1 Background

Sepsis is an organ dysfunction that can be fatal and is brought on by dysregulated host responses to infection. It is a serious disease with a complex pathogenesis and contributes significantly to mortality among patients in ICUs [1,2,3]. The high lethality and refractory nature of sepsis are closely related to its pathophysiology. In patients with sepsis, an uncontrolled inflammatory response and cellular dysmetabolism are significant causes of multiple organ dysfunction and death [4]. Therefore, restoring cellular dysmetabolism to improve the depletion of cellular bioenergy while repressing the exaggerated inflammatory response may be a potential therapeutic approach to improve sepsis mortality. However, the efficacy of adjunctive therapies other than basic therapy (mainly including antibiotics, vasopressors, and fluid resuscitation) is supported by insufficient evidence. Marik has proposed a vitamin C and related combination therapy, which is expected to inhibit the inflammatory response of sepsis, redress cellular dysmetabolism, and thus better the outcomes of patients with sepsis [5]. Subsequent randomized controlled studies and cohort studies of the vitamin C combination therapy proposed by Marik almost did not enhance the prognosis of patients with sepsis [6,7,8]. Researchers have since shifted their focus from the combination therapy to high-dose vitamin C monotherapy. The most commonly used therapeutic dose in subsequent RCTs was 12 g daily. In the LOVIT trial, which involved 863 patients, intravenous vitamin C (200 mg/kg per day) for four days was not found to improve death or persistent organ dysfunction at 28 days in adult patients with sepsis [9]. However, in the exploratory analysis of the CITRIS–ALI trial by Fowler III, high-dose vitamin C alone (50 mg/kg, every 6 h for 96 h) could improve 28-day mortality in patients with sepsis and acute respiratory distress syndrome (ARDS) [10]. A meta-analysis showed that vitamin C might reduce in-hospital mortality of adults with severe infection, but the results were not statistically significant (21 RCTs; RR, 0.88; 95% CI 0.73–1.06; low-certainty evidence) [11]. In another randomized controlled pilot study, the investigators compared the higher dose intravenous vitamin C administered at 12g every 12 h (24g per day) for seven days and placebo groups in treating critically ill patients with coronavirus disease 2019 (COVID-19). The trial results showed that a higher dose of vitamin C could effectively improve the respiratory oxygenation function in patients with COVID-19 and reduce inflammatory mediators, such as interleukin (IL)-6. Subgroup analysis of patients diagnosed with sepsis and sequential organ failure assessment (SOFA) score ≥ 3 showed that high-dose vitamin C even lowered ICU mortality [12]. However, in the CITRIS–ALI trial and the pilot trial in patients with COVID-19, the conclusion of mortality improvement came from the exploratory analysis. This needs to be verified by large-sample multicenter randomized controlled trials. Therefore, we aimed to further explore the efficacy and safety of megadose vitamin C (12g every 12 h for four days) in patients with sepsis through a multicenter, single-blind, randomized, placebo-controlled trial. It may provide higher-quality evidence for the use of megadose vitamin C in patients with sepsis.

2 Method

The ethics committee or relevant regulatory authorities will conduct a quality control review for each study center. The protocol was reviewed by the clinical trial committee of Zhujiang Hospital of Southern Medical University (2020-KY-069-05) and registered at www.clinicaltrials.gov (NCT05194189). Patients and their families did not participate in the study design. We have reported the Clinical Efficacy of Megadose Vitamin C in Sepsis (CEMVIS) trial protocol according to the SPIRIT guidelines [13].

2.1 Design and Study Setting

The CEMVIS trial was a multicenter, single-blind, randomized, placebo-controlled trial. The trial will be performed in four study centers: the Department of Critical Care Medicine of Zhujiang Hospital of Southern Medical University, Department of Critical Care Medicine of Nanfang Hospital of Southern Medical University, Department of Critical Care Medicine of Zhongshan People’s Hospital, and Department of Critical Care Medicine of Yunfu People’s Hospital from February 2022. These hospitals are large in Guangdong Province, China, and span a huge geographic area. This assures the broad representativeness of the recruited patients. The trial will be terminated after the last patient follow-up.

2.2 Study Procedures

The study procedures are illustrated in Fig. 1. Briefly, eligible patients meeting the diagnostic criteria of sepsis 3.0 and having a procalcitonin (PCT) level ≥ 2 ng/mL will be enrolled and randomized to receive intravenous administration of either 12 g vitamin C or an equivalent volume of 5% glucose injection every 12 h for four days or up to the moment they are discharged from intensive care unit (ICU). Relevant clinical data and adverse events (AEs) will be collected during the trial intervention, while patient survival at day 28 will be ascertained through telephone follow-up.

Fig. 1
figure 1

Study procedures. VC vitamin C, GS glucose, PI principal investigator. Data safety monitoring board (DSMB), Data and safety monitoring board; q12h, every 12 h

Full size image

2.3 Population

We will screen patients with sepsis who meet the diagnostic criteria of 2016 sepsis 3.0 as potential participants. Participants will be defined as patients who successfully complete the inclusion and exclusion screening procedures and sign a signed informed consent form. The inclusion criteria for participants are as follows: (1) be between the ages of 18 and 80; (2) meet the diagnostic criteria of 2016 international sepsis guidelines, which require the patient to be suspected or confirmed to have an infection and show evidence of acute organ dysfunction (SOFA ≥ 2 for patients who did not have previous chronic organ dysfunction and SOFA increased ≥ 2 for patients who have previous chronic organ dysfunction); (3) procalcitonin (PCT) ≥ 2 ng/mL at enrollment; and (4) voluntarily signing the informed consent form and taking part in the study.

The exclusion criteria are as follows: (1) pregnant or lactating patients; (2) patients with solid organ or bone marrow transplantation; (3) myocardial infarction throughout the last three months; (4) advanced lung fibrosis; (5) patients after cardiopulmonary resuscitation; (6) patients with human immunodeficiency virus; (7) patients with agranulocytosis; (8) hematologic or lymphatic malignancies in non-remission; (9) patients with nursing restrictions; (10) patients with immunodeficiency or long-term use of immunosuppressive medications; (11) patients who have advanced tumors; (12) those complicated by non-infectious conditions resulting in fatality (e.g. uncontrollable severe hemorrhage and cerebral herniation); (13) patients with an infection that cannot be surgically removed, and (14) patients who are allergic to the study medicine or possess conditions that contraindicate its use (such as a deficiency in glucose-6-phosphate dehydrogenase).

2.4 Screening and Informed Consent

Every patient admitted to the ICU will be screened by skilled research workers. Study candidates will be defined as patients satisfying all inclusion criteria and not meeting any of the exclusion criteria. Upon candidate identification, the research workers will conduct an informed consent conversation with the candidate or his/her family, acquainting them with the study's potential advantages and possible hazards. When written informed consent is obtained, the candidate will be formally registered in this trial. The enrollment date and basic information of the successfully enrolled participants will be meticulously documented. Furthermore, excluded patients will be recorded in detail with the date of exclusion and the reason for exclusion.

2.5 Randomization and Allocation Concealment

This study will use central randomization: the random sequence will be generated with the use of SAS, version 9.4, and saved to a cloud server. The patients will be randomized using the block randomization method with three different block sizes: 4, 6, and 8. Stratification will be performed according to whether septic shock is present. When the patient is successfully enrolled in the study, the trial investigator will log in to the electronic medical record system to fill in the information, and the interactive web response system will automatically complete the randomization. When more than one participants are simultaneously successfully enrolled, the order of randomization will be determined by the patient’s registration time in the ICU.

2.6 Blinding

A single-blind design was used in the CEMVIS study. After written informed consent is signed, central randomization will be used to randomly assign recruited enrolled patients in a 1:1 ratio to either the treatment (vitamin C injection) or placebo control (5% glucose injection) groups. Specialized nurses will prepare the medicine used in this trial. It will be uniformly filled using 50-mL syringes with identical appearance, labeled with “CEMVIS,” and infused at the same frequency and rate. Patients, their families, and statisticians will be blinded throughout the trial.

2.7 Interventions

Screening will be generally performed within 24 h after patients are admitted to the ICU. Successfully enrolled participants will be immediately randomized and promptly start the treatment in this trial. Patients in the treatment group will receive a 12-g vitamin C injection (manufactured by Guangzhou Baiyunshan Tianxin Pharmaceutical Co. Ltd.) using an intravenous pump at a uniform rate of 8 mL/h every 12 h. The same manner, frequency, and volume of 5% glucose injection will be administered to the patients in the control group. The treatment course will be 4 days or until the patient is transferred out of the ICU. Moreover, patients will receive routine treatment and care according to the 2021 Surviving Sepsis Campaign international guidelines, including the early use of antibiotics, fluid resuscitation, vasoactive medications, blood transfusion, and blood products as indicated, and enteral or parenteral nutrition. Patients with organ dysfunction with supportive indications will also be provided standardized supportive treatment, including continuous renal replacement therapy (CRRT), invasive or noninvasive mechanical ventilation (MV), and extracorporeal membrane oxygenation (ECMO) [14]. Enrolled patients could only be administered open-label vitamin C for parenteral nutritional needs; however, the daily dose should not exceed 6 g. In other cases, clinicians should avoid administering vitamin C therapy to patients during the study. When vitamin C is used in the non-trial treatment plan of clinicians, the reason, time, and dose of use should be recorded in detail.

2.8 Data Collection

Special electronic case report forms (CRFs) will be designed using EpiData software for data collection. One research worker will first perform data collection, and at least one other worker will examine the recorded data. To protect patient privacy, only initials and serial numbers will be recorded in the CRFs as identifiers.

Study data will be collected from patients on the day of screening (D0) and days 1, 2, 4, and 28 after enrollment (Table 1). Data to be collected on D0 include demographic data, past medical history, concomitant medications affecting the results, time from first medication to ICU admission, heart rate, oxygen saturation, respiratory rate, body temperature, blood pressure, infection indicators (white blood cell [WBC], and PCT levels), kidney injury indicators (blood urea nitrogen [BUN], cystatin C and serum creatinine [SCr] levels), circulatory function (blood lactate level), liver injury indicators (alanine aminotransferase [ALT], aspartate aminotransferase [AST], and total bilirubin levels), inflammation indicators (IL-6 levels and C-reactive protein [CRP]), lung injury indicators (oxygenation index), etiological indicators, the condition of organ function support (whether CRRT, MV, and vasoactive agents are used), and severity score (APACHE II and SOFA scores). The 28-day survival data for the patient will be acquired through telephone follow-up. Safety indicators including the incidence of AEs and serious adverse events (SAEs) will be recorded throughout the trial duration. Patients who withdraw from the trial will be recorded in detail and followed up. The central laboratory of each study site will provide all laboratory test results, and trained research workers will collect the abovementioned data.

Table 1 Schedule of clinical or laboratory assessments
Full size table

Secondary outcomes will mainly include the following: (1) Lung injury indicators: changes in the oxygenation index (PO2/FiO2) evaluated on days 0, 1, 2, and 4 following randomization but not collected for patients using ECMO. (2) Liver injury indicators: changes in total bilirubin levels and serum transaminases (AST and ALT) on days 0, 1, 2, and 4 following randomization. (3) Kidney injury indicators: changes in SCr, BUN and cystatin C on days 0, 1, 2, and 4 following randomization. (4) Circulatory function evaluation: blood lactate levels assessed on days 0, 1, 2, and 4 following randomization. (5) Organ injury score: SOFA score changes evaluated on days 0, 1, 2, and 4 following randomization. The scores will be calculated by collecting the patient’s oxygenation index, platelet count, bilirubin level, SCr level, blood pressure, and Glasgow coma score. (6) Infection indicators: changes in WBC count and PCT levels assessed on days 0, 1, 2, and 4 following randomization. (7) Inflammatory markers: IL-6 and CRP levels assessed on days 0, 1, 2, and 4 following randomization. (8) Organ function support evaluation indicators: time to successful withdrawal from MV (defined as withdrawal from MV support for > 48 h and assessed only in patients who have received MV); time to a successful withdrawal of vasoactive agents (defined as maintaining the clinician-specified mean arterial pressure target for > 24 h without the need for vasoactive agents and assessed only in patients receiving vasoactive agents); and duration of CRRT use. (9) Other measures: days of ICU stay (from enrollment to ICU discharge).

2.9 Adverse and Serious Adverse Events

Allergy, hypernatremia, calcium oxalate stones, and calcium oxalate nephropathy are common adverse reactions to vitamin C. High vitamin C doses over several months to years may produce calcium oxalate nephropathy or calcium oxalate stones. However, short-term studies of vitamin C use have not reported on calcium oxalate stones [15]. A recent document issued by a panel of experts from the United States National Institutes of Health also clearly states that 1.5-g/kg vitamin C intravenous administration is safe without obvious adverse effects [12]. A high vitamin C dose (0.2–1.5 g/kg) has been used in adjuvant chemotherapy regimens for patients with metastatic colon or gastric cancer [16]. No SAEs occurred in the above-mentioned studies. In general, there are limited studies on the AEs of high-dose vitamin C in patients with sepsis in the ICU. Therefore, this trial will concentrate on the AEs of megadose vitamin C in patients with sepsis to explore its safety. Research workers will dynamically record the AEs deemed to be associated with the medicine in the trial every day. When the clinician in charge considers that some events are related to this study, research workers will also record them in detail and judge the correlation between them and the study intervention.

An event that occurs within 28 days of enrollment and fits one or more of the following criteria will be considered an SAE: death, endangering life, prolongation of patient hospitalization, and persistent or severe disability. Any SAE will be carefully recorded. The trial will be stopped immediately if a serious adverse event is considered to be associated with the intervention, and the serious adverse event will be treated and submitted to the Clinical Research Ethics Committee. Members of the CEMVIS team and clinicians should be trained to identify SAEs before the trial.

2.10 Data and Safety Review

The Data and Safety Monitoring Board (DSMB) team will independently monitor the progress and safety of this study. When the trial starts enrollment, the DSMB subgroup will review the raw data of the trial every six months, including routine treatment measures and standard care measures, dropout events, AEs, and SAEs. To ensure that the trial is scientific and ethical, the team could stop the trial at any stage.

3 Statistical Analysis

3.1 Sample Size

In this study, the primary outcome will be 28-day all-cause mortality. Based on the exploratory analysis outcome of the CITRIS–ALI trial, we expected that the relative risk of mortality between the intervention and control groups would be 0.49 [10]. Based on the clinical experience of our centers, the average mortality of patients with sepsis in the control group is approximately 34.7% [17]. Therefore, the estimated average mortality of patients in the intervention group is calculated as follows: 34.7% × 0.49 = 17.003%. The treatment and placebo control groups will be allocated in a 1:1 ratio, and a two-sided test will be adopted (α = 0.05, β = 0.2, power = 80%). The sample size was calculated using the PASS11 software, with each group having 93 participants. Considering the dropout rate, the sample size was increased by approximately 20% and finally calculated to be 117 in each group. In summary, the total sample size for this study was 234 patients.

3.2 Data for Statistical Analysis

Data from the adjusted intent-to-treat population will be used for the analysis. Participants who used the medicine at least once and had at least one valid evaluation data record will be included for statistical analysis.

3.3 Basic Principles of Statistics

Unless otherwise noted, a two-sided test will be used for all statistical inferences. The test level with statistical significance will be set as 0.05, and the 95% confidence interval will be used for interval estimation of parameters. Parametric methods are used as much as possible. Nonparametric methods will be considered when the data do not meet the conditions of parametric methods.

3.4 Handling of Missing Data

Missing data of secondary outcomes will be imputed by the last observation carried forward method that is, the final test result was derived by transferring the data from the last observation.

3.5 Analysis of Dropout Data

The Pearson chi-square test will be used to compare the total dropout rate and dropout due to AEs in each group.

3.6 Descriptive Statistics

Means and standard deviations or medians and interquartile ranges will be used to report descriptive continuous variables, and frequencies and proportions will be used to present categorical variables.

3.7 Analysis of Baseline Data

Baseline data included demographic indicators, general conditions before treatment, and secondary outcomes. For data with a normal distribution, the t-test or t′ test (when the variance is not homogeneous) will be used to analyze the measurement data. The Wilcoxon rank sum test will be used if the data present a non-normal distribution. Counting data will be analyzed by the Pearson chi-square test.

3.8 Compliance Analysis

An analysis of compliance will be carried out based on the duration of medication and dose.

3.9 Analysis for Primary Outcome

The primary outcome will be 28-day all-cause mortality. Logistic regression analysis will be used for comparison between the intervention and control groups. Besides, the difference in survival time between the two groups will be evaluated with the cox proportional hazards model.

3.10 Analysis for Secondary Outcomes

The differences in quantitative variables between the groups will be compared by the Wilcoxon rank sum test or a two-sample t-test. Repeated measure variables between the groups will be compared by the mixed linear model. The Pearson chi-square test was employed for categorical variables.

3.11 Center Effect Analysis

The primary outcome will be to evaluate whether there is a center effect. The evaluation and adjustment will be conducted using a logistic regression model.

3.12 Safety Analysis

AEs in the two groups will be closely observed, and their incidence will be compared by the Pearson chi-square test. Changes in laboratory test results related to AEs before and after the study will be compared, and their relationship with the trial medication will be analyzed.

3.13 Interim Analysis

No interim analysis will be conducted in this study.

3.14 Subgroup Analysis

The following criteria will be used in prespecified subgroup analysis: (1) APACHE II score ≤ 25, > 25; (2) age ≤ 65, > 65 years; (3) sepsis, septic shock. To explore the efficacy of the study intervention in different subgroups, reasonable clinical subgroups will be divided for post hoc analysis.

4 Discussion

Vitamin C is a significant natural antioxidant that contributes significantly to preserving the normal function of cells and mitochondria. Vitamin C levels rapidly decline in critically ill patients. Besides, patients with sepsis have lower plasma vitamin C levels than those without. Low plasma concentrations of vitamin C are closely related to the severity of organ failure and mortality [18]. A retrospective study conducted by Professor Marik in 2017 showed that vitamin C combined with vitamin B1 and hydrocortisone reduced the mortality of patients with sepsis, improved organ dysfunction, and reduced the dosage of vasoactive agents [19]. In January 2020, a study of vitamin C combined with vitamin B1 and hydrocortisone published in the journal JAMA reported that the combination of the three drugs neither improved the shock remission time nor reduced the 28-day mortality in patients with septic shock [6]. However, in the CITRIS–ALI trial, therapy with high-dose vitamin C alone reduced the 28-day mortality of patients with sepsis and ARDS by approximately 17% [10]. A recent study showed that, in critically ill patients with COVID-19 who met the sepsis criteria, high-dose vitamin C administered at 12g every 12 h (24g per day) reduced mortality compared with placebo [12]. Based on the abovementioned evidence, we suggest that megadose vitamin C alone can be more beneficial in reducing mortality in sepsis. However, in the two abovementioned studies, the conclusion of mortality improvement came from exploratory analyses that did not adjust for multiple comparisons and should be verified by large-sample multicenter randomized controlled trials. Therefore, we aimed to conduct a multicenter randomized placebo-controlled trial to further explore the efficacy and safety of high-dose vitamin C in patients with sepsis.