We conducted an observational study at the King Faisal Specialist Hospital and Research Centre (KFSHRC), a large (~1.000.000 outpatient visits/year, 1.600 beds, ~1.000 physicians and ~13.500 employees), non-profit, tertiary referral hospital, located in the city center of Riyadh, Saudi Arabia, where patients are referred from other hospitals from across Saudi Arabia and adjacent regions. Participants were included consecutively in the period from March 1^st to March 31^st, 2020. All patients were Saudi nationals and were self-referrals, as these participants were long-term patients and therefore have direct access to health care in this hospital. The diagnosis of COVID-19 was suspected clinically and confirmed through the detection of SARS-Cov-2 in a nasopharyngeal sample with specific PCR (RealStar^® SARS-CoV-2 RT-PCR Kit RUO altona-diagnostics, Germany), which was performed in the Section of Medical Microbiology of the Department of Pathology and Laboratory Medicine at KFSHRC. Upon testing positive, the patients were admitted and isolated in negative pressure rooms. Severity of disease, i.e., mild, moderate, or severe was determined on admission and modified according to the clinical course using the WHO categories of COVID-19 disease severity 18. These are defined as follows: mild disease, no evidence of viral pneumonia or hypoxia; moderate disease, clinical signs of pneumonia (fever, cough, dyspnea, fast breathing), but not severe pneumonia, including SpO2 ≥ 90% on room air, cautioning that a SpO2 of >90–94% on room air is abnormal in a patient with normal lung and can be an early sign of severe disease. Severe disease, clinical signs of pneumonia (fever, cough, dyspnea, fast breathing) plus one of the following: respiratory rate >30 breaths/min, severe respiratory distress or SpO2 <90% on room air. Chest imaging (radiograph, CT scan, ultrasound) may assist in diagnosis and identify or exclude pulmonary complications in moderate and severe disease. For comparison, we also determined IL-1RA, MCP-1 and IP-10 levels and other parameters in a group of approximately age-sex matched controls without evidence of any infection who were recruited from hospital personnel, trainees, or patients.

Clinical, laboratory and radiographic data were handled viathe electronic medical records system (PowerChart; Cerner, USA), and included demographic details, vital signs, admission notes with chief complaint(s), history of present illness, previous diagnoses, medications, laboratory tests, radiography, and progress notes. These data were extracted from the electronic medical records, deposited, and further processed using REDCap 19. Within this project, each participant patient was assigned a unique research-specific ID number that was password-protected. Data were exported from REDCap into a Microsoft Excel spreadsheet.

For each patient with COVID-19, the following data at the time of admission to the hospital were obtained and recorded electronically: age, sex, vital signs including SpO2, presenting symptom(s), comorbidities, medications, laboratory tests and chest X-ray. Laboratory investigations included complete blood count (CBC), absolute counts of CD3+, CD3+CD4+ and CD3+CD8+ T cells, CD19+ B cells, CD56+CD16+ NK cells, Beta-2 microglobulin, IL-1RA, MCP-1, IP-10, Tumor Necrosis Factor alpha (TNF-α), ferritin, D-dimer, CRP, estimated glomerular filtration rate (eGFR, CKD-EPI equation) 20. In the control group, after exclusion of an infectious disease, the same hematologic and biochemical parameters were measured.

The tests for IL-1RA, MCP-1, IP-10, ferritin, D-dimer, and CRP were performed within two days of admission in the Medical Laboratory of the Department of Pathology and Laboratory Medicine at KFSHRC. Serum IL-1RA, MCP-1, and IP-10 levels were quantified using a highly standardized Luminex assay. Serum levels of ferritin were determined by electrochemiluminescence immunoassay (Elecsys Ferritin1) using streptavidin-coated microparticles, biotinylated mouse monoclonal anti-ferritin antibody and ruthenium-complex-labeled mouse monoclonal anti-ferritin antibody on the Roche/Hitachi cobas1 e 801 immunoassay analyzers (measuring range: 0.50–2000 μg/l; reference range for men: 30–400 μg/l; for women: 13–150 μg/l). D-dimer levels were determined in plasma with an immunoturbidometric assay (STA1-Liatest1 D-DI PLUS) using latex microparticles coated with two different mouse monoclonal anti-D-dimer antibodies (normal level < 0.5 μg/ml FEU) and analyzed on the STA-R1 Max2 instrument. CRP levels were measured in serum using an immunoturbidometric assay with latex particles coated with mouse monoclonal anti-CRP antibody (CRPHS1) on the Roche/Hitachi cobas1 c system (measuring range: 0.15–20.0 mg/l). For this high-sensitivity CRP assay, levels >10 mg/l indicate systemic inflammation.

Demographic characteristics, main comorbidities, clinical manifestations, medications, and vital signs on presentation are shown in Table 1. Twenty-one consecutive participants (mean age 49 ± 22; 9 m, 12 f) presented to the emergency department with one or more of the following chief complaints (in descending order of frequency): fever, dry cough, sore throat, rhinorrhea, fatigue, headache, diarrhea, anosmia, productive cough, dyspnea, ear pain, ageusia, anorexia, abdominal pain, nausea, emesis, seizure, syncope, myalgia, rash, and no symptoms. Fever and dry cough were the principal manifestations in all patients, whereas other symptoms varied among severity groups. Among the patients, comorbidities were as follows: hypertension, diabetes mellitus, dyslipidemia, coronary artery disease, congestive heart failure, atrial fibrillation, chronic obstructive pulmonary disease, cerebrovascular disease, leukemia in remission, colorectal cancer in remission, post-renal transplant, Hodgkin’s lymphoma in remission, and hypothyroidism.

To compare cytokine expression levels between COVID-19 patients and controls, plasma cytokine levels were measured using Luminex in 4 patient groups: severe COVID-19 (n = 6), moderate COVID-19 (n = 9), Mild COVID-19 (n = 6), and controls (n = 4). Leukocyte profile, CRP, Ferritin and Beta 2 microglobulin were also measured (figure 1). Beta-2 microglobulin was elevated in severe patients, compared to moderate, mild and controls (P <0.001) Figure 2. IP-10 level was significantly elevated in severe COVID-19 patients, in comparison to controls, mild and moderate (P< 0.0103), (P< 0.0188), (P< 0.0448) respectively. IL-1RA was also significant elevated in severe patients in comparison with other groups (figure 3). TNFA was significantly elevated in the severe COVID-19 patients’ group, compared to controls, mild, and moderate groups (P< 0.001) (Figure 4). Those biomarkers were obtained before the clinical deterioration and development of cytokine storm and ARDS, which indicate that those cytokines may play a role in early detection and recognition of severe COVID-19 disease and risk of development of cytokine storm.

KFSHRC: King Faisal Specialist Hospital and Research Centre; COVID-19: Coronavirus Disease 2019; IL-6: Interleukin-6; IL-10: Interleukin-10; ARDS: Acute Respiratory Distress Syndrome; IL-1RA: Interleukin-1 receptor antagonist; MCP1: Monocyte chemoattractant protein 1; IP-10: Interferon gamma-induced protein 10; IFN-γ: Interferon-Gamma; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; CBC: complete blood count; TNF-α: Tumor Necrosis Factor alpha; eGFR: estimated glomerular filtration rate; SD: Standard Deviation.