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RESEARCH ARTICLE

High-dose vitamin D versus placebo to

prevent complications in COVID-19 patients:

Multicentre randomized controlled clinical

trial

Javier MarianiID 1,2*, Laura AntoniettiID

1,2, Carlos Tajer1,2, León Ferder3, Felipe InserraID 3,

Milagro Sanchez Cunto4, Diego Brosio5, Fernando Ross6, Marcelo Zylberman7, Daniel

Emilio López8, Cecilia Luna Hisano9, Sebastián Maristany Batisda1, Gabriela Pace10,

Adrián Salvatore11, Jimena Fernanda Hogrefe12, Marcela Turela13, Andrés Gaido14,

Beatriz Rodera15, Elizabeth Banega16, Marı́a Eugenia Iglesias17, Mariela Rzepeski18, Juan

Manuel Gomez PortilloID 19, Magalı́ Bertelli4, Andrés Vilela6, Leandro Heffner7, Verónica

Laura Annetta5, Lucila Moracho4, Maximiliano Carmona11, Graciela Melito3, Marı́a

José Martı́nez1, Gloria Luna1, Natalia Vensentini1, Walter Manucha20

1 Hospital de Alta Complejidad en Red El Cruce—Néstor Kirchner, Florencio Varela, Buenos Aires,

Argentina, 2 Universidad Nacional Arturo Jauretche, Florencio Varela, Buenos Aires, Argentina,

3 Maimónides University, Ciudad Autónoma de Buenos Aires, Buenos Aires, Argentina, 4 Hospital de

Infecciosas Francisco Javier Muñiz, Ciudad Autónoma de Buenos Aires, Buenos Aires, Argentina, 5 Hospital

General de Agudos Dr. Enrique Tornú, Ciudad Autónoma de Buenos Aires, Buenos Aires, Argentina,

6 Clı́nica Santa Isabel, Ciudad Autónoma de Buenos Aires, Buenos Aires, Argentina, 7 Hospital General de

Agudos Dr. Cosme Argerich, Ciudad Autónoma de Buenos Aires, Argentina, 8 Hospital General de Agudos

Dr. Teodoro Álvarez, Ciudad Autónoma de Buenos Aires, Buenos Aires, Argentina, 9 Hospital General de

Agudos Dr. Juan A. Fernández, Ciudad Autónoma de Buenos Aires, Buenos Aires, Argentina, 10 Hospital

General de Agudos Parmenio Piñero, Ciudad Autónoma de Buenos Aires, Buenos Aires, Argentina,

11 Hospital Luis Lagomaggiore, Mendoza, Argentina, 12 Sanatorio Güemes, Ciudad Autónoma de Buenos

Aires, Buenos Aires, Argentina, 13 Hospital Regional Antonio J. Scaravelli, Tunuyán, Mendoza, Argentina,

14 Sanatorio Allende, Sede Cerro, Córdoba, Argentina, 15 Hospital Zonal General de Agudos “Dr. Isidoro

Iriarte”, Quilmes, Buenos Aires, Argentina, 16 Hospital Interzonal Especializado en Agudos y Crónicos,

Neuropsiquiátrico Dr. Alejandro Korn, Melchor Romero, Buenos Aires, Argentina, 17 Sanatorio Allende

Nueva Córdoba, Córdoba, Argentina, 18 Hospital Modular de Florencio Varela, Florencio Varela, Buenos

Aires, Argentina, 19 Hospital El Carmen, Godoy Cruz, Mendoza, Argentina, 20 Consejo Nacional de

Investigaciones Cientı́ficas y Técnicas, Universidad Nacional de Cuyo, Instituto de Medicina y Biologı́a

Experimental de Cuyo (IMBECU), Mendoza, Argentina

* [email protected]

Abstract

Background

The role of oral vitamin D3 supplementation for hospitalized patients with COVID-19 remains

to be determined. The study was aimed to evaluate whether vitamin D3 supplementation

could prevent respiratory worsening among hospitalized patients with COVID-19.

Methods and findings

We designed a multicentre, randomized, double-blind, sequential, placebo-controlled clini-

cal trial. The study was conducted in 17 second and third level hospitals, located in four

provinces of Argentina, from 14 August 2020 to 22 June 2021. We enrolled 218 adult

PLOS ONE

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OPEN ACCESS

Citation: Mariani J, Antonietti L, Tajer C, Ferder L,

Inserra F, Sanchez Cunto M, et al. (2022) High-

dose vitamin D versus placebo to prevent

complications in COVID-19 patients: Multicentre

randomized controlled clinical trial. PLoS ONE

17(5): e0267918. https://doi.org/10.1371/journal.

pone.0267918

Editor: Alessandro Putzu, Geneva University

Hospitals, SWITZERLAND

Received: December 29, 2021

Accepted: April 17, 2022

Published: May 27, 2022

Peer Review History: PLOS recognizes the

benefits of transparency in the peer review

process; therefore, we enable the publication of

all of the content of peer review and author

responses alongside final, published articles. The

editorial history of this article is available here:

https://doi.org/10.1371/journal.pone.0267918

Copyright: © 2022 Mariani et al. This is an open

access article distributed under the terms of the

Creative Commons Attribution License, which

permits unrestricted use, distribution, and

reproduction in any medium, provided the original

author and source are credited.

Data Availability Statement: All relevant data are

within the manuscript and it’s Supporting

Information files.

patients, hospitalized in general wards with SARS-CoV-2 confirmed infection, mild-to-mod-

erate COVID-19 and risk factors for disease progression. Participants were randomized to a

single oral dose of 500 000 IU of vitamin D3 or matching placebo. Randomization ratio was

1:1, with permuted blocks and stratified for study site, diabetes and age (�60 vs >60 years).

The primary outcome was the change in the respiratory Sepsis related Organ Failure

Assessment score between baseline and the highest value recorded up to day 7. Secondary

outcomes included the length of hospital stay; intensive care unit admission; and in-hospital

mortality. Overall, 115 participants were assigned to vitamin D3 and 105 to placebo (mean

[SD] age, 59.1 [10.7] years; 103 [47.2%] women). There were no significant differences in

the primary outcome between groups (median [IQR] 0.0 [0.0–1.0] vs 0.0 [0.0–1.0], for vita-

min D3 and placebo, respectively; p = 0.925). Median [IQR] length of hospital stay was not

significantly different between vitamin D3 group (6.0 [4.0–9.0] days) and placebo group (6.0

[4.0–10.0] days; p = 0.632). There were no significant differences for intensive care unit

admissions (7.8% vs 10.7%; RR 0.73; 95% CI 0.32 to 1.70; p = 0.622), or in-hospital mortal-

ity (4.3% vs 1.9%; RR 2.24; 95% CI 0.44 to 11.29; p = 0.451). There were no significant dif-

ferences in serious adverse events (vitamin D3 = 14.8%, placebo = 11.7%).

Conclusions

Among hospitalized patients with mild-to-moderate COVID-19 and risk factors, a single high

oral dose of vitamin D3 as compared with placebo, did not prevent the respiratory

worsening.

Trial registration

ClincicalTrials.gov Identifier: NCT04411446.

Introduction

Vitamin D3 supplementation has been proposed as a potential therapeutic strategy among

patients with coronavirus disease 2019 (COVID-19) [1–3]. Effects of vitamin D that could

favourably affect the outcomes of patients with infectious diseases include immunomodulatory

and anti-inflammatory actions [4–6]. Acute respiratory disease syndrome is the main cause of

death among hospitalized patients with COVID-19, and pro-inflammatory cytokines play a

central pathogenic role [7, 8]. Vitamin D reduces pro-inflammatory cytokines, increases those

with anti-inflammatory actions, and also upregulates angiotensin-converting enzyme 2 recep-

tor, which is the surface receptor for the entry of severe acute respiratory syndrome coronavi-

rus 2 (SARS-CoV-2) to the alveolar epithelial cells [1, 4–6, 9, 10]. These actions could

potentially improve clinical outcomes of patients with COVID-19 pneumonia.

Furthermore, epidemiological studies have suggested a relationship between low vitamin D

levels and COVID-19 risk and adverse COVID-19 outcomes, and an open-label pilot clinical

trial suggested that supplementation with calcidiol significantly reduced the need for intensive

care unit admissions [11–15]. However, a randomized clinical trial involving hospitalized

patients with moderate to severe COVID-19 showed no differences in length of hospital stay

between oral supplementation with vitamin D3 and placebo [16]. Therefore, the evidence sup-

porting the role of vitamin D supplementation to treat patients with COVID-19 remains

inconclusive, particularly among patients with mild to moderate COVID-19 [17–20].

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Funding: This study was supported by the National

Agency for the Promotion of Research,

Technological Development and Innovation (grant

FONCyT IP COVID-19-931). Vitamin D3 and

placebo were donated by Raffo S.A., an argentinian

pharmaceutical company. The funders had no role

in the design and conduct of the study; collection,

management, analysis, and interpretation of the

data; preparation, review, or approval of the

manuscript; and decision to submit the manuscript

for publication.

Competing interests: The authors have declared

that no competing interests exist. Raffo S.A.

provided support in the form of vitamin D3 and

placebo capsules. This does not alter our

adherence to PLOS ONE policies on sharing data

and materials. There are no patents, products in

development or marketed products associated with

this research to declare.

The objective of this randomized, double-blind, placebo-controlled clinical trial was to eval-

uate whether vitamin D3 supplementation, given as a single high dose, could prevent respira-

tory worsening among hospitalized patients with mild-to-moderate COVID-19 and risk

factors for disease progression.

Materials and methods

Study design

The CholecAlcifeRol to improvE the outcomes of patients with COVID-19 (CARED) trial was

a multicentre, randomized, double-blind, sequential, placebo-controlled trial, designed by

independent investigators and supported by the National Agency for the Promotion of

Research, Technological Development and Innovation. The study was conducted in 17 hospi-

tals located in four provinces of Argentina, and the local ethics committees of the participating

institutions approved the protocol. Written informed consent was obtained from all partici-

pants. The study was conducted in compliance with local regulations on research on human

subjects, the Declaration of Helsinki and Good Clinical Practice guidelines. The study is regis-

tered in ClinicalTrials.gov (Identifier number NCT04411446). Study protocol has been pub-

lished elsewhere and is available in S1 File [21]. An independent Data and Safety Monitoring

Board (DSMB) monitored the trial. The Consolidated Standards for Reporting Trials (CON-

SORT) statement was followed to report the study results [22].

The sequential design consisted in an adaptative design with two stages. In the first stage,

the study aimed to assess the effects of vitamin D on respiratory Sepsis related Organ Failure

Assessment (rSOFA), and the second stage aimed to evaluate the effects of vitamin D on clini-

cal events. The protocol specified that the decision to proceed to the second stage would be

made conditioned by the results of the primary endpoint analysis of the first stage.

Participants

The participants were adults aged 18 or older patients and either gender, who had been admitted

to general wards in the last 24 hours, with SARS-CoV-2 confirmed infection by reverse transcrip-

tase–polymerase chain reaction, an expected hospitalization for at least 24 hours, oxygen satura-

tion�90% (measured by pulse oximetry) breathing ambient air, and at least one of the following

conditions: age 45 or older or hypertension, diabetes, chronic obstructive pulmonary disease or

asthma (at least moderate), cardiovascular disease (history of myocardial infarction, percutaneous

transluminal coronary angioplasty, coronary artery bypass grafting or valve replacement surgery)

or body mass index�30 (S1 File). Age 45 or older was selected as inclusion criterion to ensure a

baseline risk of respiratory worsening that allow to detect a therapeutic effect of the treatment,

and to preserve the power of the study. Obesity was added as risk condition on October 13, 2020,

since it was recognised as risk factor after the study begun. Main exclusion criteria were�72

hours since admission, women in childbearing age, requirement for>5 litres/minute of oxygen

or mechanical ventilation, chronic kidney disease requiring haemodialysis or chronic liver failure,

chronic supplementation with pharmacological vitamin D, treatment with anticonvulsants, sar-

coidosis, malabsorption syndrome, known hypercalcemia, life expectancy<6 months, allergy to

study medication, or any condition at discretion of investigator impeding to understand the

study and give informed consent (S1 File).

Randomization and intervention

After giving informed consent, patients were randomly assigned in a 1:1 ratio to receive vita-

min D3 (cholecalciferol) or matching placebo, using an interactive web response system with

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permuted blocks of size 16 and 24. Randomization was stratified by study site, diabetes (yes vs

no) and age (�60 vs>60 years). Castor1, and electronic data capture plataform that has

online randomization capability, was used for randomization and data collection (https://

www.castoredc.com).

Study interventions consisted in a single oral dose of 500 000 IU of vitamin D3 soft gel cap-

sules (5 capsules of 100 000 IU) or matching placebo, given as soon as possible after

randomization.

The study medication was packaged, labelled and shipped to the research sites by pharma-

cists from the Faculty of Pharmacy of Maimonides University. The pharmacy staff had no

other role in the study, nor did they have contact with the sites.

Outcomes and follow-up

Follow-up was limited to hospitalization. During the first seven days blood pressure, heart

rate, pulse oximetry (SpO2), temperature, inspired fraction of oxygen (FiO2), respiratory rate,

and clinical and adverse events were recorded. In the cases that remained hospitalized for

more than seven days, clinical and adverse events were recorded from day 8 until day 30, the

discharge or death, whichever occurred first.

The primary outcome was the change in the rSOFA between baseline and the highest

rSOFA recorded up to day 7. The rSOFA was calculated by using the SpO2 instead the partial

pressure of oxygen in arterial blood (PaO2), since was expected that most patients would not

have arterial blood draws during hospitalization [23–25]. The rSOFA score was calculated

with participant breathing room air, however, for participants with oxygen supplementation

requirement and for whom treating physician judged inapproppriate to temporary interrupt, a

guide for FiO2 estimation was provided to investigators (S2 File). Values of ratios SpO2/FiO2

for rSOFA calculations were as follows: > = 400, rSOFA 0; <400 and> = 300, rSOFA 1;<300

and> = 200, rSOFA 2;<200 and> = 100, rSOFA 3; <100, rSOFA 4.

Secondary outcomes included the change in SpO2 between baseline and the lower value

recorded during the first seven days; desaturation, defined as SpO2� 90%; the combined end-

point of oxygen supplementation >40%, non-invasive mechanical ventilation or invasive

mechanical ventilation (this was the primary outcome of the second stage in the case the study

proceed); the change in the quick SOFA between baseline and the highest value recorded dur-

ing the first 7 days [26]; the requirement of invasive mechanical ventilation; the intensive care

unit admission (ICU); the length of hospital stay; the ICU length of stay; acute kidney injury;

and the in-hospital mortality.

Serious adverse events were defined as any occurrence in a participant that caused death,

was life-threatening, prolonged the hospitalization, caused significant or persistent disability

and/or was judged by investigators to represent a significant risk for participant.

A sample of 16 participants from two study sites had blood samples draws for measurement

of serum 25-hydroxyvitamin D (25-OH VitD), at baseline and after 3 to 7 days after treatment.

Serum 25-OH VitD levels were determined quantitatively by chemiluminescence immunoas-

say in a central laboratory (A98856, Access 25(OH) Vitamin D Total, Beckman Coulter Inc.,

USA) [27].

Statistical analysis

For the first stage, it was estimated that 168 patients would give the trial 80% power to detect a

between study groups difference of one point in the change of rSOFA, assuming a standard

deviation (SD) of 2, and a type I error of 5%. The sample size was increased to 200 patients to

account for non-adherence with the protocol.

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Analyses were conducted according to the intention to treat principle.

Continuous data are expressed as means and SD in cases where normal distribution held,

and medians and interquartile ranges otherwise. Categorical data are presented as frequencies

and percentages. To compare continuous variables, the Student’s T-test or the Mann-Whitney

U test, as appropriate, was used. Normality assumption was assessed using histograms and

Shapiro-Wilk’s test. Categorical variables were compared using Pearson’s Chi2 test or Exact

Fisher’s test, as appropriate. Continuous outcomes are presented as differences in medians

with the corresponding 95% confidence intervals (95% CI). Differences in medians and the

confidence intervals for these differences were generated using smoothed bootstrap with 5000

replicates. Categorical outcomes are presented as risk ratios and 95% CIs. For primary out-

come, the Wilcoxon-Mann-Whitney odds (WMWOdds) with the corresponding 95% CIs was

computed [28].

Pre-specified subgroups included age (�60 vs<60 years), gender, diabetes, hypertension,

cardiovascular disease, body mass index (>30 vs�30) and smoking status (current vs former

or never). Subgroup analyses were carried out using ordinal regression models with an interac-

tion term of the subgroup indicator variable by treatment.

For primary outcome, a sensitivity analysis using ordinal regression models was carried

out, adjusting the estimated treatment effects for stratification variables (site, diabetes and

age). Also, a post-hoc adjusted analysis using a ordinal regression model to account for imbal-

ances in COPD and asthma distribution was carried out.

All tests are 2-sided and a p value <0.05 was considered as statistically significant.

Analyses were conducted using R version 4.1.0 (R: A language and environment for statisti-

cal computing. R Foundation for Statistical Computing, Vienna, Austria).

Termination of the trial

As prespecified in the protocol, a blinded analysis was carried out after the recruitment of the

first 200 participants, the estimated sample size for the initial stage of the study. After a revision

of the results, the Steering Committee decided to stop the recruitment and terminate the trial

on 7th July 2021. This decision was based on that the differences between groups, either on the

primary outcome (i.e., the change in rSOFA) and the secondary outcomes, did not meet the

prespecified criteria to proceed to the second stage (the minimum difference considered in the

protocol was 0.3 points in rSOFA -S1 File). The decision was communicated thereafter to the

DSMB and to the local investigators.

Ethics

The Ethics Committee of the Hospital El Cruce (Comité de Ética en Investigación Hospital de

Alta Complejidad El Cruce) approved the study on 23 June 2020 (reference 36/2020). The

local ethics committees of the participating institutions approved the study protocol before the

start of the trial in each sites.

Results

Two hundred eighteen participants were included in the study between August 2020 and June

2021, at 17 research sites located in four provinces of Argentina (S2 File). One hundred fifteen

and one hundred three patients were randomly assigned to vitamin D3 and to placebo, respec-

tively. One patient with SpO2<90% at admission was erroneously recruited and randomly

assigned to vitamin D3 group, this participant was included in final analysis (Fig 1).

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Baseline characteristics

Mean age was 59.1 (10.6) years and 103 (47.2%) patients were women. Risk factors included

diabetes in 58 (26.6%) patients, hypertension in 94 (43.1%) patients, obesity in 87 (39.9%),

chronic respiratory disease in 26 (11.9%) patients, and cardiovascular disease in 10 (4.6%).

Median time from onset of the symptoms to admission was 7.0 (IQR 5.0 to 10.0) days, 194

(89.0%) patients had pneumonia, mean SpO2 was 95.3% (2.0%), 47 (21.6%) patients required

oxygen supplementation at enrolment (Table 1). Median time from hospital admission to ran-

domization was 1.0 (IQR 1.0 to 2.0) days. There were no significant differences between treat-

ment groups in baseline characteristics (Table 1).

Fig 1. Flow of the patients in the CARED study.

https://doi.org/10.1371/journal.pone.0267918.g001

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Vitamin D supplementation

Study medications were adequately tolerated and there were no reports of immediate adverse

reactions after study capsules intake. A subset of 16 patients had 25-OH VitD levels measured

at baseline and after study treatment (median 6 [IQR 3.4 to 6.0] days). Baseline measurements,

were 32.5 ng/ml (IQR 27.2 to 44.2) and 30.5 ng/ml (IQR 22.5 to 36.2), for the vitamin D3 and

placebo group, respectively (p = 0.416). Post-treatment 25-OH VitD levels were 102.0 ng/ml

(IQR 85.2 to 132.2) and 30.0 ng/ml (IQR 27.5 to 31.0), for vitamin D3 and placebo group,

respectively (p = 0.001).

Table 1. Participants characteristics.

Variables Vitamin D3 Placebo

No. (%) with data 115 (100) 103 (100)

Time from admission to randomizationa, days 1.0 (1.0–2.0) 2.0 (1.0–2.0)

Age, mean (SD), y 59.8 (10.7) 58.3 (10.6)

Women, No. (%) 51 (44.3) 52 (50.5)

Body mass indexa 28.4 (25.8–32.8) 27.7 (25.6–31.6)

Hypertension, No. (%) 47 (40.9) 47 (45.6)

Diabetes, No. (%) 32 (27.8) 26 (25.2)

Smoking, No. (%)

Never 80 (69.6) 74 (71.8)

Former 30 (26.1) 26 (25.2)

Current 5 (4.3) 3 (2.9)

Asthma or Chronic obstructive pulmonary disease, No. (%) 17 (14.8) 9 (8.7)

Cardiovascular disease, No. (%) 6 (5.2) 4 (3.9)

Hypothyroidism, No. (%) 14 (12.2) 11 (10.7)

Neoplasm, No. (%) 0 (0.0) 2 (1.9)

COVID-19 symptoms

Dyspnea, No. (%) 55 (47.8) 45 (43.7)

Fever, No. (%) 80 (69.6) 68 (66.0)

Symptoms onset to admissiona, days 7.0 (5.0–10.0) 8.0 (5.5–10.0)

Anosmia, No. (%) 38 (33.0) 34 (33.0)

Pneumonia, No. (%) 105 (91.3) 89 (86.4)

Diarrhea, No. (%) 29 (25.2) 23 (22.3)

Myalgia, No. (%) 65 (56.5) 42 (40.8)

Physical examination

Heart ratea, beats/min 78.0 (72.0–90.0) 79.0 (70.5–90.0)

Respiratory ratea, breaths/min 18.0 (18.0–20.0) 18.0 (18.0–20.0)

Pulse oximetrya, % 95.0 (94.0–97.0) 96.0 (94.0–97.0)

Oxygen supplementation, No. (%) 27 (23.5) 20 (19.4)

Laboratory values

White cell counta, /mm3 5725 (4775–7522) 5950 (4800–7900)

Calciuma, mg/dL 8.8 (8.5–9.0) 8.7 (8.5–8.9)

Creatinine clearancea, mg/ml/1.73 m2 86.1 (73.2–102.4) 85.6 (70.6–111.1)

25-hydroxyvitamin Vitamin Dab, ng/mL 32.5 (27.2–44.2) 30.5 (22.5–36.2)

Abbreviations: IQR, interquartile range, COVID-19, coronavirus disease 2019.

SI conversion factors: to convert calcium to mmol/L, multiply by 0.25; 25-hydroxyvitamin vitamin D to nmol/L,

multiply by 2.496. aMedian (IQR). b16 participants with data.

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Primary outcome

A change in the rSOFA up to day 7 was recorded in 65 participants (29.8%), in 64 patients

increased in at least one point, and in one patient decreased one point. There were no signifi-

cant differences between groups in the distribution of changes in rSOFA (Fig 2A). Among par-

ticipants in vitamin D3 group, 0.9%, 70.4%, 13.0%, 4.3%, 8.7% and 2.6% had a change in

rSOFA of -1, 0, 1, 2, 3 and 4 points, respectively; theses percentages for participants in the pla-

cebo group were 0.0%, 69.9%, 15.5%, 5.8%, 4.9%, 3.9%. The median (IQR) of the change in the

rSOFA was 0.0 (0.0 to 1.0) points in the vitamin D3 group and 0.0 (0.0 to 0.0) in the placebo

group (between-group difference 0.00; 95% CI -0.18 to 0.15; p = 0.825) (WMWOdds 0.97; 95%

CI 0.76–1.24) (Table 2).

Secondary outcomes

The median (IQR) of the change in SpO2 between baseline and the lowest value recorded dur-

ing the first 7 days was -1.0% (-3.0 to 0.0) and -1% (-4.0 to 0.0) among participants in the vita-

min D3 group and placebo group, respectively (p = 0.952) (Fig 2B). Median length of hospital

stay was 6.0 (IQR 4.0 to 9.0) days and 6.0 (IQR 4.0 to 10.0) days in the vitamin D3 and placebo

groups, respectively (p = 0.614).

Overall, 20 (9.2%) patients were admitted to the ICU and 7 (3.2%) died, without significant

differences between groups (Table 2). There were no significant differences between groups in

other secondary outcomes (Table 2).

Subgroups

Results for the primary outcome were consistent across all prespecified subgroups. There were

no significant interactions between subgroup indicators and treatment effects (Fig 3). These

analyses should be carefully interpreted since the number of participants in each subgroup is

small.

Sensitivity analysis

The analysis adjusting the estimated effects of treatment for stratification variables gave similar

results to main analysis (OR 0.96; 95% CI 0.70 to 1.31; p = 0.805).

The results of a post-hoc analysis adjusting for the imbalance in the distribution of COPD

or asthma between groups, were similar to main results (OR 0.99; 95% CI 0.69 to 1.41;

p = 0.950).

Safety

Overall, 45 serious adverse events among 29 participants were reported. There were no signifi-

cant differences between groups in either the occurrence of at least one serious adverse event

or in the incidence of specific events by organ (Table 3).

Discussion

The results of the present study show that a single, high, oral dose of vitamin D3 among a non-

selected population of patients with mild-to-moderate COVID-19 did not prevent the respira-

tory worsening. Also, there were no significant effects on the length of hospital stay or other

outcomes. Furthermore, overall results were consistent across all pre-specified subgroups.

Previous studies have suggested a role of vitamin D supplementation for the prevention of

acute respiratory disease, particularly among individuals with low serum 25-OH VitD levels

[29–31]. Several mechanisms have been proposed for the potential beneficial effects of vitamin

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Placebo Vitamin D3

Baseline Highest rSOFA Baseline Highest rSOFA

0

25

50

75

100

Pa rt

ic ip

an ts

(% ) rSOFA

0

1

2

3

4

A

85

90

95

100

Baseline Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Visit

Sp O

2 ( %

, [ m

ed ia

n, IQ

R ])

Group Placebo

Vitamin D3

B

Fig 2. Changes in rSOFA scale from baseline to wort value recorded (A), and SpO2 during first week (B).

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D among COVID-19 patients, including the modulation of immune response [1, 7, 9, 32]. Fur-

thermore, previous studies have shown that low vitamin D levels are associated with higher

SARS-Cov-2 infection risk, severity and mortality [11–13, 15]. Also, an open-label randomized

controlled trial has found that the supplementation with oral calcifediol (0.532 mg at admis-

sion, 0.266 on day 3 and 7, and 0.266 weekly until discharge) could improve clinical outcomes

among hospitalized patients [14]. However, a double-blind trial among moderate to severe

COVID-19 patients, showed that a single oral dose supplementation with 200 000 IU of vita-

min D3 did not reduce the length of hospital stay [16]. The present study is in line with these

results and extends them to mild-to-moderate COVID-19 patients, and to other relevant out-

comes (i.e., the degree of respiratory worsening).

The study used a single dose of 500 000 IU of oral vitamin D3 since it was previously dem-

onstrated that this scheme rapidly increases plasma levels of 25-OH VitD, and that achieved

levels are maintained for at least 4 weeks, covering the period of highest risk for respiratory

worsening, with an adequate security profile [33]. Although, an increase in 25-OH VitD

plasma levels was achieved, there were no effects of treatment on the study outcomes. These

results are consistent with another trial showing that there were no significant effects of vita-

min D3 supplementation despite an increase in plasma 25-OH VitD, neither in all cohort nor

in 25-OH VitD deficient participants [16]. It has been hypothesized that a supplementation

with a single high dose of vitamin D, although it serves to raise plasma levels of 25-OH VitD,

does not improve the immune response, and that chronic supplementation, with daily or

weekly doses of vitamin D would produce better clinical results instead [30, 34]. Perhaps, the

stage of the disease at hospial admission in the present study was too late for treatment to

express benefial effects, since vitamin D needs several days to induce the mechanisms immune,

Table 2. Study outcomes.

Outcomes Vitamin D3 (n = 115) Placebo (n = 103) Between-group difference (95% CI)ac P

Median (IQR) Median (IQR)

Change in rSOFA from baselineb 0.0 (0.0–1.0) 0.0 (0.0–1.0) 0.00 (-0.18 to 0.15) 0.825

Change in SpO2, % -1.0 (-3.0–0.0) -1.0 (-4.0–0.0) 0.00 (-0.87 to 1.02) 0.952

Change in quick SOFA 0.0 (0.0–1.0) 0.0 (0.0–1.0) 0.00 (-0.25 to 0.30) 0.990

Length of stay, days 6.0 (4.0–9.0) 6.0 (4.0–10.0) 0.00 (-1.84 to 0.95) 0.632

ICU length of stay, daysc 9.0 (5.0–11.1) 9.0 (4.0–10.8) 0.00 (-8.31 to 9.71) 0.909

No. with events (%) No. with events (%) Risk ratio (95% CI)

Desaturation 22 (19.1) 14 (13.6) 1.40 (0.76 to 2.60) 0.359

Oxygen >40%, NIV or MV 17 (14.8) 15 (14.6) 1.02 (0.53 to 1.93) 1.00

Mechanical ventilation 5 (4.3) 6 (5.8) 0.75 (0.23 to 2.37) 0.851

Acute kidney injury 2 (1.7) 2 (1.9) 0.90 (0.12 to 6.24) 1.00

Myocardial infarction 0 (0.0) 0 (0.0) - -

Stroke 0 (0.0) 0 (0.0) - -

Pulmonary Embolism 0 (0.0) 0 (0.) - -

ICU admission 9 (7.8) 11 (10.7) 0.73 (0.32 to 1.70) 0.622

In-hospital Death 5 (4.3) 2 (1.9) 2.24 (0.44 to 11.29) 0.451

Abbreviations: rSOFA, Sepsis related Organ Failure Assessment; SpO2, pulse oximetry; ICU, Intensive Care Unit; NIV, non-invasive ventilation; MV, mechanical

ventilation. aBetween-group differences are differences in medians and 95% CIs. bPrimary outcome. cDifferences in medians with their corresponding 95% CIs were obtained using smoothed bootstrap. dAmong 20 patients that were admitted to ICU.

https://doi.org/10.1371/journal.pone.0267918.t002

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Subgroup

Age

>60 years

<=60 years

Gender

Males

Females

Diabetes

Yes

No

Body Mass Index

>30

<=30

Cardiovascular disease

Yes

No

Smoking

Current

Former/Never

Overall

OR (95% CI)

0.98 ( 0.64 to 1.5 )

0.95 ( 0.65 to 1.4 )

0.98 ( 0.66 to 1.46 )

0.97 ( 0.64 to 1.47 )

0.79 ( 0.46 to 1.37 )

1.05 ( 0.75 to 1.47 )

1.02 ( 0.65 to 1.61 )

0.97 ( 0.67 to 1.4 )

0.41 ( 0.1 to 1.75 )

1.01 ( 0.75 to 1.35 )

0.85 ( 0.15 to 4.91 )

0.98 ( 0.73 to 1.31 )

0.98 ( 0.74 to 1.3 )

P for interaction

0.922

0.995

0.430

0.890

0.408

0.929

0.896

00.51 2 5 OR

Fig 3. Subgroup analyses.

https://doi.org/10.1371/journal.pone.0267918.g003

PLOS ONE Vitamin D to treat COVID-19

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metabolics, antioxidant, endocrine leading to antiviral effects [20]. However, well-designed

clinical trials are necessary to evaluate the effects of different regimens of vitamin D

supplementation.

Additionally, it is worthwhile that the baseline 25-OH VitD plasma levels were higher than

in other studies [11, 16, 17]; these differences could be due to differences in populations stud-

ied and to the fact that in our study the 25-OH VitD measurements were conducted mainly

during the summer and early autumn, but the causes remain speculative [35]. Although results

of vitamin D3 supplementation for treatment of patients with COVID-19 could be theoreti-

cally modified by the serum vitamin D status, with deficient populations obtaining the most

benefits, this remains speculative. Moreover, two clinical trial that included critically ill

patients -most of them with infectious diseases- with vitamin D defficiency (�20 ng/mL) and

randomized them to high vitamin D3 doses or placebo showed no beneficial effect of the treat-

ment [36, 37].

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

centre inclusion of participants with representation of a broad sociocultural background from

the country, and the high adherence to study protocol and follow-up.

The present study has several limitations. A single high dose of vitamin D3 was chosen to

ensure rapid and persistent high plasma levels of 25-OH VitD, it is possible that multiple dos-

ing regimens could have different biological effects [30]. The primary outcome assessed the

effects of the treatment on the respiratory system, precluding to detect other potentially rele-

vant effects. The follow-up was limited to hospital stay, longer follow-up would be necessary to

detect relevant effects on recovery after discharge. Also, the study was underpowered to detect

differences between groups on clinically important events (i.e., intensive care unit admission,

mechanical ventilation, mortality). Participants were admitted with a median of 7 days from

symptoms onset and most of them with established pneumonia; whether treatment earlier in

the course of disease could modify the subsequent clinical course has yet to be determined

[38]. In the present study, the measured serum 25-OH VitD levels among the participants with

blood samples were sufficient, whether different results would be obtained among a vitamin D

deficient population remains to be determined.

The SpO2/FiO2 ratio used as primary outcome have been validated as surrogate of PO2/

FiO2. Although validation studies of SpO2/FiO2 ratio did not included patients with COVID-

19, the absence of effects on other measures of respiratory worsening besides rSOFA, gives

reassurance to study results [24, 25, 39]. Since women of childbearing age were excluded from

the study our results are not generalizable to this population.

Table 3. Serious adverse events.

Vitamin D3 Placebo P

No. with data 115 103

At least one seriuos adverse event, No. (%) 17 (14.8) 12 (11.7) 0.631

Cardiovascular, No. (%) 6 (5.2) 4 (3.9) 0.884

Metabolic, No. (%) 3 (2.6) 2 (1.9) 1.00

Infectious, No. (%) 5 (4.3) 3 (2.9) 0.840

Respiratory, No. (%) 2 (1.7) 2 (1.9) 1.00

Hematologic, No. (%) 2 (1.7) 1 (1.0) 1.00

Gastrointestinal, No. (%) 7 (6.1) 5 (4.9) 0.920

Neurological, No. (%) 3 (1.4) 0 (0.0) 0.249

https://doi.org/10.1371/journal.pone.0267918.t003

PLOS ONE Vitamin D to treat COVID-19

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Conclusions

Supplementation with a single, high dose of vitamin D3 at admission to patients hospitalized

with mild-to-moderate COVID-19 did not prevent respiratory worsening as compared with

placebo.

Supporting information

S1 Checklist.

(DOC)

S1 File. Protocol of the study.

(PDF)

S2 File. Guide for FiO2 estimation for participants with oxygen supplementation.

(PDF)

S3 File. Sites, principal investigators and number of participants recruited in the study.

(PDF)

S4 File. Dataset of the primary analysis.

(XLSX)

Author Contributions

Conceptualization: Javier Mariani, Laura Antonietti, Carlos Tajer, León Ferder, Felipe

Inserra, Walter Manucha.

Data curation: Javier Mariani, Laura Antonietti, Milagro Sanchez Cunto, Fernando Ross,

Lucila Moracho.

Formal analysis: Javier Mariani.

Funding acquisition: Javier Mariani, Laura Antonietti, Carlos Tajer, León Ferder, Felipe

Inserra, Walter Manucha.

Investigation: Javier Mariani, Laura Antonietti, Carlos Tajer, León Ferder, Felipe Inserra,

Milagro Sanchez Cunto, Diego Brosio, Fernando Ross, Marcelo Zylberman, Daniel Emilio

López, Cecilia Luna Hisano, Sebastián Maristany Batisda, Gabriela Pace, Adrián Salvatore,

Jimena Fernanda Hogrefe, Marcela Turela, Andrés Gaido, Beatriz Rodera, Elizabeth

Banega, Marı́a Eugenia Iglesias, Mariela Rzepeski, Juan Manuel Gomez Portillo, Magalı́

Bertelli, Andrés Vilela, Leandro Heffner, Verónica Laura Annetta, Lucila Moracho, Maxi-

miliano Carmona, Graciela Melito, Marı́a José Martı́nez, Gloria Luna, Natalia Vensentini,

Walter Manucha.

Methodology: Javier Mariani, Laura Antonietti, Carlos Tajer, León Ferder, Felipe Inserra,

Graciela Melito, Natalia Vensentini, Walter Manucha.

Project administration: Javier Mariani, Laura Antonietti, Maximiliano Carmona, Natalia

Vensentini, Walter Manucha.

Resources: Javier Mariani, Laura Antonietti, León Ferder, Felipe Inserra, Maximiliano Car-

mona, Graciela Melito, Marı́a José Martı́nez, Natalia Vensentini, Walter Manucha.

Software: Javier Mariani.

Supervision: Javier Mariani, Laura Antonietti, Carlos Tajer, Maximiliano Carmona, Marı́a

José Martı́nez, Gloria Luna, Natalia Vensentini, Walter Manucha.

PLOS ONE Vitamin D to treat COVID-19

PLOS ONE | https://doi.org/10.1371/journal.pone.0267918 May 27, 2022 13 / 16

Validation: Javier Mariani, Laura Antonietti, Milagro Sanchez Cunto, Diego Brosio, Fernando

Ross, Marcelo Zylberman, Marı́a Eugenia Iglesias, Mariela Rzepeski, Juan Manuel Gomez

Portillo, Magalı́ Bertelli, Andrés Vilela, Leandro Heffner, Verónica Laura Annetta, Lucila

Moracho, Maximiliano Carmona, Graciela Melito, Marı́a José Martı́nez, Gloria Luna, Nata-

lia Vensentini, Walter Manucha.

Visualization: Javier Mariani, Laura Antonietti, Carlos Tajer, León Ferder, Felipe Inserra,

Milagro Sanchez Cunto, Diego Brosio, Fernando Ross, Marcelo Zylberman, Daniel Emilio

López, Cecilia Luna Hisano, Sebastián Maristany Batisda, Gabriela Pace, Adrián Salvatore,

Jimena Fernanda Hogrefe, Marcela Turela, Andrés Gaido, Beatriz Rodera, Elizabeth

Banega, Marı́a Eugenia Iglesias, Mariela Rzepeski, Juan Manuel Gomez Portillo, Magalı́

Bertelli, Andrés Vilela, Leandro Heffner, Verónica Laura Annetta, Lucila Moracho, Maxi-

miliano Carmona, Graciela Melito, Marı́a José Martı́nez, Gloria Luna, Natalia Vensentini,

Walter Manucha.

Writing – original draft: Javier Mariani, Laura Antonietti, Carlos Tajer, León Ferder, Felipe

Inserra, Milagro Sanchez Cunto, Diego Brosio, Fernando Ross, Marcelo Zylberman, Daniel

Emilio López, Cecilia Luna Hisano, Sebastián Maristany Batisda, Gabriela Pace, Adrián Sal-

vatore, Jimena Fernanda Hogrefe, Marcela Turela, Andrés Gaido, Beatriz Rodera, Elizabeth

Banega, Marı́a Eugenia Iglesias, Mariela Rzepeski, Juan Manuel Gomez Portillo, Magalı́

Bertelli, Andrés Vilela, Leandro Heffner, Verónica Laura Annetta, Lucila Moracho, Maxi-

miliano Carmona, Graciela Melito, Marı́a José Martı́nez, Gloria Luna, Natalia Vensentini,

Walter Manucha.

Writing – review & editing: Javier Mariani, Laura Antonietti, Carlos Tajer, León Ferder,

Felipe Inserra, Milagro Sanchez Cunto, Diego Brosio, Fernando Ross, Marcelo Zylberman,

Daniel Emilio López, Cecilia Luna Hisano, Sebastián Maristany Batisda, Gabriela Pace,

Adrián Salvatore, Jimena Fernanda Hogrefe, Marcela Turela, Andrés Gaido, Beatriz

Rodera, Elizabeth Banega, Marı́a Eugenia Iglesias, Mariela Rzepeski, Juan Manuel Gomez

Portillo, Magalı́ Bertelli, Andrés Vilela, Leandro Heffner, Verónica Laura Annetta, Lucila

Moracho, Maximiliano Carmona, Graciela Melito, Marı́a José Martı́nez, Gloria Luna, Nata-

lia Vensentini, Walter Manucha.

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