Non-invasively measuring small airway RNA load & inflammation in acute COVID-19 at resource-limited primary care

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SARS-CoV-2 RNA was not detectable in end-tidal oral exhaled breath condensates (EBC) depleted of saliva-containing large droplets (LD)1. We evaluated if sampling by singing impacts saliva contamination, virus detection sensitivity, & cytokine profiling in EBC. Symptomatic cases (0-5 days from onset, n=30; 10 healthy controls) positive by nasopharyngeal swab (NPS) rapid antigen test were recruited at a 1ry care centre in a Brazilian favela observational study (UFMG IRB approval 54358021.1.0000.5149). NPS, saliva, PBM-HALETM (PulmoBioMedTM) EBC & LD from tidal breathing (30min) & singing (15min) were obtained with informed consent. Cytokines (Luminex) & α-amylase activity (Salimetrix) was measured in 0.02mL aliquots. Purified RNA (PureLinkTM) was measured by RT-PCR (N1/N2/RP (USCDC), 18S rRNA Taqman®). Singing did not alter EBC yield (0.102±0.05mL/min vs. tidal breathing 0.123±0.04mL/min; p=0.124). All saliva & 20 LD samples had α-amylase activity; EBC was saliva-free. Singing increased EBC 18S rRNA by 90.6x to Ct 27.8±1.90 (no RP present), enabling SARS-CoV-2 RNA measurement (Ct 31.8±3.30) in all patients without false positives/negatives. EBC viral RNA (18S rRNA-normalised) was 85.1x higher when singing vs paired saliva, & 1.47x higher vs paired NPS. Singing EBC had elevated IL-4 (58.6x, p=1e-5.56), IL-12(p40) (31.7x, p=1e-1.63) & TNF-α (4.29x, p=1e-1.44), but reduced IL-8 (7.63x, p=1e-1.56), comparable to tidal EBC sampling. During acute COVID-19, singing EBC FA has 1.93 log10 higher viral load than saliva, & small airway inflammation consistent with nasal responses2. 1. Moschos SA et al. ERJ 2022; 60(66):4515 2. Baker JR et al. Lancet Resp. Med. 2022; 6:545-560

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