Non-Invasive Infections Diagnosis in Lung Transplant Recipients in Exhaled Breath Condensate

Abstract

Purpose The purpose of this research was to identify diagnostic volatile organic compounds (VOCs) in exhaled breath condensate (EBC) from lung transplant recipients (LTR) colonized or infected by Aspergillus spp., Staphylococcus aureus and Pseudomonas aeruginosa, in order to develop a cheap, accurate and quick non-invasive diagnostic test. To this end we performed metabolomic analysis in EBC using gas chromatography-time-of-flight mass spectrometry (GC-QTOF) in the high-resolution mode. Methods Between January 2018-June 2019, 40 LTR were recruited in the Lung Transplant Unit, from whom 71 EBC and respiratory samples (sputum, bronchoalveolar lavage or oropharyngeal swabs) were obtained. Bacteria and fungi identification were performed according to routine laboratory standards for respiratory samples. The ECOScreen2 device was used for EBC collection at -20 °C during 15 minutes, and samples were stored at −80°C in 200 μL and 500 μL aliquots until analysis. EBC samples from patients colonized either with P. aeruginosa, S. aureus, Aspergillus spp. or none of the above (controls) where selected for headspace Solid Phase Micro Extraction (SPME) with DVBP fibres in order to concentrate VOCs before their injection in the Agilent GCMS 7200 Accurate Mass Q-TOF equipment for the metabolomic analysis. GC-QTOF untargeted analysis (deconvolution, alignment, and recursive analysis) was performed with the MassHunter and Mass Profiler Software, and statistical analysis with R software. Results We selected 27 samples for SPME-GC/QTOF analysis: nine controls, seven from patients colonized by Aspergillus spp., six with P. aeruginosa, three with S. aureus and two with both P. aeruginosa and S. aureus. The PLS-DA analysis clearly differentiated patients colonized with S. aureus or P. aeuruginosa from those who were not, with a very little overlap in patients colonized with A. fumigatus from the controls. Volcano plot analysis found two possible biomarkers for Aspergillus spp. detection, six for S. aureus and none for P. aeruginosa, may be because of the small n in our study. Conclusion SPME-GC/QTOF in EBC is a promising tool for non-invasive diagnosis of fungal and bacterial infections in LTR. However, the presumptive identification of these biomarkers needs to be confirmed with targeted metabolomic studies and their sensitivity and specificity should be studied in future research.