The West African Ebola virus outbreak underlined the importance of delivering mass diagnostic capability outside the clinical or primary care setting.

Rationale: Exhaled breath condensate (EBC) promises a valuable, non-invasive, and easy to obtain clinical sample. However, it is not currently used diagnostically due to poor reproducibility, sample contamination, and sample loss. Objective: We evaluated whether a new, hand-held EBC collector (PBM-HALETM) that separates inertially impacted large droplets (LD) before condensing the fine aerosol (FA) fraction, 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 reporter 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 (R2=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 or beta actin mRNA, and 141 non-volatile metabolites including 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. No SARS-CoV-2 genomes were detected in convalescent or acute COVID-19 patient tidal breath FA EBC.