2.1. Trail design
This research is a double-blind, randomized, two-arm parallel, phase-2, controlled trial that was performed from July 10 till October 10, 2020. 40 cases from Imam Khomeini and Shariati Hospital, Tehran University of Medical Sciences (TUMS) were included in this research. The trial protocol has been designed and mediated in adherence to the Consolidated Standards of Reporting Trials (CONSORT) guideline (18) and was approved by the Iranian Registry of Clinical Trials (ID: IRCT20081027001411N4). on 9 July 2020. According to the contradictory results about the effect of tocilizumab on COVID-19 patients, the lowest sample size with a 10% drop-out rate was estimated 40 patients according to the effect size of tocilizumab on the pulmonary disease (19). The trial was conducted under the Declaration of Helsinki guidelines and was agreed by the Ethics Committee of the Tehran University of Medical Science (Approval Number: IR.TUMS.VCR.REC1399.290).
The confirmation of COVID-19 infection in patients was confirmed by the following specifications: 1. Confirmation of SARS-CoV-2 in nasopharyngeal swab via polymerase chain reaction (PCR) and 2. Atypical computed tomography (CT) features (subpleural, bilateral, peripheral ground-glass opacity). 18 years or older patients were eligible for enrollment in the trial if they complied with the following conditions: 1. Confirmed COVID-19 infection; 2. Elevated C-reactive protein (CRP higher than 10mg/L) or IL-6 (higher than 18 pg/ml) or lymphopenia (lymphocyte count under 1100/ MCL); 3. At the pulmonary stage of the disease with blood oxygen saturation <93% or respiratory rate (RR) higher than 24; 4. Not connecting to the mechanical ventilator; 5. Not responding to standard COVID-19 treatment. All participants provided informed consent before enrollment. Patients were not eligible for enrollment in the trial if they complied with the following conditions: 1. Were allergic or intolerant to any therapeutic factors used in this study; 2. With positive pro-calcitonin (PCT) and had an active infection (including latent or active tuberculosis (TB) infection); 3. Had a history of receiving immunosuppressive drugs and corticosteroids; 4. With a history of active malignancies.
2.3 Randomization and treatment
Eligible patients (one or two days after hospitalization) were randomly distributed to the control and intervention groups by block randomization (1:1 ratio). One patient in the standard care arm and three patients in the tocilizumab arm refused to participate and were excluded from the study and overall analysis before baseline measurements. Patients allocated to receive usual care alone or usual care plus 8mg/kg tocilizumab, (Actemra, Roche) (if the patient’s conditions were not stable, 2 doses by 12 hours were administrated, maximum dose: 800 mg). All patients received usual care for the disease based on the Iranian protocol for diagnosis and treatment of COVID-19. The major standard medical cares that were received by COVID-19 patients are summarized in Table 1. In this study, patients, investigators, and outcome assessors did not inform which group received an intervention. Besides, a placebo was not used in the control group.
The clinical, demographic, and laboratory data of the patients were recorded before their enrollment. Participants were followed through day 0 to 5, improvement, discharge, or death. The primary outcome was improvement and discharge or death after administration of intervention whichever came first. The 28 days’ mortality rate, time to improvement, and time to the event were also evaluated in each group.
Clinical characteristics of the patients such as RR, heart rate (HR), blood pressure, fever, and general symptoms including gastrointestinal involvement (GI), thorax pain, dyspnea, cough, weakness, and myalgia were assessed at enrollment and after 3-5 days of starting the treatment. Besides, the degree of oxygen saturation (SO2) and the necessity and type of oxygen supports including 1. Nasal cannula, 2. Simple oxygen mask, 3. Reservoir mask, 4. Noninvasive ventilation (NIV) and 5. Invasive ventilation were assessed at enrollment and after 3-5 days of starting the intervention. CT scan findings including pulmonary involvement percent and radiological properties were also evaluated at baseline and 6 weeks after treatment (only in participants who consented). Laboratory examinations to assess the level of complete blood count (CBC), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) liver enzymes, CRP, erythrocyte sedimentation rate (ESR), fasting blood sugar (FBS), vein blood gas (VBG) (containing PH, the partial pressure of carbon dioxide (PCO2), and bicarbonate (HCO3)), IL-6, D-dimer, troponin, creatine phosphokinase (CPK) and ferritin were done at baseline and by 3-5 days of starting the treatment and also at hospital discharge time.
Improvement and discharge were determined by the patients’ clinical, general, and laboratory conditions and were determined by consciousness, ameliorated dyspnea, stopped fever for 3 days, SO2 upper than 93%, normal range of urinary output, tolerated oral regimen (PO), blood pressure more than 10 millimeters of mercury (mm Hg), a respiratory rate less than 20 breaths per minute, heart rate less than 90 beats per minutes, and reduced CRP amount and no any other side effects. All data were assessed during the study were registered on case report forms (CRFs) and the Excel database.
2.5. Side events
All unfavorable adverse effects experienced by participants during this trial, whether or not related to tocilizumab intervention, were assessed and stored.
2.6. Statistical analysis
All continuous variables were reported as the mean ± standard deviation (SD) and categorical variables are expressed as N (%). The Kolmogorov–Smirnov (K-S) normality test was used for continuous variables. Repeated measures ANOVA was performed for comparison between both groups over time. Besides, Mann Whitney or Student's t-tests was applied to assess the statistical contrasts between two arms in each time point. Moreover, Wilcoxon signed-rank or paired t-tests was performed to test for statistical comparison between two-time points in each of the studied intervention arms. Two-sided Chi-square/Fisher’s exact tests were applied to test the associations between two intervention arms and the categorical variables. To analyze and draw the time-to-death curve between two intervention arms, Kaplan–Meier curve and the log-rank test were applied. Using the Benjamini-Hochberg correction method for multiple comparisons, the false discovery rate was corrected. Statistical significance was defined at p<005 and all statistical analysis was done by STATA software (Versions 11.2).