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Background:   Aspergillus fumigatus  is a ubiquitous saprophytic airborne fungus responsible for more than one million deaths every year. The siderophores of  A. fumigatus  represent important virulence factors that contribute to the microbiome-metabolome dialog in a host. From a diagnostic point of view, the monitoring of  Aspergillus  secondary metabolites in urine of a host is promising due to the non-invasiveness, rapidity, sensitivity, and potential for standardization.   Methods:  Using a model of experimental aspergillosis in immunocompromised Lewis rats, the fungal siderophores ferricrocin (FC) and triacetylfusarinine C (TAFC) were monitored in rat urine before and after lung inoculation with  A. fumigatus  conidia. Molecular biomarkers in high-dose (HD) and low-dose (LD) infection models were separated using high performance liquid chromatography (HPLC) and were detected by mass spectrometry (MS). In the current work, we corroborated the  in vivo  MS infection kinetics data with micro-positron emission tomography/computed tomography (μPET/CT) kinetics utilizing  68 Ga-labeled TAFC.   Results:  In the HD model, the initial FC signal reflecting aspergillosis appeared as early as 4 h post-infection. The results from seven biological replicates showed exponentially increasing metabolite profiles over time. In  A. fumigatus , TAFC was found to be a less produced biomarker that exhibited a kinetic profile identical to that of FC. The amount of siderophores contributed by the inoculating conidia was negligible and undetectable in the HD and LD models, respectively. In the μPET/CT scans, the first detectable signal in HD model was recorded 48 h post-infection. Regarding the MS assay, among nine biological replicates in the LD model, three animals did not develop any infection, while one animal experienced an exponential increase of metabolites and died on day 6 post-infection. All remaining animals had constant or random FC levels and exhibited few or no symptoms to the experiment termination. In the LD model, the TAFC concentration was not statistically significant, while the μPET/CT scan was positive as early as 6 days post-infection.   Conclusion:  Siderophore detection in rat urine by MS represents an early and non-invasive tool for diagnosing aspergillosis caused by  A. fumigatus . μPET/CT imaging further determines the infection location  in vivo  and allows the visualization of the infection progression over time.

Early and Non-invasive Diagnosis of Aspergillosis Revealed by Infection Kinetics Monitored in a Rat Model