Airborne Pathogen Detection in Fine Aerosol Exhaled Breath Condensates

Rationale Exhaled breath condensate (EBC) promises a valuable, non-invasive, and easy to obtain clinical sample. However, it’s not currently used diagnostically due to poor reproducibility, sample contamination, and sample loss. Objective We evaluated whether a new, hand-held EBC collector (PBM-HALE TM ) that separates inertially impacted large droplets (LD) before condensing fine aerosols (FA) in distinct, self-sealing containers, overcomes current limitations. Methods Sampling consistency was determined in healthy volunteers by microbial culture, 16S phylogenetics, spectrophotometry, RT-PCR, and HILIC-MS. Capture of aerosolised polystyrene beads, liposomes, virus-like particles, or pseudotyped virus was analysed by nanoparticle tracking analysis, reporter expression assays, and flow cytometry. Acute symptomatic COVID-19 case tidal FA EBC viral load was quantified by RT-qPCR. Exhaled particles were counted by laser light scattering. Measurements and Main Results Salivary amylase-free FA EBC capture was linear (R 2 =0.9992; 0.25-30 min) yielding RNA (6.03 μg/mL) containing eukaryotic 18S rRNA (RT-qPCR; p<0.001) but not human GAPDH, RNase P, or beta actin mRNA;141 non-volatile metabolites included eukaryotic cell membrane components, and cuscohygrine 3 days after cocaine abuse. Culturable aerobe viability was condensation temperature-dependent. Breath fraction-specific microbiota were stable, identifying Streptococcus enrichment in a mild dry cough case. Nebulized pseudotyped virus infectivity loss <67% depended on condensation temperature, and particle charge-driven aggregation. SARS-CoV-2 RNA genomes were detected only by forced expiration FA EBC capture, in 100% of acute COVID-19 patients. Conclusions High purity, distal airway FA EBC can reproducibly and robustly inform contamination-free infectious agent emission sources, and be quantitatively assayed for multiple host, microbial, and lifestyle biomarker classes.