25-Hydroxyvitamin D Concentrations Are Lower in

Patients with Positive PCR for SARS-CoV-2

Antonio D’Avolio 1,* , Valeria Avataneo 1, Alessandra Manca 1, Jessica Cusato 1 ,

Amedeo De Nicolò 1, Renzo Lucchini 2, Franco Keller 2 and Marco Cantù 2

1 Laboratory of Clinical Pharmacology and Pharmacogenetics, Amedeo di Savoia Hospital,

Department of Medical Sciences, University of Turin, 10126 Turin, Italy; valeria.avataneo@unito.it (V.A.);

alessandra.manca@unito.it (A.M.); jessica.cusato@unito.it (J.C.); amedeo.denicolo@unito.it (A.D.N.)

2 Department of Laboratory Medicine EOLAB, Ente Ospedaliero Cantonale, 6500 Bellinzona, Switzerland,

Renzo.Lucchini@eoc.ch (R.L.); Franco.Keller@eoc.ch (F.K.); Marco.Cantu@eoc.ch (M.C.)

* Correspondence: antonio.davolio@unito.it; Tel.: +39-011-4393867; Fax: +39-011-4393996

Received: 20 April 2020; Accepted: 7 May 2020; Published: 9 May 2020

Abstract: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease

2019 (COVID-19), with a clinical outcome ranging from mild to severe, including death. To date,

it is unclear why some patients develop severe symptoms. Many authors have suggested the

involvement of vitamin D in reducing the risk of infections; thus, we retrospectively investigated the

25-hydroxyvitamin D (25(OH)D) concentrations in plasma obtained from a cohort of patients from

Switzerland. In this cohort, significantly lower 25(OH)D levels (p = 0.004) were found in PCR-positive

for SARS-CoV-2 (median value 11.1 ng/mL) patients compared with negative patients (24.6 ng/mL);
his was also confirmed by stratifying patients according to age >70 years. On the basis of this
preliminary observation, vitamin D supplementation might be a useful measure to reduce the risk of
infection. Randomized controlled trials and large population studies should be conducted to evaluate
these recommendations and to confirm our preliminary observation.

Keywords: vitamin D; SARS-CoV-2; concentrations; COVID-19; coronavirus; deficiency

1. Introduction

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019

(COVID-19), with clinical outcomes ranging from mild to severe, including death. To date, there is no specific recommended treatment, with COVID-19- and SARS-COV-2-a
ected patients targeted to

receive supportive care to help relieve symptoms.

However, only a fraction of infected people show clinical symptoms, and an even lower percentage

require medical attention [1,2]. To date, it is not yet known why some patients develop more

severe symptoms.

Recently, some articles have suggested the possible involvement of vitamin D in reducing the risk

of respiratory tract infections, especially in the influenza and COVID-19 context. Furthermore, the

role of vitamin D supplementation in reducing the risk of infection [3–6] is still under investigation,

however, no clinical evidence has been reported yet.

For these reasons, we retrospectively described the 25-hydroxyvitamin D (25(OH)D) plasma

concentrations in a cohort of patients from Switzerland.

Nutrients 2020, 12, 1359; doi:10.3390/nu12051359 www.mdpi.com/journal/nutrients

Nutrients 2020, 12, 1359 2 of 7

2. Methods

2.1. Data Collection

We retrospectively evaluated the repository data for patients who underwent a nasopharyngeal

swab PCR analysis for SARS-CoV-2 and a 25(OH)D measurement at “Ente Ospedaliero Cantonale”

(Canton of Tessin, Switzerland), during the period from 1 March to 14 April 2020.

According to the Swiss federal o ce for public health’s (Bundesamt für Gesundheit (BAG)) rules,

patients selected for the SARS-CoV-2 PCR analysis had to have symptoms of an acute airway disease

(e.g., cough, sore throat, breathing di culties), with or without fever, feeling of fever, muscle pain,

or sudden anosmia or ageusia.

The vitamin D analysis was required to be conducted within seven weeks of the SARS-CoV-2 PCR

result. As an additional control cohort, all patients with a 25(OH)D measurement during the same

period (1 March to 14 April) of 2019 were evaluated.

The duration of sunshine, expressed as total sun hours, into the analyzed period (1 March

to 14 April) were compared between 2019 and 2020. Data were provided by the federal o ce of

meteorology and climatology MeteoSwiss. Reported data refer to the Locarno-Monti measuring station.

2.2. Sample Processing for Vitamin D Quantitation

Sampleswere processedwithMassChrom® 25-OH-Vitamin D3/D2 in serum/plasma (Chromsystems,

Germany) on a MassStar liquid handler (Hamilton, Switzerland) according to the manual’s procedures.

The extracted samples analysis was carried out with a liquid chromatography coupled with a tandem

mass spectrometry (LC-MS/MS) instrument G6490A (Agilent Technologies, Santa Clara, CA, USA)

equipped with a 1290 Infinity LC Systems UHPLC.

2.3. PCR Analysis for COVID-19 Identification

Samples were collected using the Copan® FLOQSwabs® UTM® Nasopharyngeal Sample

Collection Kit composed of the Flexible Minitip Flock Swab + 3 mL UTM® Viral Transport Medium

(COPAN, Italy). An amount of 200 L of media was extracted with the MagPurix® Viral/Pathogen

Nucleic Acid Extraction Kit B using an Automated Nucleic Acid Purification System MagPurix 12s

(Zinexts, Taiwan) and amplified with a Sars-COV (COVID19) E-gene and RdRp Gene kit (TIB MOLBIOL,

Germany). Data acquisition was performed with an ABI 7500 Fast Real-Time PCR (Applied Biosystems,

Foster City, CA, USA).

2.4. Statistical Analysis

For the descriptive statistics, the continuous variables are summarized as the median (25th–75th

percentile, interquartile range (IQR)). The categorical variables are described as frequencies and

percentages. All data were assessed for normality using a Shapiro–Wilk test and the categorical data

were compared using Mann–Whitney or Kruskal–Wallis statistical tests.

Spearman’s rank correlation was utilized to determine the continuous data.

Statistical analyses were carried out using the SPSS software package, version 26.0 (IBM, Armonk,

NY, USA).

3. Results

The 2020 cohort of 107 total patients (male = 54.2%; median age = 73 years (IQR 63–81); median

25(OH)D = 22.0 ng/mL (IQR 8.9–30.5)) included 27 SARS-CoV-2 PCR-positive (male = 70.4%; median

age = 74 years (IQR 65–81) with median 25(OH)D = 11.1 ng/mL (IQR 8.2–21.0)), and 80 SARS-CoV-2

PCR-negative (male = 48.8%; median age = 73 years (IQR 61–82) with median 25(OH)D = 24.6 ng/mL

(IQR 8.9–30.5)) patients. The measurement of 25(OH)D was generally performed three days after the

molecular PCR test (overall median days away = 􀀀3.0 (IQR 􀀀7.0–0.0)), and a not statistically significant

Nutrients 2020, 12, 1359 3 of 7

di
erence in days away was found: 􀀀2.0 days (IQR 􀀀7.0–1.75) vs. 􀀀3.0 (IQR 􀀀6.0 to 􀀀1.0) (p = 0.119)

within the PCR-negative and PCR-positive patients, respectively.

As an additional control cohort, without a SARS-CoV-2 PCR test, all patients with at least one

measurement for 25(OH)D in the corresponding period (1 March to 14 April) of 2019 (before Covid-19

pandemic) were evaluated, with a total of 1377 patients (male = 45.3%; median age = 63 years

(IQR 46–76); median 25(OH)D = 24.6 ng/mL (IQR 16.2–33.0)). The total sun hours in the analyzed

period (1 March to 14 April) are 333.4 h and 349.4 h in 2019 and 2020, respectively, with a total increment

of 16 h in 2020 (+4.8%).

As depicted in Figure 1, we observed statistically significant (p = 0.004) lower 25(OH)D levels

(11.1 ng/mL) in patients positive for the SARS-CoV-2 PCR compared with the negative patients

(24.6 ng/mL). By comparing the 2020 and 2019 cohorts, we observed an even stronger statistically

significant di
erence (p < 0.001) in 25(OH)D levels in patients with a positive PCR for SARS-CoV-2

compared with the 2019 patients (24.6 ng/mL); however, no significant di
erence (p = 0.076) between

the 2019 and 2020 negative PCR cohorts was observed.

Nutrients 2020, 12, x FOR PEER REVIEW 4 of 7

Figure 1. 25-hydroxyvitamin D concentrations in the three evaluated groups (patients from 1 March

to 14 April of 2019 and 2020 with a negative PCR, and of 2020 with a positive PCR for SARS-CoV-2.

*: significant results.

Figure 1. 25-hydroxyvitamin D concentrations in the three evaluated groups (patients from 1 March

to 14 April of 2019 and 2020 with a negative PCR, and of 2020 with a positive PCR for SARS-CoV-2.

*: significant results.

As depicted in Figure 2A, when dividing the 2020 cohort according to gender and PCR result,

a non-statistically significant di
erence in vitamin D concentrations was found: 24.8 ng/mL (IQR

14.5–30.9) vs. 9.3 ng/mL (IQR 7.3–20.5) (p = 0.062) and 23.8 ng/mL (IQR 7.13–32.7) vs. 11.4 ng/mL

(IQR 8.9–23.6) (p = 0.131) within women (41 vs. 8) and men (39 vs. 19), respectively. Nevertheless,

vitamin D concentrations were significantly di
erent when comparing patients from the 2019 and 2020

PCR-positive cohorts, stratified by gender: 25.6 ng/mL (IQR 17.3–33.3) vs. 9.3 ng/mL (IQR 7.3–20.5)

(p = 0.019) in women (n = 753 vs. n = 8) and 22.9 ng/mL (IQR 14.7–33.1) vs. 11.4 ng/mL (IQR 8.9–23.6)

(p = 0.005) in men (n = 624 vs. n = 19).

Nutrients 2020, 12, 1359 4 of 7

Figure 1. 25-hydroxyvitamin D concentrations in the three evaluated groups (patients from 1 March

to 14 April of 2019 and 2020 with a negative PCR, and of 2020 with a positive PCR for SARS-CoV-2.

*: significant results.

(A)

Nutrients 2020, 12, x FOR PEER REVIEW 5 of 7

(B)

Figure 2. (A) 25-hydroxyvitamin D concentrations in the three evaluated groups divided by gender

(patients from 1 March to 14 April of 2019 and 2020 with a negative PCR, and of 2020 with a positive

PCR to SARS-CoV-2; (B) 25-hydroxyvitamin D concentrations in the three evaluated groups divided

by age (0–70 years vs. >70 years) (patients from 1 March to 14 April of 2019 and 2020 with a negative

PCR, and of 2020 with a positive PCR for SARS-CoV-2. *: significant results.

4. Discussion

The world is in the grip of the COVID-19 pandemic. Public health measures to reduce the risk

of infection and death, in addition to quarantines, are desperately needed. In this paper, we describe

for the first time that the 25(OH)D level is significantly lower in SARS-CoV-2 PCR-positive patients

than in PCR-negative patients (Figure 1).

Figure 2. (A) 25-hydroxyvitamin D concentrations in the three evaluated groups divided by gender

(patients from 1 March to 14 April of 2019 and 2020 with a negative PCR, and of 2020 with a positive

PCR to SARS-CoV-2; (B) 25-hydroxyvitamin D concentrations in the three evaluated groups divided by

age (0–70 years vs. >70 years) (patients from 1 March to 14 April of 2019 and 2020 with a negative PCR,

and of 2020 with a positive PCR for SARS-CoV-2. *: significant results.

Nutrients 2020, 12, 1359 5 of 7

As depicted in Figure 2B, when stratifying the 2020 patients by age (0–70 years and >70 years)

and PCR positivity, the vitamin D concentrations are not significantly di
erent (p = 0.277) among

the two groups (n = 37 vs. n = 9), with median values of 25.9 ng/mL (IQR 15.9–32.1) vs. 17.2 ng/mL

(IQR 11.7–31.6), respectively. Nevertheless, when considering only patients with age >70 years (n = 43

vs. n = 18), the vitamin D concentrations are significantly di
erent (p = 0.037), with median values

of 23.1 ng/mL (IQR 8.5–31.7) in PCR-negative patients vs. 9.3 ng/mL (IQR 8.1–19.9) in PCR-positive

patients. Moreover, when comparing patients enrolled in 2019 with the 2020 PCR-positive patients,

the vitamin D concentrations are even more significantly di
erent (p < 0.001): in patients with age

>70 years (n = 501 vs. n = 18), the median was 26.4 ng/mL (IQR 15.7–36.4) in the 2019 cohort vs.

9.3 ng/mL (IQR 8.1–19.9) in the 2020 cohort. The same di
erence was not observed when comparing

patients with an age lower than 70 years (n = 876 vs. n = 9), with median values of 23.9 ng/mL (IQR

16.4–31.6) vs. 17.2 ng/mL (IQR 11.7–31.6) (p = 0.287), respectively.

4. Discussion

The world is in the grip of the COVID-19 pandemic. Public health measures to reduce the risk of

infection and death, in addition to quarantines, are desperately needed. In this paper, we describe for

the first time that the 25(OH)D level is significantly lower in SARS-CoV-2 PCR-positive patients than

in PCR-negative patients (Figure 1).

Despite the relatively low numbers, this evidence is particularly strong, since the group of

PCR-negative patients had a risk of infection and symptoms of respiratory tract infections as indications

for the PCR testing. Since the risk of symptomatic upper respiratory tract infection is suggested to be

associated with low 25(OH)D levels [7], its concentration is expected to be quite low in PCR-negative

patients (partially confirmed by the trend of a di
erence with the 2019 cohort, p = 0.076), making this

control group even more stringent. Therefore, the significantly lower 25(OH)D concentrations in the

PCR-positive group could indicate that the risk of SARS-CoV-2 infection has a stronger relationship

with the 25(OH)D concentration, rather than other respiratory tract infections.

Nevertheless, no precise information regarding the clinical conditions of the PCR-negative patients

was available in this study, making further stratification impossible.

On the other hand, when stratifying the 2020 patients by gender and PCR positivity, the vitamin

D concentrations were di
erent, with a clear trend, but were not statistically significant, probably

due to the low number in each cluster. In fact, when we consider the 2019 patients, the vitamin D

concentrations were significantly di
erent for both the 2020 PCR-positive women (753 vs. 8) and men

(624 vs. 19), respectively (Figure 2A).

In our cohort, the 25(OH)D concentrations were significantly lower in patients with a positive

SARS-CoV-2 PCR assay when compared with the PCR-negative patients and patients from the 2019

cohort, when the age was >70 years (Figure 2B). There were no di
erences when we compared patients

with an age <70 years. These data may be important if we consider that age is a well-known predictor

of disease severity in COVID-19. Based on this evidence, we could hypothesize that higher 25(OH)D

levels (perhaps around 30 ng/mL as a possible target) could reduce the risk if severe disease in the

elderly (>70 years).

Our data have several possible biases: the data represented a relatively low number of patients from

a single hospital center, no clinical information was available about the severity of COVID-19 symptoms

for PCR-positive patients, no clinical information was available about symptoms in PCR-negative

patients (without a possible clinical stratification), etc. Moreover, other potential confounding variables

linking SARS-CoV-2 PCR positivity and lower vitamin D levels could be diet or possible vitamin D

supplementation (data not available). Finally, the PCR for SARS-CoV-2 and the 25(OH)D quantification

could be performed on di
erent days, but the median days away were close in both the PCR-positive

and PCR-negative groups, with no statistical di
erence between them. Therefore, the half-life of

25(OH)D (approximately 2–3 weeks) should not influence our results.

Nutrients 2020, 12, 1359 6 of 7

The exposure to the sun could be important. The total sun hours in the analyzed period (1 March

to 14 April) were 333.4 h and 349.4 h in 2019 and 2020, respectively. The di
erence in sunshine was

4.8% and it seems to be not significant. Eating behavior and the possible vitamin D supplementation

are more likely to be important factors to consider [3]. Another critical issue that we could consider

is the possible reverse causality, where patients with COVID-19 could have a drop in their 25(OH)D

levels due to the SARS-CoV-2 infection. However, with a relatively short disease, this issue can be

considered unlikely and no data are available. In conclusion, this study represents a preliminary

observation justified by several described mechanisms through which 25(OH)D can reduce the risk of

infections [3]. These mechanisms include the induction or transcription of cathelicidins and defensins

that can reduce viral replication rates and concentrations of pro-inflammatory cytokines responsible

for producing inflammation and injuring the lining of lungs, leading to pneumonia, as well as the

capability of vitamin D to increase the concentrations of anti-inflammatory cytokines. Several pieces

of evidence support the role of vitamin D in reducing the risk of COVID-19, including, as indicated

by other authors, that the outbreak occurred in winter, a time when 25(OH)D concentrations are low;

that the number of cases in the Southern Hemisphere near the end of summer is low; that vitamin

D deficiency has been found to contribute to acute respiratory distress syndrome [3]; and that case

fatality rates increase with age (>70 years) and with chronic disease comorbidity, both of which are

associated with a lower 25(OH)D concentration [3].

As suggested by Grant et al., it is recommended that people at risk of COVID-19 consider taking

10,000 IU/day of vitamin D3 for a few weeks to rapidly increase their 25(OH)D concentrations, followed

by 5000 IU/day to reduce the risk of infection. The goal should be to raise 25(OH)D concentrations

above 40–60 ng/mL (100–150 nmol/L) [3], or at least 30 ng/mL, considering our preliminary data.

It is probable that vitamin D3 supplementation would be useful in the treatment of COVID-19

infection, in preventing a more severe symptomatology and/or in reducing the presence of the virus in

the upper respiratory tract and making the patients less infectious (justifying negative PCR in people

with higher 25(OH)D). Randomized controlled trials and large population studies should be conducted

to evaluate these recommendations and to confirm our preliminary observations and hypothesis.

Author Contributions: Conceptualization, A.D., and M.C.; methodology, A.D. and M.C.; statistical analysis, A.D.,

M.C., and A.D.N.; resources, M.C., R.L., and F.K.; data curation, M.C., writing—original draft preparation, A.D.,

V.A., A.D.N. and M.C.; writing—review and editing, A.D., V.A., A.M., J.C., A.D.N., R.L., F.K. and M.C. All authors

have read and agreed to the published version of the manuscript.

Funding: This research received no external funding.

Conflicts of Interest: The authors declare that they have no conflict of interest and no competing financial interests.

Disclaimer: PHASE I AIFA, UNI EN ISO 9001 and 13485 Certificate Laboratory; Certificate No. IT-64386 and

DM/17/154/S; Certification for: “Design, development and application of determination methods for clinical

analytes and drugs, also with reference to in vitro dyagnostics. pharmacogenetic analyses.” and “Design and

development and application of quantification and detection methods of clinical analytes and drugs, finalized to

the production of in vitro diagnostics” www.tdm-torino.org.

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