Podium Presentation in Room 4 on Thursday at 9:40 (Chair: Chris Cox)
Authors: Dobias R. (1), Skriba A. (2), Pluhacek T. (2), Luptakova D. (2), Havlicek V. (2)
Background: The detection and quantitation of siderophores in urine represents an innovative early indication of pathogenic microorganisms in a host [1-3]. In this presentation we document why the general application of siderophores in invasive infectious diagnostics will soon represent the same clinical breakthrough like ribosomal protein typing 10 years ago. In addition to handling co-infections or polymicrobial frenemies, with mass spectrometry we monitor the antimicrobial drug metabolism and microbial resistance in a single analysis.
Methods: A database of 700 microbial siderophores was used for screening patient’s status with invasive infections caused by Aspergillus fumigatus, Mycobacterium avium, Pseudomonas aeruginosa and various candidi and zygomycetes. Human urine samples were examined by liquid chromatography and Fourier-transform ion cyclotron resonance (FTICR) mass spectrometer. The biomarkers were quantified against matrix-matched standards with ferrioxamine E as internal standard and our in-house tool called CycloBranch (https://ms.biomed.cas.cz/cyclobranch) was used for compound dereplication.
Results: For the diagnosis of aspergillosis we used an array of ferri-triacetylfusarinine C (Fe-TAFC)/ferri-ferricrocine (Fe-FC)/gliotoxin (GTX) in a cohort of 28 patients (14 positive and 14 negative controls). LOD for TAFC in human urine was 0.1 ng/mL. TAFC exceeding 10 ng/mL could be lethal, max experienced concentration was 1.6 ug/mL. In the working dataset of critically ill, mostly non-neutropenic humans, the TAFC sensitivity in urine was 93% compared to standard GM in serum (36%). The kinetic profiles upon azole treatment showed a quick drop in siderophore production in vivo. In the same cohort, ferri-pyoverdine E (Fe-PVD) was detected as urinal biomarker in a patient with Pseudomonas aeruginosa. The LOD for Fe-PVD in urine was 10 ng/mL. In serum samples the LOD and LOQs were 1 and 5 ng/mL, respectively. Co-infecting frenemies, Mycobacterium/Aspergillus and Pseudomonas/Aspergillus, were also detected in human and rat samples, respectively. In Mycobacterium the mycobactins were used as disease biomarkers and matched against commercial standards. Most recent results obtained with zygomycetes and Scedosporium will also be reported.
Conclusion: Urinal samples are analytically attractive due to less chemical background compared to serum and clinically interesting due to non-invasive application. Optimized sample preparation could soon move siderophore analysis from FTICR equipment to standard MALDI-Biotyper-type labs.
Acknowledgement: Ministry of Education, Youth and Sports of the Czech Republic (LO1509) and Czech Science Foundation (19-10907S).
1. Pluhacek T., et al. In Methods in Molecular Biology, Bhattacharya, S. K., Ed.; Springer Nature, 2019.
2. Skriba A., et al.: Frontiers in Microbiology 9, 2356 (2018).
3. Luptakova D., et al.: Scientific Reports 7, 16523 (2017).
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