Severe COVID-19 not fought by vitamin D when given too late - RCT

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Vitamin D levels increased to avg of 43ng?

All participants appear to have been in hospital a while BEFORE getting Vitamin D

"The mean time between the onset of symptoms and randomization was 10.2 days (SD,4.3);
89.6% required supplemental oxygen at baseline "

The above is a subset of the table in the PDF

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Effect of Vitamin D3 Supplementation vs Placebo on Hospital Length of Stay in Patients with Severe COVID-19: A Multicenter, Double-blind, Randomized Controlled Trial

Trial Registration: ClinicalTrials.gov Identifier: NCT04449718

Igor H. Murai, PhD; Alan L. Fernandes, PhD; Lucas P. Sales, MSc; Ana J. Pinto, BSc;

Karla F. Goessler, PhD; Camila S. C. Duran, MD; Carla B. R. Silva, MD; Andre S.

Franco, MD; Marina B. Macedo, MD, MSc; Henrique H. H. Dalmolin, MD; Janaina

Baggio, MD; Guilherme G. M. Balbi, MD; Bruna Z. Reis, PhD; Leila Antonangelo,

MD, PhD; Valeria F. Caparbo, PhD; Bruno Gualano, PhD; Rosa M. R. Pereira, MD,

PhD.

Corresponding author (to whom reprint requests should be addressed):

Rosa Maria Rodrigues Pereira, MD, PhD.

Rheumatology Division, Faculdade de Medicina FMUSP, 3° andar, Universidade de

Sao Paulo, Sao Paulo, SP, BR.

Av. Dr. Arnaldo, 455 - Pacaembu - Sao Paulo, SP - Brasil

Postal code: 01246-903. Phone: + 55 11 3061.7490 e-mail: rosamariarp@yahoo.com

Key points:

Question: Can vitamin D3 supplementation reduce hospital length of stay in hospitalized patients with severe COVID-19?

Findings: In this double-blind, randomized, placebo-controlled trial involving 240

hospitalized patients with severe COVID-19, a single dose of 200,000 IU of vitamin D3

supplementation was safe and effective in increasing 25-hydroxyvitamin D levels, but

did not significantly reduce hospital length of stay (hazard ratio, 1.12) or any other

clinically-relevant outcomes compared with placebo.

Meaning: Vitamin D3 supplementation does not confer therapeutic benefits among hospitalized patients with severe COVID-19.

Importance: Patients with COVID-19 may exhibit 25-hydroxyvitamin D deficiency,but the beneficial effects of vitamin D3 supplementation in this disease remain to be proven by randomized controlled trials.

Objective: To investigate the efficacy and safety of vitamin D3 supplementation in

patients with severe COVID-19.

Design, Setting, and Participants: This is a multicenter, double-blind, randomized,

placebo-controlled trial conducted in two centers (a quaternary hospital and a field

hospital) in Sao Paulo, Brazil. The trial included 240 hospitalized patients with severe

COVID-19. The study was conducted from June 2, 2020 to October 7, 2020.

I

nterventions: Patients were randomly allocated (1:1 ratio) to receive either a single

oral dose of 200,000 IU of vitamin D3 or placebo.

Main Outcomes and Measures: The primary outcome was hospital length of stay,

defined as hospital discharge from the date of randomization or death. Secondary

outcomes were mortality, admission to ICU, mechanical ventilation requirement, and

serum levels of 25-hydroxyvitamin D, creatinine, calcium, C-reactive protein, and D-

dimer.

Results: Of 240 randomized patients (mean age, 56 years; 56% men), 232 (96.7%)

were included in the primary analysis. Log-rank test showed that hospital length of stay

was comparable between the vitamin D3 supplementation and placebo groups (7.0 days

[95% CI, 6.1 to 7.9] and 7.0 days [95% CI, 6.2 to 7.8 days]; hazard ratio, 1.12 [95% CI,

0.9 to 1.5]; P = .379; respectively). The rate of mortality (7.0% vs 5.1%; P = .590),

admission to ICU (15.8% vs 21.2%; P = .314), and mechanical ventilation requirement

(7.0% vs 14.4%; P = .090) did not significantly differ between groups. Vitamin D3

supplementation significantly increased serum 25-hydroxyvitamin D levels compared to

placebo (difference, 24.0 ng/mL [95% CI, 21.0% to 26.9%]; P = .001). No adverse

events were observed.

Conclusions and Relevance: Among hospitalized patients with severe COVID-19,

vitamin D3 supplementation was safe and increased 25-hydroxyvitamin D levels, but

did not reduce hospital length of stay or any other relevant outcomes vs placebo. This

trial does not support the use of vitamin D3 supplementation as an adjuvant treatment of

patients with COVID-19.

Introduction

A growing body of evidence has indicated that vitamin D may enhance the innate1-3 and

adaptive immunity.4, 5 Since antigen-presenting cells have the ability to synthesize 1,254 dihydroxyvitamin D (the active form of vitamin D) from 25-hydroxyvitamin D, it has

been postulated that vitamin D supplementation could improve the function of

macrophages and dendritic cells, thereby ameliorating overall immune response.6 In

fact, insufficient vitamin D status has been suggested as a potential risk factor for non-

communicable7 and acute respiratory tract diseases,8, 9 including viral infections.10

In this context, it has been recently conjectured that optimal levels of vitamin D could

play important immunomodulatory and anti-inflammatory roles, thereby benefiting

patients with COVID-19.11, 12 However, the putative benefits of supplementary vitamin

D3 to patients with COVID-19 remain speculative and partially supported by limited

data from observational studies and one small-scale, non-randomized clinical trial.13-15

To our knowledge, this is the first randomized, double-blind, placebo-controlled trial to

investigate the safety and efficacy of vitamin D3 supplementation on hospital length of

stay and other relevant clinical outcomes in hospitalized patients with severe COVID-

19. Our main a priori hypothesis was that a single dose of 200,000 IU of vitamin D3

supplementation would increase 25-hydroxyvitamin D levels and shorten hospital

length of stay among these patients.

Methods

The study was approved by the Ethics Committee of Clinical Hospital of the School of

Medicine of the University of Sao Paulo and by the Ethics Committee of Ibirapuera

Field Hospital. All the procedures were conducted in accordance with the Declaration of

Helsinki. The participants provided written informed consent before being enrolled in

the study (Ethics Committee Approval Number 30959620.4.0000.0068). The trial

protocol and statistical plan are included in Supplement 1. This manuscript was written

according to the recommendations by the Consolidated Standards of Reporting Trials

(CONSORT) guidelines (see Supplement 2).

Participants

Hospitalized patients were recruited from Clinical Hospital of the School of Medicine

of the University of Sao Paulo (a quaternary referral teaching hospital), and from

Ibirapuera Field Hospital, both located in Sao Paulo, Brazil. Enrollment started on June

2, 2020, to August 27, 2020, with the final follow-up on October 7, 2020.

Inclusion criteria

Inclusion criteria were: 1) adults aged 18 years or older; 2) diagnosis of COVID-19 by

either polymerase chain reaction (PCR) for severe acute respiratory

syndrome coronavirus 2 (SARS-CoV-2) from nasopharyngeal swabs or computed

tomography scan findings (bilateral multifocal ground-glass opacities > 50%)

compatible with the disease; 3) diagnosis of flu syndrome with hospitalization criteria

on hospital admission, presenting respiratory rate > 24 breaths per minute, saturation <

93% on room air or risk factors for complications, such as heart disease, diabetes

mellitus, systemic arterial hypertension, neoplasms, immunosuppression, pulmonary

tuberculosis, and obesity, followed by COVID-19 confirmation before randomization.

Exclusion criteria

Exclusion criteria were: 1) patient unable to read and sign the written informed consent;

2) patient already admitted under invasive mechanical ventilation; 3) previous vitamin

D3 supplementation (> 1000 IU/day); 4) renal failure requiring dialysis or creatinine >

2.0 mg/dL; 5) hypercalcemia defined by total calcium > 10.5 mg/dL; 6) pregnant or

lactating women; and 7) patients with expected hospital discharge in less than 24 hours. 10

Study design and treatment

This was a multicenter, double-blind, parallel-group, randomized placebo-controlled

trial. Eligibility screening was performed between June 2, 2020 to July 21, 2020 at

Clinical Hospital of the School of Medicine of the University of Sao Paulo (n = 122),

and from July 22, 2020 to August 27, 2020 at Ibirapuera Field Hospital (n = 118). The

final follow-up in both centers was on October 7, 2020. Eligible patients were assigned

in a 1:1 ratio into either the vitamin D3 supplementation group or the placebo group.

The randomization list was created using a computer-generated code, which was

managed by a staff member who had no role in the study. We assessed patients5 clinical

status, coexisting chronic diseases, demographic characteristics, self-reported body

weight and height, and ethnicity on hospital admission. Outcomes were assessed at

baseline and on hospital discharge or death records.

The vitamin D3 supplementation group received an oral, single dose of 200,000 IU of

vitamin D3 dissolved in a 10 mL of peanut oil solution on the same day of

randomization. The selected dose is within the recommended range for effectively

promoting vitamin D sufficiency.16 Patients in the placebo group received 10 mL of

peanut oil solution. The vitamin D3 and placebo solutions were identical in color, taste,

smell, consistency, and container. Both were prepared by the pharmacy unit of Clinical

Hospital and labeled by a staff member who did not participate in the study. Allocation

blindness was kept until the final statistical analysis.

Outcome measures

The primary outcome was hospital length of stay, defined as the total number of days

that patients remained hospitalized from the date of study admission until the date of

hospital discharge or death. The criteria used for patient discharge were: 1) no need for

supplemental oxygen in the last 48 hours; 2) no fever in the last 72 hours; and 3)

oxygen saturation > 93% in room air without respiratory distress.

The secondary outcomes were: 1) mortality; 2) number of patients admitted to the

intensive care unit (ICU); 3) number of patients who needed mechanical ventilation and

duration of mechanical ventilation; and 4) serum levels of 25-hydroxyvitamin D

(assessed by a chemiluminescent immunoassay), calcium (assessed by a NM-BAPTA

method), creatinine (assessed by a colorimetric assay based on kinetic Jaffe’s reaction),

and C-reactive protein and D-dimer (both assessed by an immunoturbidimetric assay).

The biochemical analyses were carried out in an accredited laboratory from Clinical

Hospital.

Statistical Analysis

Considering the lack of data available for sample size determination based on the

primary outcome (i.e., hospital length of stay after vitamin D3 supplementation in

patients with severe COVID-19), the number of participants was chosen on the basis of

feasibility, such as resources, capacity of research staff and facility, and available

patients, in line with current recommendations.17, 18 Subsequently, we calculated sample

size assuming a 50% between-group difference in hospital length of stay (considering 7

days as a median time of stay, with an expected variability of 9 days). By considering a

power of 80% and a 2-sided significance level of 5% (a = .05), the total sample was

estimated to be 208 patients (104 in each arm). Considering possible dropouts, and to

increase the power for secondary outcomes, we opted by increasing the sample size by

approximately 15%.

All analyses were carried out following the intention-to-treat principle for all

randomized patients, with no imputation for any missing data. Proportions were

compared between groups using %2 test and Fisher's exact test. Student's t-tests were

used for comparing continuous variables at baseline. The log-rank test was used to

compare the Kaplan-Meier estimate curves the number of days for hospital length of

stay, the primary outcome. Cox regression models for hospital length of stay, admission

to ICU and mechanical ventilation requirement were adjusted by potential confounders

that were not fully balanced by randomization (P < .2) to estimate hazard ratios (HR),

with corresponding 2-sided 95% CI. Generalized estimating equations (GEE) for

repeated measures were used for testing possible differences in laboratory parameters,

assuming group and time as fixed factors, with marginal distribution, and a first-order

autoregressive correlation matrix to test the main and interaction effects. Post-hoc tests

with Bonferroni5s adjustment were performed for multiple comparisons. The

aforementioned statistical procedures were also carried out in post-hoc sensitivity

analyses involving patients exhibiting 25-hydroxyvitamin D deficiency (i.e., < 20

ng/mL).

Statistical analyses were performed with IBM-SPSS software, version 20.0.

Significance level was set at a = .05.

Results

Patients

Of 1208 patients assessed for eligibility, 240 were eligible and randomly assigned to

either the vitamin D3 group or the placebo group. Patients were non-eligible due to the

following reasons: 284 were at ICU, 263 had hospital discharge within 24 hours, 217

did not have COVID-19 confirmation, 95 had renal dysfunction, 37 had dementia or

severe mental confusion hampering their ability to provide the inform consent for

participation, 30 were pregnant or lactating women, 14 had hypercalcemia due to

metastatic neoplasm, 11 were receiving vitamin D3 (> 1000 IU/day), 9 were younger

than 18 years, 6 were illiterate and, therefore, unable to read and sign the informed

consent, and 2 died before randomization.

Of the 120 patients who were randomized to the vitamin D3 group, 3 did not receive

intervention (1 withdrew the consent, 1 vomited immediately after ingesting the

supplement, and 1 was admitted to the ICU before taking vitamin D3) and 3 were lost to

follow-up. Of the 120 patients who were randomized to the placebo group, 2 withdrew

the consent. Thus, of the 240 patients randomized, 232 (96.7%) completed the follow-

up (Figure 1).

Overall, patients5 age was 56.3 years (SD, 14.6), BMI was 31.6 kg/m2 (SD, 7.1), 56.3%

were men, 55% were white, 52.5% had hypertension, 35% had diabetes, 13.3% had

cardiovascular diseases, and 6.3% had asthma. The mean time between the onset of

symptoms and randomization was 10.2 days (SD, 4.3); 89.6% required supplemental

oxygen at baseline (183 were on oxygen therapy and 32 were on non-invasive

ventilation), and 59.6% had computed tomography scan findings suggestive of COVID-

19. Demographic and clinical characteristics did not significantly differ between groups,

except for sore throat, which was more prevalent in the vitamin D3 group vs placebo

(38.3% vs 24.2%, P = .026), and PTH, which was higher in the vitamin D3 group vs

placebo (50.1 vs 42.6 pg/mL, P = .025) (Table 1).

Primary Outcome

Hospital length of stay (Figure 2) was comparable between the vitamin D3 group and

the placebo group (7.0 days [95% CI, 6.1 to 7.9] and 7.0 days [95% CI, 6.2 to 7.8 days],

HR, 1.12, [95% CI, 0.9 to 1.5]; P = .379; respectively). The Cox regression model did

not show any significant associations between this outcome and potential confounders.

Secondary Outcomes

There were no significant differences between the vitamin D3 group and the placebo

group for mortality (7.0% vs 5.1%; P = .590), admission to ICU (15.8% vs 21.2%; P =

.314) and mechanical ventilation requirement (7.0% vs 14.4%; P = .090) (Figure 3).

Duration of mechanical ventilation was also comparable between the vitamin D3 group

(18.1 days [95% CI, 3.5 to 32.7]) and the placebo group (11.4 days [95% CI, 7.1 to

15.6]; P = .549, respectively).

The Cox regression model did not show significant associations between secondary

outcomes and potential confounders.

Vitamin D3 supplementation significantly increased 25-hydroxyvitamin D levels vs

placebo (difference, 24.0 ng/mL [95% CI, 21.1- 26.9]; P < .001) (Figure 3). Following

the intervention, 86.7% of the patients in the vitamin D3 group showed 2522 hydroxyvitamin D levels above 30 ng/mL (vs 10.9% in the placebo group), and only 6.7% of the patients in the vitamin D3 group exhibited 25-hydroxyvitamin D deficiency

(vs 51.5% in the placebo group).

Post-hoc Sensitivity Analyses

In a sensitivity analysis involving patients with 25-hydroxyvitamin D deficiency at

baseline (n = 116) (Supplementary Table 1), vitamin D3 supplementation significantly

increased 25-hydroxyvitamin D levels vs placebo (difference, 22.7 ng/mL [95% CI,

19.3 to 26.1]; P < .001) (Figure 3). Among the patients with 25-hydroxyvitamin D

deficiency, no between-group differences were observed in length of hospital stay

(Figure 2). In addition, there were no significant differences between the vitamin D3

group and the placebo group for mortality (7.0% vs 1.7%; P = .206), admission to ICU

(17.5% vs 15.5%; P = .806), and mechanical ventilation requirement (7.0% vs 8.6%; P

.999) (Figure 3). Duration of mechanical ventilation did not differ between the

vitamin D3 group (15.0 days [95% CI, -12.0 to 42.0]) and the placebo group (12.6 days

[95% CI, -7.6 to 26.0]; P = .730).

Safety and Adverse Events

There were no changes in any health-related laboratory markers following the

intervention (Table 2). Vitamin D3 supplementation was well tolerated and no severe

adverse events were reported throughout the trial, with the exception of one patient who

vomited following vitamin D3 administration.

Discussion

This is the first randomized, double-blind, placebo-controlled trial to show that vitamin

D3 supplementation is safe and increases 25-hydroxyvitamin D levels, but is ineffective

to improve hospital length of stay or any other clinical outcomes among hospitalized

patients with severe COVID-19.

Vitamin D has been postulated to play an important role on immune system, acting as a

regulator of both innate and adaptative responses.6, 19 Observational studies have shown

that 25-hydroxyvitamin D levels are associated with better clinical outcomes in

respiratory diseases.20 Positive associations between low 25-hydroxyvitamin D levels

and poor prognosis among patients with COVID-19 have also been observed.21

Furthermore, a small-scale, non-randomized trial demonstrated that the administration

of regular boluses of vitamin D3 before the infection was associated with better survival

and less severe disease among older, frail patients with COVID-19.22 In the current trial,

however, a single dose of 200,000 IU of vitamin D3 supplementation failed to promote

any clinically relevant effects among hospitalized patients with severe COVID-19,

contesting the utility of supplementary vitamin D3 as a treatment in this disease.

The lack of clinical benefits seen in this study was independent of the ability of vitamin

D3 supplementation to increase serum 25-hydroxyvitamin D levels. In fact, following

the intervention, 86.7% of the patients in the supplementation arm achieved vitamin D

sufficiency (> 30 ng/mL) vs 11% only in the placebo group. In a sensitivity analysis

confined to the patients exhibiting 25-hydroxyvitamin D deficiency, vitamin D3

supplementation remained effective in increasing 25-hydroxyvitamin D levels vs

placebo; yet, no clinical improvements were noted. Collectively, these analyses indicate

that a single oral dose of 200,000 IU of supplementation can rapidly increase 2520 hydroxyvitamin levels, in agreement with our hypothesis, so that the present null findings cannot be attributed to the failure of increasing serum 25-hydroxyvitamin D

levels.

Despite the clinical inefficacy of vitamin D3 supplementation, the intervention was not

associated with any important adverse events or meaningful changes in laboratory

parameters, suggesting that a relatively high-dose of vitamin D3 can be well tolerated in

general and free of adverse effects in patients with COVID-19.

The strengths of this study include the randomized, double-blind, placebo-controlled

experimental design, the adequate power, particularly for the primary analysis, the very

low attrition rate (3.3%), the concomitant assessment of 25-hydroxyvitamin D levels

along with clinical outcomes, and the assessment of hospitalized patients with severe

COVID-19.

Limitations

This trial has several limitations. First, the sample size could have been underpowered

to detect significant changes for the secondary outcomes. Second, as the patients had

several coexisting diseases and were subjected to a diverse medication regimen, the

results could have been affected by the heterogeneity of the sample and its treatment.

Third, the proportion of patients with 25-hydroxyvitamin D deficiency enrolled in this

study was considerably lower than those reported in other cohorts,23 possibly as a

consequence of differences in geographic locations. Although we conduced sensitivity

analyses involving patients with 25-hydroxyvitamin D deficiency, one could argue that

they could have been underpowered, as previously pointed out. Therefore, caution

should be exercised in generalizing these findings to patients from other geographical

regions. Finally, the findings should be also confined to the dose and supplementation

strategy used in this trial. Further studies should determine whether preventive or

early vitamin D3 supplementation could be useful in the treatment of patients with COVID-

19, especially those with a mild or moderate disease.

Conclusions

Among hospitalized patients with severe COVID-19, a single dose of 200,000 IU of

vitamin D3 supplementation was safe and increased 25-hydroxyvitamin D levels, but

did not reduce hospital length of stay or any other clinically relevant outcomes vs

placebo. Thus, this trial does not support the use of vitamin D3 supplementation as an

adjuvant treatment of patients with COVID-19.

Article information

Author Affiliations: Rheumatology Division, Hospital das Clinicas HCFMUSP,

Faculdade de Medicina da Universidade de Sao Paulo, Sao Paulo, Brazil (Murai,

Fernandes, Sales, Duran, Silva, Franco, Macedo, Dalmolin, Baggio, Balbi, Reis,

Caparbo, Pereira); Applied Physiology & Nutrition Research Group; Faculdade de

Medicina da Universidade de Sao Paulo, Sao Paulo, Brazil (Pinto, Goessler, Gualano);

Clinical Pathology Division, Hospital das Clinicas HCFMUSP, Faculdade de Medicina

da Universidade de Sao Paulo, Sao Paulo, Brazil (Antonangelo); Food Research Center,

Universidade de Sao Paulo, Sao Paulo, Brazil (Gualano).

Author Contributions: Dr. Pereira had full access to all of the data in the study and

take responsibility for the integrity of the data and the accuracy of the data analysis.

Concept and design: Murai, Fernandes, Pinto, Goessler, Gualano, Pereira.

Acquisition, analysis and interpretation: All authors.

Drafting of the manuscript: Murai, Fernandes, Gualano, Pereira.

Critical revision of the manuscript for important intellectual content: AW authors.

Statistical analysis: Murai, Fernandes, Pinto, Reis, Gualano, Pereira.

Obtained funding: Gualano, Pereira.

Supervision: Gualano, Pereira.

Administrative, technical, or material support: Sales, Antonangelo, Caparbo.

Conflict of Interest Disclosures: The authors have nothing to disclose.

Funding/Support: This study was supported by Sao Paulo Research Foundation

(FAPESP) (grants 20/05752-4; 19/24782-4; 20/11102-2; 16/00006-7; 17/13552-2;

15/26937-4; 19/18039-7) and Conselho Nacional de Desenvolvimento Cientifico e

Tecnologico (305556/2017-7).

Data Sharing Statement: See Supplement 3.

Additional Contributions: The authors are thankful to Dr. Monica Pinheiro and Dr.

Roberta Costa for the assistance at Ibirapuera Field Hospital; Dr. Rogerio Ruscitto do

Prado for conducting statistical analyses; Mayara Diniz Santos for the technical support;

all the staff members from both centers; all the patients who participated in this study.

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