Clinical sample collection, SARS-CoV-2 viral load quantification, and gene expression analysis

Nasopharyngeal swabs were collected and deposited in 2 ml of saline solution at diverse hospitals and clinical centers in the Buenos Aires area, Argentina. These clinical samples were processed at the Instituto de Investigaciones Biomédicas en Retrovirus y SIDA (INBIRS, Buenos Aires, Argentina), a specialized center dedicated to SARS-CoV-2 diagnosis by RT-qPCR. The demographic and clinical characteristics of the patients are shown in Table 3. RNA was extracted from 300 µl of swab samples using a Chemagic 360-D automated extraction equipment (Perkin-Elmer). SARS-CoV-2 RNA was quantified using GeneFinder SARS-CoV-2 RT-PCR kit (Osang Health Care) which allows multiplex detection of viral genes N, E and RdRp and human gene RRP30. Viral load was quantified using Ct values of the N gene and a standard curve. After the determination of SARS-CoV-2, the remaining RNA was used for gene expression studies. cDNA was transcribed using the High-Capacity cDNA Reverse Transcription Kit (Life Technologies, 4368813). Gene expression was then measured by qPCR using SYBR Green I Master Mix (Roche). A list of primers used is provided (Supplementary Table ¹).

Demographic and clinical characteristics of patients with COVID-19.

For the purpose of this work, we used the remaining volume of anonymized samples that had been collected for clinical diagnosis of SARS-CoV-2 and therefore the IRB (Comite de Bioetica, Fundacion Huesped) deemed unnecessary to obtain informed consent from the patients. The authors were not involved in sample collection.