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 68Ga-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.

Department of Analytical Chemistry Regional Centre of Advanced Technologies and Materials Faculty of Science Palacký University Olomouc Olomouc Czechia

Institute of Microbiology of the Czech Academy of Sciences Prague Czechia

Institute of Molecular and Translational Medicine Faculty of Medicine and Dentistry Palacký University Olomouc Olomouc Czechia

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Carroll C. S., Amankwa L. N., Pinto L. J., Fuller J. D., Moore M. M. (2016). Detection of a serum siderophore by LC-MS/MS as a potential biomarker of invasive Aspergillosis. PubMed DOI PMC

Davies G., Rolle A. M., Maurer A., Spycher P. R., Schillinger C., Solouk-Saran D., et al. (2017). Towards translational immunoPET/MR imaging of invasive pulmonary Aspergillosis: the humanised monoclonal antibody JF5 detects PubMed DOI PMC

Goncalves S. M., Lagrou K., Duarte-Oliveira C., Maertens J. A., Cunha C., Carvalho A. (2017a). The microbiome-metabolome crosstalk in the pathogenesis of respiratory fungal diseases. PubMed DOI PMC

Goncalves S. M., Lagrou K., Rodrigues C. S., Campos C. F., Bernal-Martinez L., Rodrigues F., et al. (2017b). Evaluation of bronchoalveolar lavage fluid cytokines as biomarkers for invasive pulmonary Aspergillosis in at-risk patients. PubMed DOI PMC

Haas H., Eisendle M., Turgeon B. G. (2008). Siderophores in fungal physiology and virulence. PubMed

Haas H., Petrik M., Decristoforo C. (2015). An iron-mimicking, Trojan horse-entering fungi–has the time come for molecular imaging of fungal infections? PubMed DOI PMC

Havlicek V., Lemr K., Schug K. A. (2013). Current trends in microbial diagnostics based on mass spectrometry. PubMed DOI

Koo S., Thomas H. R., Daniels S. D., Lynch R. C., Fortier S. M., Shea M. M., et al. (2014). A breath fungal secondary metabolite signature to diagnose invasive Aspergillosis. PubMed DOI PMC

Luptakova D., Pluhacek T., Petrik M., Novak J., Palyzova A., Sokolova L., et al. (2017). Non-invasive and invasive diagnoses of Aspergillosis in a rat model by mass spectrometry. PubMed DOI PMC

Moore C. B., Novak-Frazer L., Muldoon E., Dunn K. W., Masania R., Richardson M. D., et al. (2017). First isolation of the pan-azole-resistant PubMed DOI

Pappalardo L., Hoijemberg P., Pelczer I., Bailey T. (2014). NMR-Metabolomics study on falcons affected by Aspergillosis. DOI

Petrik M., Franssen G. M., Haas H., Laverman P., Hortnagl C., Schrettl M., et al. (2012). Preclinical evaluation of two PubMed DOI PMC

Petrik M., Haas H., Dobrozemsky G., Lass-Florl C., Helbok A., Blatzer M., et al. (2010). PubMed DOI PMC

Pluhacek T., Lemr K., Ghosh D., Milde D., Novak J., Havlicek V. (2016a). Characterization of microbial siderophores by mass spectrometry. PubMed DOI

Pluhacek T., Petrik M., Luptakova D., Benada O., Palyzova A., Lemr K., et al. (2016b). PubMed DOI

Ramirez-Garcia A., Pellon A., Rementeria A., Buldain I., Barreto-Bergter E., Rollin-Pinheiro R., et al. (2018). PubMed DOI

Savelieff M. G., Pappalardo L. (2017). Novel cutting-edge metabolite-based diagnostic tools for Aspergillosis. PubMed DOI PMC

Schalk I. J., Guillon L. (2013). Fate of ferrisiderophores after import across bacterial outer membranes: different iron release strategies are observed in the cytoplasm or periplasm depending on the siderophore pathways. PubMed DOI

Schrettl M., Haas H. (2011). Iron homeostasis-Achilles’ heel of Aspergillus fumigatus? PubMed DOI PMC

Szigeti Z. M., Talas L., Palicz Z., Szentesi P., Hargitai Z., Csernoch L., et al. (2018). Murine model to follow hyphal development in invasive pulmonary Aspergillosis. PubMed DOI

Trevethan R. (2017). Sensitivity, specificity, and predictive values: foundations, pliabilities, and pitfalls in research and practice. PubMed DOI PMC

Ullmann A. J., Aguado J. M., Arikan-Akdagli S., Denning D. W., Groll A. H., Lagrou K., et al. (2018). Diagnosis and management of PubMed DOI

Zhao Y., Nagasaki Y., Paderu P., Sugrue M. W., Leather H. L., Wingard J. R., et al. (2018). Applying host disease status biomarkers to therapeutic response monitoring in invasive Aspergillosis patients. PubMed DOI

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