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

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.

Triacetylfusarinine C: A urine biomarker for diagnosis of invasive aspergillosis

“…30 TAFC was also detected in urine and blood samples of rats with reported LODs and LOQs of 0.28 and 0.85 ng/mL for serum and 0.02 and 0.05 ng/mL for urine, respectively. 28 However, these methods do not allow absolute matrix-independent TAFC determination. The technical LOQ of the CE-ESI-MS method reported here is 0.28 ng/mL, which increases for clinical samples due to the applied enrichment to ≥ 0.01 ng/mL, which is the most sensitive method reported so far.…”

“…In agreement, TAFC was recently detected in urine in a rat model for IA employing mass spectrometric methods. 28 In humans, TAFC has previously been detected in BALF, 29 , and in serum samples from patients with IA using ultra performance liquid chromatography tandem mass spectrometry. 30 The objective of this study was to evaluate the potential of TAFC as a urine biomarker for human IA.…”

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

“…The progression of A. fumigatus infection was studied in a rat pulmonary infection model as described previously ( Luptakova et al, 2017 ). Briefly, to induce neutropenia, the rats received repeated (5 days and 1 day before inoculation; 75 mg/kg) intraperitoneal injections of DNA-alkylating agent cyclophosphamide (Endoxan, Baxter, Prague, Czechia).…”

“…Mass spectrometry experiments were performed using 7 high dose (HD1–HD7), 9 low dose (LD1–LD9) and 3 control (CTRL1–CTRL3) animals (biological replicates) in the HD and LD models, respectively. The siderophore extractions from urine were performed according to a protocol adapted for metabolite profiling ( Luptakova et al, 2017 ). Briefly, the urine samples (20 μL) were mixed with the FOX-MIX standard (EMC Microcollections GmbH, Tübingen, Germany; 10 μL; 1 μg/mL in 5% ACN, ferrioxamines B, D, G, and E), which served as an internal standard, and were diluted with methanol (110 μL) to provide a final methanolic solution of approximately 80%.…”

“…Methods routinely used in clinical practice to detect IPA, such as culture, serology, molecular, and radiology techniques, are often slow, invasive, and lack specificity or sensitivity ( Luptakova et al, 2017 ). The identification of patients at high risk, appropriate prophylaxis, diagnostic surveillance, and early and reliable diagnostic tests remain important for improving patient management and underline the need for specific and sensitive diagnostic tools for the identification of IPA ( Zhao et al, 2018 ).…”