Noninvasive Combined Diagnosis and Monitoring of Aspergillus and Pseudomonas Infections: Proof of Concept

Radim Dobiáš, Anton Škríba, Tomáš Pluháček, Miloš Petřík, Andrea Palyzová, Marcela Káňová, Eva Čubová, Jiří Houšť, Jiří Novák, David A Stevens, Goran Mitulovič, Eva Krejčí, Petr Hubáček, Vladimír Havlíček, Radim Dobiáš, Anton Škríba, Tomáš Pluháček, Miloš Petřík, Andrea Palyzová, Marcela Káňová, Eva Čubová, Jiří Houšť, Jiří Novák, David A Stevens, Goran Mitulovič, Eva Krejčí, Petr Hubáček, Vladimír Havlíček

Abstract

In acutely ill patients, particularly in intensive care units or in mixed infections, time to a microbe-specific diagnosis is critical to a successful outcome of therapy. We report the application of evolving technologies involving mass spectrometry to diagnose and monitor a patient's course. As proof of this concept, we studied five patients and used two rat models of mono-infection and coinfection. We report the noninvasive combined monitoring of Aspergillus fumigatus and Pseudomonas aeruginosa infection. The invasive coinfection was detected by monitoring the fungal triacetylfusarinine C and ferricrocin siderophore levels and the bacterial metabolites pyoverdin E, pyochelin, and 2-heptyl-4-quinolone, studied in the urine, endotracheal aspirate, or breath condensate. The coinfection was monitored by mass spectrometry followed by isotopic data filtering. In the rat infection model, detection indicated 100-fold more siderophores in urine compared to sera, indicating the diagnostic potential of urine sampling. The tools utilized in our studies can now be examined in large clinical series, where we could expect the accuracy and speed of diagnosis to be competitive with conventional methods and provide advantages in unraveling the complexities of mixed infections.

Keywords: Aspergillus fumigatus; Pseudomonas aeruginosa; coinfection; invasive infection; noninvasive diagnosis; quorum-sensing molecules; siderophores; virulence factor.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
ARF patients’ chest radiography and CT scans. (A,B) Radiographs showing increasing bilateral, inhomogeneous blurring, likely caused by inflammatory infiltrations (Patient 1). (C,D) Atelectasis in both lower lung lobes and irregular multiple ground glass opacities revealed by the CT scan of Patient 2. Right-sided pneumothorax and small amount of fluid in bilateral pleural cavities are also discernable.
Figure 2
Figure 2
Noninvasive detection of Aspergillus and Pseudomonas biomarkers returned by CycloBranch. (A) Chemical structures of the siderophores (1–6) and HHQ (9). Compound numbering is described in the associated table. (B) Patient 1’s urine profile compiled from reconstructed ion chromatograms recorded in the low- and high-mass experiments (see Materials and Methods for details). TafC was detected as a singly charged species (m/z 906.3304, see the right inset for Peak 6, indicating the presence of 54Fe and 56Fe stable isotopes). The left inset (Peak 1) indicates the isotopic profile of FoxE. (C) Patient 1’s breath condensate profile sampled on the same Day 12 of illness with a mixture of doubly charged pyoverdines. Peak 5 in the left inset is represented by an isotopic pattern, indicating two charges (54Fe/56Fe isotopes are separated by approximately one Dalton). The right inset belongs to pyochelin singly protonated molecule. M in the table stands for neutral molecule; Ret. time, retention time. Fluconazole and its glucuronide structures are reported in Supplementary Materials Figure S8.
Figure 3
Figure 3
MALDI-FTICR-MS annotation of Patient 1’s breath condensate (sampled Day 12). Pch (A), PvdE (B), and HHQ (C) were all detected by MALDI MS as protonated forms. The structures of these molecules are shown. The annotation in red was returned by CycloBranch software [9] when running the MALDI mass spectrum against the library of P. aeruginosa secondary metabolites. The red color was added to an examined spectrum by the software automatically. The color indicates which isotope features have been used by the computing algorithm for the annotation process.

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