Study objectives
To investigate the preventive effect of vitamin D2 on COVID-19 and the improvement of symptoms after COVID-19 infection in health care workers.
Study Design
This was a multicenter, open-label, randomized controlled trial. The subjects provided written informed consent before participation. They were randomly assigned to receive 200,000 IU vitamin D2 fortnightly or no intervention. This study was conducted on December 23, 2022 and was closed to recruitment on December 26, 2022, and a total of 262 participants were enrolled. The trial protocol is available in See supplement file2.
Ethical approval
This study has been registered in a clinical trial registry (clinicaltrial.gov, ID: NNCT05673980 on 22/12/2022). This study protocol was reviewed and approved by the Peking University Third Hospital Medical Science Research Ethics Committee, approval number was M2022828. This manuscript was written in accordance with the CONSORT 2010 guidelines for reporting randomized controlled trials. Signed informed consent must be obtained from participants before any operational procedures related to clinical trials. The informed consent was dated and signed by the subjects.
Patient and public involvement
Patient or the public did not be involved in the design, conduct, reporting, or dissemination plans of this study.
Participants
The study included 248 health care workers from Peking University Third Hospital, Beijing Haidian Hospital, Chongli People Hospital and Yanqing People Hospital. All subjects were off vitamin D or received only preventive doses before signing the informed consent. The inclusion criteria consisted of having a negative PCR or antigen test and being asymptomatic for COVID-19. The exclusion criteria consisted of pregnant and lactating women, long-term vegetarians, and a history of serious concomitant diseases, chronic diarrhea, subtotal gastrectomy, biliary obstruction, and pancreatic disease.
All the subjects followed up regularly by PCR or antigen test for COVID-19 infection and completed seven electronic questionnaires during this trial. The information collected included the name, age, height, weight, vaccine, antigen or PCR test results, the time of positive PCR test or antigen test, the time of positive to negative PCR test or antigen test, the extent and duration of COVID-19 symptoms (fever, cough, sore throat, headache, joint pain, diarrhea, and vomiting), concomitant diseases, and smoking history of subjects.
Randomization and Study Interventions
The subjects were randomly allocated into different groups by local investigators using a randomization envelope, which concealed the allocation sequence. The random sequence was generated with the SAS 9.4 procedure using a size block of 4, and the ratio between the two groups was 1:1.
Subjects in the vitamin D2 group were given 200,000 IU containing 5 mg of calciferol orally on the first day and the fourteenth day after enrollment, while the other group subjects received no intervention during the whole trial.
Outcome Assessment
The primary outcome was the incidence of COVID-19 infection as detected by laboratory PCR or antigen test within one month. The secondary outcomes were the duration of a positive to negative PCR or antigen test after COVID-19 infection, the duration of fever, cough, runny nose, stuffy nose, sore throat, headache, fatigue, muscle aches, joint aches, loss of taste or smell, vomiting, diarrhea and other symptoms after COVID-19 infection, the proportion of subjects whose COVID-19 infection symptoms were confirmed by PCR or antigen testing for more than 7 days, the proportion of subjects who tested positive by PCR or antigen test but had no symptoms of COVID-19, the proportion of subjects with mild, moderate and severe COVID-19 symptoms, and the change in 25(OH)D concentration.
At baseline, serum 25(OH)D, CRP, complement component C1q and inflammatory cytokines (IL-1β, IFN-α, IFN-γ, TNF-α, MCP-1, IL-6, IL-8, IL-10, IL-12, IL-17A, IL-18, IL-23, and IL-33) were measured in a group of 214 subjects, and subsequently, on the thirtieth day of the study, the subjects completed the second laboratory assessment. All the blood samples were analyzed at the Peking University Third Hospital. Seven follow-up visits were conducted during the trial.
Blinding
This was a open-label controlled trial, the blinding method was not applicable.
Sample Size Calculation and Power
The primary objective of the study was to compare the incidences of COVID-19 infection in the two groups. According to our preliminary study, the expected incidence of COVID-19 infection was 60.0% in the treatment group and 70.0% in the control group. Given an α of 0.05 and β of 0.2 (i.e., statistical power of 80%), a total of 103 subjects per group (total sample size: 206 subjects) was needed. Assuming a dropout rate of 5%, 218 patients were required for screening to provide an adequate sample size.
Statistical Analysis
The statistician who conducted the analysis was blinded to group allocation. Primary and secondary outcomes were analyzed according to an intention-to-treat (ITT) principle in the ITT dataset. The per-protocol (PP) sets were used for sensitivity analysis. The safety analysis sets included all enrolled subjects who received treatment and were evaluated for safety. Primary analyses were conducted using multiple imputation methods for missing observations based on the baseline, post-intervention and follow-up, assuming that missing data were missing at random.
For the primary outcome and categorical secondary outcomes, the chi-square test or Fisher’s exact test was used to detect the differences between groups. The Kaplan-Meier survival curve and log-rank test were used to analyze the primary outcome as supplements. The generalized linear mixed model (GLMM) was used to control the baseline imbalance and central effect, and the different centers were set as fixed effects.
For continuous secondary outcomes, normally distributed data were presented as the means with standard deviations and compared between groups using the independent t test, while non-normally distributed variables were presented as medians with interquartile ranges (IQRs) and compared using the Mann-Whitney U test. For secondary outcomes and subgroup analyses, the analyses were performed as exploratory tests.
All the statistical analyses were conducted using SPSS 26.0 (IBM, Armonk, NY, USA) and R 4.2.2 software (www.r-project.org). The statistical significance was defined as P <0.05 with two-sided testing.