Study design and population
Adult patients (≥18 years old) hospitalized between March 6th and April 14th with confirmed SARS-CoV-2 infection by positive real-time polymerase chain reaction test targeting the E- gene of SARS-CoV-2 were consecutively recruited from Oslo University Hospital, Ullevål and Drammen Hospital, Vestre Viken Hospital Trust, Norway to a multi-center prospective cohort study (Norwegian SARS-CoV-2 study; NCT04381819). Clinical information and laboratory samples were for most cases collected within 48 hours after hospitalization. Peripheral blood was drawn at inclusion, day 2-5 and day 7-10 during hospitalization.
Data collection
Clinical and routine diagnostics were abstracted from electronic medical records using a modified version of the International Severe Acute Respiratory and emerging Infection Consortium (ISARIC, isaric.tghn.org) /World Health Organization (WHO) Clinical Characterization Protocol (CCP) and entered into a secure web-based REDCap database (Research Electronic Data Capture, Vanderbilt University, TN, hosted by University of Oxford, UK).
Clinical outcomes
Clinical outcomes were: 1) development of respiratory failure (RF) during hospitalization defined as an arterial partial pressure of oxygen (PaO2) to fraction of inspired oxygen (FiO2) ratio (P/F) of less than 40 kPa (<300 mm Hg), corresponding to the threshold-value for ARDS by the Berlin definition (21), and 2) need of treatment in ICU. Where PaO2 was not available from the records, PaO2 values were calculated from SaO2 (22).
Ethical considerations
Informed consents were obtained from all patients or patients' family members. The study was approved by the Regional Committee for Medical and Health Research Ethics in South- Eastern Norway (reference number 106624, clinicaltrial.gov NCT04381819).
Sample processing
Peripheral blood was collected into 4mL Vacuette ® (Greiner bio-one International) containing EDTA to avoid coagulation. Samples were immediately stored on ice, processed within 30 minutes and plasma were isolated by centrifugation at 2000g for 20 minutes at 4°C. Plasma were immediately frozen at - 80°C in several aliquots and thawed only once prior to multiplex analysis.
Multiplex analyses
EDTA plasma samples from 34 hospitalized patients (Drammen hospital [n=16] and Oslo University Hospital Ullevål [n=18] were analyzed using a multiplex cytokine assay (Bio-Plex Human Cytokine 27-Plex Panel; Bio-Rad Laboratories Inc., Hercules, CA) containing the following interleukins, chemokines and growth factors: Interleukin (IL)-1β, IL-1 receptor antagonist (IL1-ra), IL-2, IL-4, IL-5, IL-6, IL-7, IL-8/CXCL8, IL-9, IL-10, IL-12 (p70), IL-13, IL-15, IL-17, eotaxin/CXCL11, basic fibroblast growth factor (bFGF), granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), interferon (IFN)-γ, interferon-inducible protein (IP-10)/CXCL10, monocyte chemotactic protein (MCP-1)/CCL2, macrophage inflammatory protein (MIP)-1α/CCL3, MIP-1β/CCL4, platelet derived growth factor-BB (PDGF-BB), regulated upon activation T cell expressed and secreted (RANTES)/CCL5, tumor necrosis factor (TNF), and vascular endothelial growth factor (VEGF). The samples were analyzed on a Multiplex Analyzer (Bio-Rad Laboratories) according to instructions from the manufacturer. Six of the 27 analytes were not detectable in more than 30% of the samples and were therefore excluded for further analysis (IL-5, IL-10, IL-15, GM-CSF, VEGF and INF-γ). A limited number of samples were below the lower detection limit and in the statistical analyses these were given a random number below the detection limit. The normal reference values for this 27-plax assay are indicated with red lines in Figure 1 (23).
Statistical analysis
Non-parametric testing was used to investigate differences between two or more groups. Mann-Whitney U test was used to compare two independent groups while Kruskal-Wallis was used comparing three groups. Friedman’s test was applied on longitudinal samples, investigating changes from baseline, day 2-5 and day 7-10. P-values were considered significant when <0.05. Due to the explorative nature of the study the statistical analyses were not corrected for multiple testing. The area under the receiver operating characteristics (ROC) curve (AUC) was estimated for patients with or without RF. Correlations were calculated using Spearman rank correlation coefficient. Statistical analysis was performed by SPSS statistical software (Macintosh version 26.0, IBM, Armonk, NY, USA).