Invasive aspergillosis (IA) caused by the fungus Aspergillus fumigatus is the most frequent fungal cause of death in immunocompromised patients. The number of patients at risk for developing IA is steadily increasing due to advances in medicine. Early and accurate diagnosis of IA is critical to patient outcome, yet is exceedingly difficult due to the lack of specific clinical symptoms, the wide range of other infectious etiologies, and the general inability to perform invasive diagnostic tests in these high-risk patients. The FDA-approved galactomannan (GM) assay is a non-invasive molecular diagnostic tool for IA, but unfortunately has several serious limitations that hamper its accuracy. In our preliminary studies, we have discovered two novel small molecular weight serum biomarkers using a murine model of IA and metabolomic analysis of serum from infected mice. These biomarkers are not present in uninfected mouse sera and are absent in normal human serum; hence they have the potential to enable the development of a novel sensitive diagnostic assay for early detection of IA. The objective of this application is to determine the specificity of thee novel markers for IA against other infections seen in these high-risk patients, to compare the performance of these new biomarkers against the GM assay in murine models of IA, and to extend these findings to patient blood samples. The GM assay is an immunoassay where structurally similar cell wall mimicking antibiotics which do not contain lipids are well-known to lead to false positive results. An innovative advantage to our application is that these new small molecular weight biomarkers are rarely found in nature and will be more specific for IA. Our approach will be divided into three aims: 1) Define the in vitro specificity of the two small molecular biomarkers for Aspergillus spp. for detecting IA. We will characterize the specificity of these two biomarkers against cultures of yeast, mold, and bacterial pathogens. 2) Define the sensitivity and kinetics of our new biomarkers in murine models of IA in direct comparison to the GM assay. We will characterize the kinetics of appearance of the two biomarkers in all stages of invasive infection to determine their value in both diagnosis and for monitoring progression of infection. We will simultaneously also compare the two biomarkers to the standard GM assay, including during mold-active antifungal therapy well-known to decrease the sensitivity of the GM assay. 3) Characterize the performance of two small molecular weight biomarkers in IA stored clinical blood samples from at-risk uninfected patients, patients with well-defined IA, and control patients. The impact of these studies will be to establish these two new biomarkers as candidates for early and sensitive diagnosis of IA that will be further advanced in the Phase II application and eventual development for use with the growing number of patients at risk for IA. PUBLIC HEALTH RELEVANCE: Invasive fungal infection caused by Aspergillus fumigatus is a leading cause of death for patients with lowered immune systems. Despite this high mortality, the early and accurate diagnosis of invasive aspergillosis is not optimal and the current standard has several major limitations. We will define the specificity and in vivo kinetics of two novel smal molecular weight biomarkers we have discovered for their eventual use in patients at risk for invasive aspergillosis.