Non-invasive and invasive diagnoses of aspergillosis in a rat model by mass spectrometry

Dominika Luptáková,Jiří Novák,Lucie Sokolová,Tomáš Pluháček,Karel Lemr,Vladimír Havlíček,Andrea Palyzová,Anton Škríba,Blanka Šedivá,Miloš Petřík

doi:10.1038/s41598-017-16648-z

Abstract

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.

Highlights

More than 1.6 million people are estimated to die of fungal diseases each year and about a billion people have cutaneous fungal infections[1]
computed tomography (CT) scans of the infected lungs indicated a pathology of an unknown etiology, the fused positron emission tomography (PET)/CT images enabled us to observe the locations where 68Ga-doped triacetylfusarinine C (TAFC) was selectively captured by the fungus (Supplementary Fig. S1)
Grocott’s methenamine silver (GMS) and eosin staining was performed as another independent ex vivo tool and revealed extensive fungal burden in the infected animals, which was manifested by dense hyphae growth from the cell walls into the lumen of the trachea, bronchi/bronchioli and alveoli

Summary

Introduction

More than 1.6 million people are estimated to die of fungal diseases each year and about a billion people have cutaneous fungal infections[1]. A standard Aspergillus diagnostic approach involves the culture of bronchoalveolar lavage samples; this method is sensitive but is invasive and time-consuming. The lower limit of quantitation (LLOQ) using a triple quadrupole mass spectrometer was 10 ng/mL in the multiple reaction monitoring mode, and gliotoxin was detected in only eight of thirty samples obtained from patients at risk of invasive aspergillosis. Another research group detected two siderophores using desorption electrospray mass spectrometry in the wings of bats suffering from white nose syndrome[14]. Both siderophores, i.e., triacetylfusarinine C (TAFC) and ferrichrome, were produced by Pseudogymnoascus destructans. An LOQ of 5 ng/mL was reported in the multiple reaction monitoring mode using a triple quadrupole mass spectrometer[15]

Methods

Results

Conclusion