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\begin{document}

\title{Detectable respiratory SARS-CoV-2 RNA is associated with low vitamin D
levels and high social deprivation}



\author[1]{Mark Livingston}%
\author[1]{Aiden Plant}%
\author[2]{Simon Dunmore}%
\author[1]{Andrew Hartland}%
\author[1]{Stephen Jones}%
\author[3]{Ian Laing}%
\author[4]{Sudarshan Ramachandran}%
\affil[1]{Walsall Manor Hospital}%
\affil[2]{University of Wolverhampton}%
\affil[3]{Royal Preston Hospital}%
\affil[4]{University Hospitals Birmingham NHS Foundation Trust}%


\vspace{-1em}



  
  \date{June 18, 2024}


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\selectlanguage{english}
\begin{abstract}
Aims: Accumulating evidence links COVID-19 incidence and outcomes with
vitamin D status. We investigated if an interaction existed between
vitamin D levels and social deprivation in those with and without
COVID-19 infection. Methods: Upper- or lower-respiratory tract samples
from 104 patients were tested for SARS-CoV-2 RNA in accordance with
Public Health England criteria (January--May 2020) using RT-PCR. The
latest serum total 25-hydroxyvitamin D(25-OHD) levels, quantified by
LC-MS/MS, was obtained for each patient (September 2019--April 2020).
Index of Multiple Deprivation (IMD) was generated for each patient.
Univariate and logistic regression analyses examined associations
between age, gender, 25-OHD, IMD score and SARS-CoV-2 result in the
total cohort and subgroups. Results: In the total cohort, a positive
SARS-CoV-2 test was significantly associated with lower 25-OHD levels
and higher IMD. A positive test was associated with higher IMD in the
male subgroup and with lower 25-OHD levels in those aged
\textgreater{}72 years. Low 25-OHD and IMD quintile 5 were separately
associated with positive COVID-19 outcome in the cohort. Patients in IMD
quintile 5 with vitamin D levels [?]34.4 nmol/L were most likely to have a
positive COVID-19 outcome, even more so if aged \textgreater{}72 years
(OR: 19.07, 95\%CI: 1.71--212.25; p=0.016). Conclusions: In this cohort,
combined low vitamin D levels and higher social deprivation were most
associated with COVID-19 infection. In older age, this combination was
even more significant. Our data supports the recommendations for
normalising vitamin D levels in those with deficient / insufficient
levels and in groups at high-risk for deficiency.%
\end{abstract}%



\sloppy




\textbf{What's already known about this topic?}

Vitamin D deficiency and insufficiency are related to a range of adverse
health outcomes. Low vitamin D levels appear related to pneumonia,
cytokine burst and acute respiratory distress syndrome, all of which are
associated with COVID-19. Accumulating evidence links COVID-19 incidence
and outcomes with vitamin D status. We investigated to see if an
interaction existed between vitamin D levels and social deprivation in
those with and without COVID-19 infection.

\textbf{What does this article add?}

Combined low vitamin D levels and higher social deprivation were most
associated with COVID-19 infection. In older age, this combination was
more significant. Our data supports normalising vitamin D levels in
those with deficient/insufficient levels and in high-risk groups.

\textbf{Introduction} Research over the past 30 years has shown that in
addition to the well-established functions as a mediator of calcium and
bone metabolism, the hormone 1,25 dihydroxyvitamin D has effects on a
range of pathological processes {[}1{]}. These include associations with
pathogenesis of neoplastic, inflammatory, demyelinating, and
cardiovascular diseases, as well as diabetes; the aetiology possibly
being due to modulation of innate and adaptive immune functions via
genes regulated by the transcription factor vitamin D receptor
{[}2,3{]}. Hypovitaminosis D, identified by low serum levels of
biologically inactive 25-hydroxyvitamin D (25-OHD), has a high worldwide
prevalence {[}4{]}. Crowe et al. {[}5{]} studied the prevalence of
vitamin D deficiency (\textless{}30 nmol/L) between 2005 and 2015 in
210,502 individuals in the UK. A third (69,515 individuals) of the
cohort had low hormone concentrations, this prevalence being higher in
individuals who were of male gender, younger age, ethnic minorities, and
the economically deprived {[}5{]}.

It can be argued that currently active measures to counter COVID-19 have
been mainly focussed on non-pharmaceutical methods, perhaps in view of
the acute explosion of the disease worldwide. The association between
hypovitaminosis D and COVID-19 appears to have some scientific merit and
must be studied further {[}6{]}. Low vitamin D concentrations appear
related to pneumonia, cytokine burst and acute respiratory distress
syndrome, all of which are associated with COVID-19 {[}7{]}.
Furthermore, low vitamin D appears prevalent in critically ill patients
in intensive care units (ICUs) and is associated with increased length
of stay, readmission and mortality {[}8{]}. Han et al. {[}8{]} carried
out a pilot randomised controlled trial of 31 subjects admitted to ICU
and ventilated and administered either placebo, 50,000 IU or 100,000 IU
of vitamin D daily for 5 consecutive days. Length of stay in hospital
was significantly correlated to the dose of vitamin D supplements
(placebo: length of stay = 36 \selectlanguage{ngerman}±19 days, day 7 vitamin D levels = 52.2
±28.0 nmol/L; 50,000 IU/day: length of stay = 25 ±14 days, day 7 vitamin
D levels = 114.3 ±49.0 nmol/L; 100,000 IU/day: length of stay = 18 ±11
days, day 7 vitamin D levels = 138.0 ±36.0 nmol/L). The other clinical
outcomes studied were not statistically different between the 3 groups.
Laird et al. {[}9{]} found a significant association between mean
vitamin D levels and mortality attributed to COVID-19 in various
European countries {[}9{]}. Interestingly, the Scandinavian countries
Norway, Finland, Sweden, although receiving lower levels of sunlight,
had higher mean vitamin D concentrations than their Southern European
counterparts (owing to widespread fortification of foods) and lower
COVID-19 infection and mortality {[}9{]}In view of the accumulating
evidence there have been recommendations that individuals at risk of
COVID-19 should consider treatment with inactive vitamin D to reduce the
risk of infection: 10,000 IU/day for a few days followed by 5,000 IU/day
to raise serum 25-OHD concentrations to 100--150 nmol/L {[}6, 10{]}.

However, although a study by Hastie et al. {[}11{]} using data from the
UK Biobank did show an association between vitamin D levels and COVID-19
infection, this relationship was not sustained when confounders were
included in the logistic regression model. Hence, the relationship
between vitamin D levels and COVID-19 infection appear complex, and
perhaps may only be seen in subgroups. This heterogeneity would make the
association very dependent on the cohort studied.

A report from the UK Office for National Statistics suggested that
COVID-19 related deaths (20,283 in England and Wales between March 1,
2020 and April 17, 2020) showed a relationship between mortality and
deprivation; the age-standardised mortality rate in the most deprived
areas of England was 55.1/100,000 population compared with 25.3/100,000
population in the least deprived areas.
(https://www.ons.gov.uk/peoplepopulationandcommunity/birthsdeathsandmarriages/deaths/bulletins/deathsinvolvingcovid19bylocalareasanddeprivation/latest
- last accessed: 19/05/2020).

Hayden et al. {[}12{]} showed that vitamin D deficiency is related to
ethnicity and social deprivation; an association (p=0.0001) was observed
between the Index of Multiple Deprivation (IMD) and the percentage of
patients with serum vitamin D levels \textless{}50~nmol/L in Manchester
and Trafford, UK. Similarly, Heald et al. {[}13{]} showed a similar
relationship between IMD and vitamin D levels in individuals aged
\textgreater{}70 years.

We obtained data from 104 individuals tested according to UK Public
Health England Guidelines for COVID-19
(https://www.gov.uk/government/publications/wuhan-novel-coronavirus-initial-investigation-of-possible-cases/investigation-and-initial-clinical-management-of-possible-cases-of-wuhan-novel-coronavirus-wn-cov-infection
- last accessed 10/05/2020); 93.3\% were tested within the hospital (the
remainder were from either residential care homes or general practice).
The aim of this study was to see whether any differences in serum
vitamin D and social deprivation, using the IMD scores, existed between
the patient groups with and without a SARS-CoV-2 detected PCR test. We
also wished to see if an interaction existed between vitamin D
concentration and deprivation with regard to COVID-19. Moreover, the
analyses were repeated in subgroups associated with a poorer prognosis,
in particular male gender and older age.

\textbf{Patients and Methods} Walsall Healthcare NHS Trust provides
acute hospital and community health services for people living in
Walsall, UK, and the surrounding areas, and serves a population of
around 270,000 people. Acute hospital services are provided from Walsall
Manor Hospital, a university-affiliated hospital, which has
\textasciitilde{}600--650 inpatient beds, and provides community health
services from over 60 sites including primary care health centres and
General Practitioner surgeries.

All 104 patients in this study were tested for COVID-19 between January
31, 2020 and May 7, 2020, in accordance with the UK Public Health
England COVID-19 testing criteria, from samples received at the
Department of Microbiology, Walsall Manor Hospital.

Clinical samples of the upper- (nasopharyngeal swabs) or
lower-respiratory tract (sputum or bronchial washings) were tested for
the presence of SARS-CoV-2 after inoculation into viral transport
medium, using initially reference laboratory investigational RdRP-gene
real-time reverse-transcriptase PCR assay until
6\textsuperscript{th}April 2020. After this date, detection of
SARS-CoV-2 was performed locally on a commercial assay detecting the
ORF-1a/b and E-genes with a reported limit of detection of
\textless{}300 copies/mL (Roche Cobas; Roche Diagnostics GmbH, Manheim,
Germany) {[}14{]}. The relative clinical sensitivity and specificity of
these targets are not yet known.

Vitamin D levels were the most recent for each patient from a period
covering 6 months (between September 1, 2019 and April 30, 2020)
obtained from the Clinical Biochemistry laboratory database. Liquid
chromatography-tandem mass spectrometry (LC-MS/MS; Waters TQD LC-MS/MS;
Waters Corporation, UK) was used to quantify serum
25-OHD\textsubscript{2} and 25-OHD\textsubscript{3} levels (combined to
give the total 25-OHD level) (coefficient of variation (CV) was 6.5\%,
5.5\% and 5.2\% for internal quality control (IQC)
25-OHD\textsubscript{3} levels of 19.4, 39.5 and 110.1 nmol/L,
respectively; the CV was 10.9\%, 5.6\% and 5.4\% for IQC
25-OHD\textsubscript{2} levels of 3.7, 37.7 and 108.8 nmol/L,
respectively).

The English Indices of Deprivation combine factors of housing, social
and economic issues to give a single deprivation score for small areas
(known as Lower-Layer Super Output Areas or LSOAs) in England. An
overall weighted aggregation index of multiple deprivation (IMD) is
generated based on thirty seven separate indicators,

organized across seven distinct domains of deprivation and each area is
ranked from the least to most deprived. The criteria and their
associated weightings are: Income deprivation, 22.5\%, Employment
deprivation, 22.5\%, Health deprivation and disability 13.5\%,
Education, skills and training deprivation 13.5\%, Barriers to housing
and services 9.3\%, Crime 9.3\% and Living environment deprivation
9.3\%. The indices are a widely used standard measure for comparing
areas across the country and help to identify areas with high levels of
overall deprivation or areas with specific concerns (health for example)
that may not be recognised from the overall index. The measures of
deprivation are collected nationally and published every 3--4 years. In
the IMD 2019, the most deprived LSOA in England is given a rank of 1 and
the least deprived is ranked 32,844. Each vitamin D result was aligned
with an associated postcode for each patient and linked to its LSOA
using the geo-convert tool (http://geoconvert.mimas.ac.uk - last
accessed on 19/05/2020). The LSOA is then linked to the 2019 IMD for the
specified full postcode {[}13{]}. This was not possible using
geo-convert for six patients included in this stud.
https://assets.publishing.service.gov.uk/government/uploads/system/uploads/attachment\_data/file/835115/IoD2019\_Statistical\_Release.pdf
- last accessed on 20/05/2020)..

\textbf{Statistics}

The association between a positive SARS-CoV-2 test compared with a
negative SARS-CoV-2 test result and serum vitamin D levels, IMD, age and
gender was initially studied using unpaired t-test (continuous
variables), rank-sum non-parametric test (ordinal data: IMD) in the
total cohort and subgroups; age, vitamin D by median values and IMD by
the quintile threshold closest to the median
(https://tools.npeu.ox.ac.uk/imd/ - last accessed on 19.05.2020).
Logistic regression analyses were then carried out with SARS-CoV-2 RNA
real time RT-PCR test result as the dichotomous outcome (detected or not
detected) and vitamin D levels and factorised IMD as independent
variables with age and gender included as confounders. The regression
analyses were carried out in the total groups and the previously
mentioned subgroups. All analyses were performed on Stata 14 (StataCorp
LLC, Texas, USA).

\textbf{Results} In the total cohort of 104 individuals tested for
SARS-CoV-2, a positive result was significantly associated with low
vitamin D concentrations and IMD, but not age and gender (Table 1). The
total cohort was then stratified by gender, median age (72 years),
vitamin D levels (34.4 nmol/L) and IMD (quintile 1-4 and 5; median IMD
was 35.70, this figure within quintile 5, the threshold separating
quintile 4 from 5 being 34.18). 57.1\% of the total cohort had an IMD in
quintile 5. In males and individuals aged \textgreater{}72 years, a
positive SARS-CoV-2 test was associated with higher IMD (greater
deprivation) and low vitamin D levels, respectively. Low vitamin D
levels were associated with a positive COVID-19 test in individuals
classified as IMD quintile 5, and whilst this was not seen in IMD
quintiles 1-4, IMD was higher in COVID-19 positive individuals with
vitamin D levels below the total cohort median value, this approaching
statistical significance (Table 1).

We then performed a logistic regression analysis with COVID-19 positive
diagnosis (reference: negative diagnosis) as the dichotomous outcome.
Low vitamin D levels and IMD quintile 5 (compared with IMD quintiles
1-4) were significantly associated with the outcome whilst age and
gender were not (Table 2: Model 1). Model 2 (Table 2) was a repeat of
Model 1, but with vitamin D stratified by the median value; once again
low vitamin D and IMD quintile 5 were significantly associated with a
positive COVID-19 test. Models 3 and 4 (Table 2) comprised the cohort
stratified by the median vitamin D levels, and IMD quintile 5 was only
significantly associated with a positive COVID-19 test in the subgroup
with low vitamin D levels (Table 2: Model 3). Vitamin D levels were
associated with a positive SARS-CoV-2 test in the IMD quintile 5
subgroup (Table 2: Model 5) but not in the IMD quintiles 1-4 subgroup
(Table 2: Model 6).

The above analyses (Table 2: Models 3 and 5) suggests the possibility of
an interaction between IMD quintile 5 and low vitamin D levels, both
associated with positive SARS-CoV-2 test (Table 2: Model 2). Hence, the
total cohort was stratified into 4 groups by IMD quintiles (quintile 1-4
and 5) and vitamin D status (\selectlanguage{english}[?] 34.4, \textgreater{}34.4 nmol/L). A
Chi-square analysis did not show an association between these 4 groups
(p=0.41). Model 7 (Table 2) shows that individuals belonging to IMD
quintile 5 with vitamin D levels \selectlanguage{english}[?] 34.4 nmol/L were more likely to have
a positive SARS-CoV-2 test (reference: vitamin D \textgreater{}
34.4nmol/L + IMD quintiles 1-4). Interestingly, the association with a
diagnosis of COVID-19 was clearly not evident in individuals with only
one risk factor (vitamin D \selectlanguage{english}[?] 34.4 nmol/L or IMD quintile 5). The
analysis with the combined IMD and vitamin D subgroups was repeated in
patients aged \selectlanguage{english}[?] 72 years (Model 8) and \textgreater{}72 years (Table 2:
Model 9). The association between the `at risk' group (IMD quintile 5 +
vitamin D \selectlanguage{english}[?] 34.4 nmol/L) appeared greater in patients aged
\textgreater{}72 years (Table 2: Model 9). Although we did not have
sufficient patient numbers to study the above associations in black and
ethnic minorities, we were able to analyse the data in the known white
Caucasian subgroup (n=59):-- individuals belonging to IMD quintile 5
with vitamin D levels \selectlanguage{english}[?] 34.4 nmol/L being the only patients
significantly associated (odds ratio: 19.00, 95\% confidence interval:
2.73--132.27; p=0.003) with a diagnosis of COVID-19 (reference: vitamin
D \textgreater{}34.4 nmol/L + IMD quintiles 1--4).

\textbf{Discussion} There is accumulating evidence on the association
between vitamin D levels and COVID-19 incidence and outcome following
infection {[}6--8, 15{]}. Ours is a relatively small study cohort
fulfilling the PCR based testing criteria for COVID-19. Univariate
analyses showed that low vitamin D levels and IMD scores were associated
with a positive test for SARS-CoV-2. It was clear that these
associations appeared strengthened in some subgroups; males (IMD
scores), age \textgreater{}72 years (vitamin D). Further, the
association between low vitamin D and a positive SARS-CoV-2 result was
seen in the IMD quintile 5 subgroup only. Logistic regression with age
and gender added as confounding variables confirmed the results of the
above mentioned univariate association between low vitamin D,
deprivation (IMD quintile 5) and a positive SARS-CoV-2 test.
Interestingly, low vitamin D and IMD quintile 5 were associated with a
positive test in patients belonging to IMD quintile 5 (Table 2: Model 5)
and low vitamin D (Table 2: Model 3), respectively, and not the
complementary subgroups.

These results are interesting as only a combination of low vitamin D
levels and deprivation (IMD quintile 5) appear to be associated with
COVID-19 (table 2: Model 7), this association perhaps more evident in
the older age group (Table 2: Model 9). Patients characterised by one of
these factors were not at any higher risk of a diagnosis of COVID-19.
Thus, associations between vitamin D levels, social deprivation and
COVID-19 infection will be dependent on cohort characteristics.
Consequently, we highlight the importance of acknowledging heterogeneity
and studying subgroups. Our results were influenced by the fact that
57.1\% with IMD scores belonged to IMD quintile 5, the most deprived
group; each quintile should consist of \textasciitilde{}20\%, but
accordingly this shows the increased social deprivation present in this
cohort. The 2019 Index of Multiple Deprivation ranks Walsall as the 25th
most deprived English local authority (out of 317), placing Walsall
within the most deprived 10\% of districts in the UK. High deprivation
is most likely a surrogate for either one or more risk factors. It is
established that rates of obesity and potentially of insulin resistance
are higher in more socially disadvantaged individuals {[}16{]}. Low
vitamin D could be a causative or a surrogate factor in COVID-19
infection. Only normalising vitamin D levels with supplements would
provide data on the role of the hormone in COVID-19 infection.
Interestingly, whilst addressing social deprivation is a long term
programme, normalising serum vitamin D concentrations can be easily
achieved in a relatively short period within existing clinical
guidelines. In our clinical practice, we have for \textgreater{}10 years
been using vitamin D supplements in patients with hormonal deficiency
(\textless{}30 nmol/L) or insufficiency (30--50 nmol/L), these
thresholds in synchrony with laboratory distribution data and at slight
odds with the UK National Institute for Health and Care Excellence
(NICE) recommendations
(https://cks.nice.org.uk/vitamin-d-deficiency-in-adults-treatment-and-prevention\#!scenario
-- last accessed on 10/05/2020). Depending on symptoms and
concentrations of serum vitamin D, a clinical decision based on the NICE
guidelines would be taken as to the dose of the hormone supplements
(https://cks.nice.org.uk/vitamin-d-deficiency-in-adults-treatment-and-prevention\#!scenario
-- last accessed on 10/05/2020). This should be followed by a repeat
test of vitamin D and a bone profile in 3--6 months to ensure adequate
treatment efficacy. Adverse effects are uncommon {[}17{]}; hence, the
importance of our findings.

In usual circumstances, ours would be considered an interesting
observation meriting further investigation. Even when a significant
association was observed, the relatively small numbers led to the odds
ratio having wide confidence intervals. We acknowledge that false
negative SARS-CoV-2 RT-PCR test results could have a significant bearing
on our study outcome. The relative clinical sensitivity and specificity
of the targets in the Roche assay are not yet fully known, but the false
negative rate for is estimated to be \textasciitilde{}30\% {[}18{]}.
However, the current situation perhaps requires the usual scientific
method processes to be accelerated, especially if any associated
therapeutic intervention is perceived as safe. Our data adds to results
from other studies suggesting a possible role for vitamin D in COVID-19
infection {[}15{]}. As described previously there is mechanistic
plausibility in view of vitamin D modulating immune function and viral
replication {[}2,3{]}. A further positive aspect of our study is the
inclusion of SARS-CoV-2 test negative patients which were suspected of a
COVID-19 diagnosis for comparison (control group). A larger, more robust
study is required to validate our findings, with the cohort size
allowing for subgroup analyses of different populations (e.g. different
ethnic groups) at varying levels of risk {[}19{]}. It would also be
interesting to compare with similar global data to see if vitamin D
levels could, at least partially account for differences in incidence /
prevalence that is evident between nations
(https://www.who.int/docs/default-source/coronaviruse/situation-reports/20200304-sitrep-44-covid-19.pdf?sfvrsn=783b4c9d\_2
-- last accessed on 10/05/2020). This should be followed by large well
designed outcome studies (either randomised controlled trials or
longitudinal cohort studies) to see if vitamin D supplementation,
especially in individuals subject to social deprivation, alters outcome
trajectory at varying stages of the infection. However, at this time of
necessary urgent action, our data adds support to the recommendations
for normalising vitamin D levels in those with deficient / insufficient
levels and in groups at high-risk for deficiency.

In conclusion, combined low vitamin D levels and higher social
deprivation were most associated with COVID-19 infection. In older age,
this combination was even more significant. The importance of studying
subgroups and acknowledging heterogeneity is highlighted, with results
being dependant on the cohort studied.

\textbf{Funding:} None to declare.

\textbf{Conflicts of Interest:} None to declare.

\textbf{Acknowledgements:}

The authors acknowledge Mr Yusuf Varachia for help with extracting the
data from the laboratory database.

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\emph{Table legends}

\textbf{Table 1:} Univariate analyses studying associations between age,
gender, serum total 25-hydroxyvitamin D levels and Index of Multiple
Deprivation (IMD) scores and SARS-CoV-2 RT-PCR test results (for
COVID-19) in the total cohort and selected subgroups.

\textbf{Table 2:} Logistic regression analyses studying associations
between serum total 25-hydroxyvitamin D levels, Index of Multiple
Deprivation (IMD) quintile 5, separately and in combination, and
COVID-19 infection in the total cohort and selected subgroups, the
analyses adjusted for age and gender. OR: odds ratio, CI: confidence
intervals.

\textbf{Hosted file}

\verb`Tables 1 and 2.docx`  available at \url{https://authorea.com/users/312296/articles/454940-detectable-respiratory-sars-cov-2-rna-is-associated-with-low-vitamin-d-levels-and-high-social-deprivation}

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