Invasive pulmonary aspergillosis results in 450,000 deaths per year and complicates cancer chemotherapy, transplantations and the treatment of other immunosuppressed patients. Using a rat model of experimental aspergillosis, the fungal siderophores ferricrocin and triacetylfusarinine C were identified as markers of aspergillosis and quantified in urine, serum and lung tissues. Biomarkers were analyzed by matrix-assisted laser desorption ionization (MALDI) and electrospray ionization mass spectrometry using a 12T SolariX Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. The limits of detection of the ferri-forms of triacetylfusarinine C and ferricrocin in the rat serum were 0.28 and 0.36 ng/mL, respectively. In the rat urine the respective limits of detection achieved 0.02 and 0.03 ng/mL. In the sera of infected animals, triacetylfusarinine C was not detected but ferricrocin concentration fluctuated in the 3-32 ng/mL range. Notably, the mean concentrations of triacetylfusarinine C and ferricrocin in the rat urine were 0.37 and 0.63 μg/mL, respectively. The MALDI FTICR mass spectrometry imaging illustrated the actual microbial ferricrocin distribution in the lung tissues and resolved the false-positive results obtained by the light microscopy and histological staining. Ferricrocin and triacetylfusarinine C detection in urine represents an innovative non-invasive indication of Aspergillus infection in a host.

Institute of Microbiology of the Czech Academy of Sciences Prague 4 142 20 Czech Republic

Institute of Molecular and Translational Medicine Palacky University Olomouc 779 00 Czech Republic

Regional Centre of Advanced Technologies and Materials Department of Analytical Chemistry Olomouc 771 47 Czech Republic

University of West Bohemia Plzen 306 14 Czech Republic

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Cole, D. C., Govender, N. P., Chakrabarti, A., Sacarlal, J. & Denning, D. W. Improvement of fungal disease identification and management: combined health systems and public health approaches. The Lancet Infect. Dis. 10.1016/S1473-3099(17)30308-0 (2017). PubMed

Del Poeta M. Special issue: Novel antifungal drug discovery. J. Fungi. 2016;2:33. doi: 10.3390/jof2040033. PubMed DOI PMC

Denning DW. Minimizing fungal disease deaths will allow the unAIDS target of reducing annual AIDS deaths below 500 000 by 2020 to be realized. Philos. Transactions of the Royal Soc. B: Biol. Sci. 2016;371:20150468. doi: 10.1098/rstb.2015.0468. PubMed DOI PMC

Lin S-J, Schranz J, Teutsch SM. Aspergillosis case-fatality rate: Systematic review of the literature. Clin. Infec. Dis. 2001;32:358–366. doi: 10.1086/318483. PubMed DOI

Denning DW, Pleuvry A, Cole DC. Global burden of allergic bronchopulmonary aspergillosis with asthma and its complication chronic pulmonary aspergillosis in adults. Med. Mycol. 2013;51:361–370. doi: 10.3109/13693786.2012.738312. PubMed DOI

Dagenais TRT, Keller NP. Pathogenesis of aspergillus fumigatus in invasive aspergillosis. Clin. Microbiol. Rev. 2009;22:447–465. doi: 10.1128/CMR.00055-08. PubMed DOI PMC

Havlicek V, Lemr K, Schug KA. Current trends in microbial diagnostics based on mass spectrometry. Anal. Chem. 2013;85:790–797. doi: 10.1021/ac3031866. PubMed DOI

Page ID, Richardson MD, Denning DW. Comparison of six aspergillus-specific IgG assays for the diagnosis of chronic pulmonary aspergillosis (CPA) J. Infect. 2016;72:240–249. doi: 10.1016/j.jinf.2015.11.003. PubMed DOI

Cerqueira LB, de Francisco TMG, Gasparetto JC, Campos FR, Pontarolo R. Development and validation of an HPLC-MS/MS method for the early diagnosis of aspergillosis. Plos One. 2014;9:e92851. doi: 10.1371/journal.pone.0092851. PubMed DOI PMC

Johnson G, et al. Biomarkers for invasive aspergillosis: the challenges continue. Biomark. Med. 2014;8:429–451. doi: 10.2217/bmm.13.129. PubMed DOI

Pluhacek T, et al. Characterization of microbial siderophores by mass spectrometry. Mass Spectrom. Rev. 2016;35:35–47. doi: 10.1002/mas.21461. PubMed DOI

Hissen AH, Wan AN, Warwas ML, Pinto LJ, Moore MM. The Aspergillus fumigatus siderophore biosynthetic gene sidA, encoding L-ornithine N5-oxygenase, is required for virulence. Infect. and Immun. 2005;73:5493–503. doi: 10.1128/IAI.73.9.5493-5503.2005. PubMed DOI PMC

Pluhacek T, et al. Aspergillus infection monitored by multimodal imaging in a rat model. Proteomics. 2016;16:1785–92. doi: 10.1002/pmic.201500487. PubMed DOI

Mascuch SJ, et al. Direct detection of fungal siderophores on bats with white-nose syndrome via fluorescence microscopy-guided ambient ionization mass spectrometry. Plos One. 2015;10:e0119668. doi: 10.1371/journal.pone.0119668. PubMed DOI PMC

Carroll CS, Amankwa LN, Pinto LJ, Fuller JD, Moore MM. Detection of a serum siderophore by LC-MS/MS as a potential biomarker of invasive aspergillosis. Plos One. 2016;11:e0151260. doi: 10.1371/journal.pone.0151260. PubMed DOI PMC

Prichystal J, Schug KA, Lemr K, Novak J, Havlicek V. Structural analysis of natural products. Anal. Chem. 2016;88:10338–10346. doi: 10.1021/acs.analchem.6b02386. PubMed DOI

Novak J, Lemr K, Schug KA, Havlicek V. CycloBranch: De novo sequencing of nonribosomal peptides from accurate product ion mass spectra. J. Am. Soc. for Mass Spectrom. 2015;26:1780–1786. doi: 10.1007/s13361-015-1211-1. PubMed DOI

Novak J, et al. Batch-processing of imaging or liquid-chromatography mass spectrometry datasets and de novo sequencing of polyketide siderophores. BBA-Proteins Proteom. 2017;1865:768–775. doi: 10.1016/j.bbapap.2016.12.003. PubMed DOI

Brandon M, Howard B, Lawrence C, Laubenbacher R. Iron acquisition and oxidative stress response in Aspergillus fumigatus. BMC Syst. Biol. 2015;9:17. doi: 10.1186/s12918-015-0163-1. PubMed DOI PMC

Schrettl M, Haas H. Iron homeostasis-achilles’ heel of Aspergillus fumigatus? Curr. Opin. in Microbiol. 2011;14:400–405. doi: 10.1016/j.mib.2011.06.002. PubMed DOI PMC

Blatzer M, et al. SidL, an Aspergillus fumigatus transacetylase involved in biosynthesis of the siderophores ferricrocin and hydroxyferricrocin. Appl. and Environ. Microbiol. 2011;77:4959–4966. doi: 10.1128/AEM.00182-11. PubMed DOI PMC

Petrik M, et al. In vitro and in vivo evaluation of selected 68Ga-siderophores for infection imaging. Nucl. Medicine Biol. 2012;39:361–369. doi: 10.1016/j.nucmedbio.2011.09.012. PubMed DOI PMC

Strohalm M, et al. Poly N-(2-hydroxypropyl)methacrylamide -based tissue-embedding medium compatible with MALDI mass spectrometry imaging experiments. Anal. Chem. 2011;83:5458–5462. doi: 10.1021/ac2011679. PubMed DOI

Petrik M, et al. Ga-68-siderophores for PET imaging of invasive pulmonary aspergillosis: Proof of principle. J. Nucl. Medicine. 2010;51:639–645. doi: 10.2967/jnumed.109.072462. PubMed DOI PMC

Yuan M, Breitkopf SB, Yang XM, Asara JM. A positive/negative ion-switching, targeted mass spectrometry-based metabolomics platform for bodily fluids, cells, and fresh and fixed tissue. Nat. Protoc. 2012;7:872–881. doi: 10.1038/nprot.2012.024. PubMed DOI PMC

Gu HD, Liu GW, Wang J, Aubry AF, Arnold ME. Selecting the correct weighting factors for linear and quadratic calibration curves with least-squares regression algorithm in bioanalytical LC-MS/MS assays and impacts of using incorrect weighting factors on curve stability, data quality, and assay performance. Anal. Chem. 2014;86:8959–8966. doi: 10.1021/ac5018265. PubMed DOI

Oide S, Berthiller F, Wiesenberger G, Adam G, Turgeon BG. Individual and combined roles of malonichrome, ferricrocin, and tafc siderophores in Fusarium graminearum pathogenic and sexual development. Front. Microbiol. 2015;5:759. doi: 10.3389/fmicb.2014.00759. PubMed DOI PMC

Silva-Baila MG, et al. Hydroxamate production as a high affinity iron acquisition mechanism in Paracoccidioides spp. Plos One. 2014;9:e105805. doi: 10.1371/journal.pone.0105805. PubMed DOI PMC

Inglis DO, et al. Comprehensive annotation of secondary metabolite biosynthetic genes and gene clusters of Aspergillus nidulans, A. fumigatus, A. niger and A. oryzae. BMC Microbiol. 2013;13:91. doi: 10.1186/1471-2180-13-91. PubMed DOI PMC

Imbert S, et al. Aspergillus pcr in serum for the diagnosis, follow-up and prognosis of invasive aspergillosis in neutropenic and nonneutropenic patients. Clin. Microbiol. Infect. 2016;22:562.e1–562.e8. doi: 10.1016/j.cmi.2016.01.027. PubMed DOI PMC

Levesque E, et al. Detection of (1,3)-beta-d-glucan for the diagnosis of invasive fungal infection in liver transplant recipients. Int. J. Mol. Sci. 2017;18:862. doi: 10.3390/ijms18040862. PubMed DOI PMC

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