A breath-based in vitro diagnostic assay for the detection of lower respiratory tract infections

Abstract

An accurate diagnosis is critical to reducing mortality in people with lower respiratory tract infections (LRTIs). Current microbiological culture is time-consuming, and nucleic acid amplification-based molecular technologies cannot distinguish between colonization and infection. Previously, we described developing a sampling system for effectively capturing biomolecules from human breath. We identified a new class of proteoform markers of protease activation, termed proteolytic products of infection, for detecting LRTIs in people with mechanical ventilation. Here, we further developed an in vitro assay by designing a specific substrate sensor for human neutrophil elastase (HNE) to detect LRTIs in breath samples. In the proof-of-concept study, we then applied this in vitro assay to breath samples collected from intubated patients and healthy volunteers. The findings revealed that the LRTI group demonstrated a significant mean differential, showing a 9.8-fold elevation in measured HNE activity compared with the non-LRTI group and a 9.2-fold compared with healthy volunteers. The in vitro assay's diagnostic potential was assessed by constructing a receiver operating characteristic curve, resulting in an area under the curve of 0.987. Using an optimal threshold for HNE at 0.2 pM, the sensitivity was determined to be 1.0 and the specificity to be 0.867. Further correlation analysis revealed a strong positive relationship between the measured HNE activity and the protein concentration in the breath samples. Our results demonstrate that this breath-based in vitro assay provides high diagnostic performance for LRTIs, suggesting that the technology may be useful in the near term for the accurate diagnosis of LRTIs.

Keywords: human breath; lower respiratory tract infection; mass spectrometry; noninvasive diagnostics.

Fig. 1.

Evaluation of the diagnostic potential of the in vitro assay using breath samples collected by BreathBiomics. A) Schematic of breath collection from intubated patients on mechanical ventilation in ICUs and the pump system. B) Schematic of breath collection from healthy volunteers using a facial mask. C) Boxplot display of the distribution of natural-log-transformed HNE measurements, mean differences, and adjusted P-values by group: LRTI (n = 13), non-LRTI (n = 15), and healthy volunteers (HV; n = 19) D) Representative MALDI-TOF spectra of breath samples collected from different groups. E) ROC curve analysis for the in vitro assay performance.

Fig. 2.

Bread-based in vitro assay overview and HNE substrate design. A) Overview of the breath-based in vitro assay to detect LRTIs. B) The concept of the HNE substrate sensor designed in this study. C) The structure of the HNE substrate sensor used in this study. D) Representative high-resolution and MALDI-TOF mass spectra of the HNE substrate sensor and its cleavage product. E) Ion fragmentation maps of HNE substrate sensor and its cleavage product characterized by DI-ESI-MS/MS.

Fig. 3.

Evaluation of the HNE substrate sensor using rmNE and rhNE. A) Representative MALDI-TOF mass spectra of the sensor and the cleavage products with different time points of reaction using rmNE. B) The ratio of cleavage products to the sensor with different time points. C) Specificity testing using a blank control, 15 µM of rhNE, 15 µM of cathepsin G (CTSG), and 15 µM of PR3. D) LOD test at 37 °C for 24 h using different concentrations of rhNE.

Fig. 4.

Identification of HNE in breath samples using bottom-up proteomics and correction analysis. A) Representative ion fragmentation map of an HNE peptide. B) Protein profiles of proteases and antiproteases identified in breath samples of intubated patients. C) Correlation analysis between HNE concentrations and protein levels in breath samples.