indicator of imminent therapeutic failure, guiding selection of appropriate antifungal therapy, and reducing the extremely high morbidity and mortality associated with ARAF IA.