This study is a multicenter, randomized trial that was approved by the ethics committee of Zhongnan Hospital of Wuhan University (#2020001). This study was conducted in the ICUs of Zhongnan Hospital of Wuhan University, Leishenshan (Thunder God Mountain) Hospital, and Taihe Hospital from February 14, 2020, to March 29, 2020. The ICUs specifically for COVID-19 from Zhongnan Hospital and Leishenshan Hospital were managed by the same team. The trial was registered on the website of ClinicalTrials.gov (ID: NCT04264533;registered February 14 2020) before patient recruitment.
Patients were screened and enrolled following admission to the three ICUs. The patients who were diagnosed as severe SARS-CoV-2 related pneumonia, appeared or had a high risk of multiple organs injury would be transferred to ICU. The following inclusion criteria were met: (1) age ≥18 and <80 years; (2) RT-PCR positive for SARS-CoV-2; (3) pneumonia confirmed by chest imaging; and admission to the ICU. Exclusion criteria were PaO2/FiO2(P/F)>300 mmHg，allergy to vitamin C, pregnancy or breastfeeding, expected survival duration <24 hours, and previous history of glucose-6-phosphate dehydrogenase deficiency or end-stage pulmonary disease. Patients who were already enrolled in other clinical trials were excluded as well. If these criteria were met within 48 hours of ICU admission, informed consent was obtained from the patients or their family members. When the patients’ actual treatment time was less than 3 days due to death or discharge from the ICU, they were removed from this trial. The reason was because the efficacies of the treatments could not be evaluated with limited times of treatment.
Randomization, allocation and blinding
Each ICU was assigned with an independent random numeric table generated by Microsoft Excel 2019 by the primary investigator alone. Each table had equal numbers of 1 and 2, which represented the placebo group (bacteriostatic water infusion) and treatment group (HDIVC), respectively. The generated random list was stored by the principal investigator who was not involved in the treatment of patients and hidden to the other investigators.When a patient was transferred to the ICU and met the enrolment criteria, the clinician on duty would inform the principal investigator and obtain a number from the list. Then, participants were enrolled in the corresponding group according to the chronological order of ICU recruitment. The grouping and intervention were unknown to the participants and investigators who were responsible for data collection and statistical analysis. VC injection and sterile water for injection were both colourless and contained in the same brown syringes with different marks and without explanations on the syringe to make sure that patients could not distinguish the treatment they receive.
Patients were randomized to receive vitamin C or placebo within 48 hours after admission to the ICU. To control the infusion rates accurately and not affect the fluid management of severe patients, we infused vitamin C or placebo via central vein catheterization controlled by a pump. The study groups in this trial were 1) HDIVC: 24 g vitamin C per day. Patients were infused with 12 g vitamin C diluted in 50 ml of bacteriostatic water every 12 hours at a rate of 12 ml/hour by infusion pump for 7 days. 2) Placebo: 50 ml of bacteriostatic water infused every 12 hours at the same rate. Study interventions were initiated on the same day as informed consent and randomization. The preparation, transportation, storage, and use of therapies (VC and bacteriostatic water for injection) were in line with the drug management protocol in each hospital.
General treatments and standard procedure of ventilation supports
In addition, other general treatments followed the latest COVID-19 guidelines. Oseltamivir and azithromycin were usually used in the general ward. After ICU admission, low weight molecular heparin was applied for the prevention deep vein thrombus. Piperacillin/tazobactam was used for patients receiving tracheal intubation.
If the patients showed the symptoms of rapid deterioration of hypoxemia, severe ARDS, or septic shock, hydrocortisone(1mg/kg/day) could be considered.
Respiratory support (IMV, NIV and HFNC) were given to patients with hypoxic respiratory failure and ARDS. If respiratory failure could not be improved or worsened continuously within a short time after using HFNC or NIV, intubation were performed and the approach of lung-protective ventilation was applied. ECMO was considered as the rescue therapy when the refractory hypoxemia was difficult to be corrected by protective lung ventilation . When patients’ respiratory functions improved and were ready for weaning from the ventilators, the spontaneous breathing test (SBT) was performed. After the SBT was passed, invasive ventilator was considered to remove with the endotracheal tube extubation.
Risks and Adverse Events
Adverse events(AEs) related to HDIVC included (1) nausea or vomiting during or after infusion of VC; (2) electrolyte disturbance; and (3) acute kidney injury, as described by Khoshnam-Rad . AEs and serious adverse events (SAEs) were observed and followed in accordance with the good clinical practice guidelines issued by the National Medical Products Administration of the People’s Republic of China. If any severe adverse events were observed during infusion, the infusion was stopped immediately, and the patient’s vital signs were carefully monitored. All the AEs and SAEs were recorded in details, and the causal relationship between the infusion and AEs was analyzed.
Data collection and management
Baseline data, which included demographics, anthropometrics, comorbid conditions, vital signs, Acute Physiology and Chronic Health Evaluation II (APACHE II) scores, and Glasgow coma scale (GCS) scores, were obtained on the day of randomization. Laboratory data, sequential organ failure assessment (SOFA) scores, PaO2/FiO2, and other treatments used were monitored on days 1, 3, and 7 (day 1 was defined as the day of the first administration of vitamin C).
The primary outcome of the study was invasive mechanical ventilation (IMV)-free days in 28 days (IMVFD28). Secondary outcomes included 28-day mortality, organ functions and inflammatory parameters, including white blood cell counts, neutrophil counts, lymphocyte counts, procalcitonin, interleukin-6 (IL-6), and C-reactive protein (CRP). Multi-organ dysfunction was assessed using SOFA scores. Additionally, vasopressor days, respiratory support days (including invasive and non invasive mechanical ventilation), IMVFD28, patient condition improvement rate, patient condition deterioration rate, length of ICU and hospital stay, ICU and in-hospital mortality were recorded as additional secondary outcomes of this research. IMVFD28s were defined as the number of days a patient was extubated after recruitment to day 28. If the patient died with MV, a value of zero was assigned. Deterioration of the patient’s condition was defined as the patient requiring HFNC or NIV on day 1 and requiring ECMO or IMV, or dying, after 7 days of treatment. Improvement of the patient’s condition was defined as the patient requiring ECMO or IMV on day 1 and switching to HFNC, NIV, or discharged from the ICU after 7 days of treatment. The P/F was calculated based on the PaO2/FiO2, and we choose the lowest values recorded on the specific day. All the data were collected from the clinical information system of three ICUs. Septic shock was identified according to International Guidelines for Management of Sepsis and Septic Shock(2016). Acute kidney injury was identified according to the Kidney Disease: Improving Global Outcomes definition. Acute cardiac injury was defined as the serum levels of troponin I were above the 99th percentile upper reference limit or new abnormalities were shown in electrocardiography and echocardiography. Acute liver failure (ALF), which is defined as coagulopathy (INR ≥ 1.5), hepatic encephalopathy, and onset less than 26 weeks in a patient without underlying chronic liver disease. Coagulation disorders was defined as the presence of D-dimer >0.24mg/L or FDP > 5mg/L.
The sample size was calculated according to primary endpoint, As this trial began at the early stage of COVID-19, such preliminary data lacked, and the sample size was finally calculated from the pervious studies on ARDS. We used the non-inferiority test formula to calculate the sample size with a one-sided error rate (α) of 2.5%, a power of 80%, and a withdrawal rate of 10%, and the anticipated sample size was140. With the control of the epidemic, this trial was stopped early, and the number of qualifying COVID-19 patients did not satisfy the anticipated sample size. Thus, we considered this trial as the pilot trial. Numerical variables are described as the mean with standard deviation (SD) or median with interquartile range (IQR) according to distribution and were compared with the t-test/Mann-Whitney U test. Category data are represented as frequencies and proportions and compared with the chi-square test and Fisher's exact test.The primary intention-to-treat analysis included all randomised participants. For the outcome variables, the hazard ratio and 95% CI were estimated by the Cox proportional risk model for mortality, and odds ratios with 95% CI were calculated by binary logistic regression for the other variables. Kaplan-Meier analysis was used to estimate the 28-day mortality to reflect the early survival differences for the two groups, and survival curves were compared with the Wilcoxon test. Survival analyses were further performed in subgroup with SOFA score more than 3. The testing was 2-sided, and a P-value<0.05 was considered statistically significant. SPSS 20.0 and GraphPad Prism 8.0 were used to complete data processing and statistical analysis.