New and minimally-invasive tools to aid the diagnosis of invasive fungal diseases (IFD) are urgently needed as the immunocompromised population at highest risk increases. Advancements in molecular technology have rendered new diagnostics more readily available for clinical use.MethodsThis case-control study utilized prospectively collected, archived plasma specimens and data from the Aspergillus Technology Consortium (AsTeC) Repository to investigate the diagnostic performance of microbial cell free DNA (mcfDNA) sequencing as a minimally-invasive diagnostic for IFDs in a population of high-risk immunocompromised hosts including hematologic malignancy, stem cell and solid organ transplants patients. The 2008 Mycoses Study Group/European Organization for the Research and Treatment of Cancer diagnostic criteria served as the gold standard for test performance.ResultsSixty-five adult subjects with proven or probable IFD and 65 controls without IFD were included. Among IFD episodes Aspergillus was the most common pathogen (70.8%, 46/65), followed by Mucorales (10.8%, 7/65). Overall, sensitivity was 47.7% and specificity was 100%. Sensitivity varied based on disease certainty and pathogen; sensitivity was higher in proven versus probable IFD (60.0% vs 37.1%, respectively) and higher for subjects with invasive mucormycosis (100%) compared with aspergillosis (45.7%).ConclusionsA positive result by mcfDNA sequencing may reduce the need for invasive sampling in patients with suspected IFD. In this exploratory analysis, its high sensitivity and specificity for invasive mucormycosis suggests it could be useful for early treatment and intervention of this IFD. Future studies should focus on understanding how specific factors impact the sensitivity of mcfDNA sequencing for invasive aspergillosis.