A Breath-Based in Vitro Diagnostics for Lower Respiratory Tract Infection

Lower Respiratory Tract Infections (LRTIs) represent the leading cause of death due to infectious diseases. Current diagnostic modalities primarily depend on clinical symptoms and lack specificity, especially in light of common colonization without overt infection. To address this, we developed a noninvasive diagnostic approach that employs BreathBiomics™, an advanced human breath sampling system, to detect protease activities induced by bacterial infection in the lower respiratory tract. Specifically, we engineered a high-sensitivity and high-specificity molecular sensor for human neutrophil elastase (HNE). The sensor undergoes cleavage in the presence of HNE, an event that is subsequently detected via Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS). Application of this methodology to clinical samples, breath specimens collected from intubated patients with LRTIs, demonstrated the detection of the cleaved sensor by MALDI-TOF MS. Our findings indicate that this novel approach offers a noninvasive and specific diagnostic strategy for people with LRTIs. Significance The potential for using human breath for noninvasive disease detection and diagnosis has long been recognized, yet the lack of effective biomolecular sampling technologies has hindered progress. To address this limitation, we developed BreathBiomics™, an advanced sampling system designed to efficiently capture biomolecules in human exhaled breath. By focusing on protease dysregulation, an established event induced by bacterial infections, we demonstrated that BreathBiomics™ can capture proteases and facilitate their subsequent activity-based detection for the diagnosis of LRTI. We verified the assay’s sensitivity and clinical applicability through empirical studies. Our work marks a significant advancement by providing the first viable pathway for the development of in vitro diagnostic assays leveraging human breath for disease detection and diagnosis. ### Competing Interest Statement D.C., K.M.K, C.R.H., E.R.C., M.S.C., W.A.B., and M.M. have competing interests. D.C., K.M.K., C.R.H., M.S.C., and E.R.C. are employed by Zeteo Tech, Inc. W.A.B. and M.M. serve as chief executive officer and chief technology officer for Zeteo Tech, Inc., respectively. M.A.M. and S.C. have no competing interests. An unpublished U.S. Provisional Patent Application assigned to Zeteo Tech, Inc. was applied based on this research. ### Funding Statement This work was supported by the National Institute of Allergy and Infectious Diseases (NIAID) of the NIH under Awards R44AI177245. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The study received approval from the Institutional Review Boards at The Johns Hopkins University School of Medicine (Application Number: IRB00249449), and all study participants provided informed consent. All experimental procedures were executed in accordance with these approved protocols. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors.