Viral load
ORF
On baseline and days 4, 7 and 14 after the initiation of the treatment, the viral load was determined by detection of three highly conserved epitope regions within SARS-CoV-2 pathogenic viral RNA strain, open reading frame (ORF) 1ab (ORF1ab), nucleocapsid N protein (N Protein) and spike S protein (S Protein), in saliva samples. These analyses were performed in a central laboratory (Arquimea Medical S.L., Leganés, Madrid, Spain). Viral RNA was obtained using the Chemagic™ Viral DNA/RNA 300 kit H96 from (PerkinElmer España, S.L., Tres Cantos, Madrid, Spain), and purification was done using the automated chemagic 360 Instrument (PerkinElmer). RT-qPCR was completed with the TagPath™ COVID-19 CE-IVD RT-PCR Kit (Thermo Fisher, Waltham, MA, USA), and detection of OFR1ab, N Protein and S Protein was completed in the 7500 Real-Time PCR Instrument (Thermo Fisher) and QuantStudio Real-Time PCR Instrument (Thermo Fisher). The sensitivity and specificity of the platform is >99% and 99.5%, respectively. The viral load was estimated as the number of amplification cycles (cycle thresholds, Ct) to detect genes encoding ORF1ab, N Protein and S Protein in a single PCR reaction. An RT-qPCR for SARS-CoV-2 was considered positive in the presence of a Ct value lower than 35 for at least two of the three genes analysed. A higher number of cycles means a lower viral load. Viral load was defined as ‘high’ for Ct values ≤25, ‘medium’ for Ct values >25 and ≤30 and ‘low’ for Ct values ≥30.