Association Between Vitamin D and Novel SARS-CoV-2 Respiratory Dysfunction – A Scoping Review of Current Evidence and Its Implication for COVID-19 Pandemic

Objectives To assess the association between vitamin D deficiency and increased morbidity/mortality with COVID-19 respiratory dysfunction. Design Scoping review. Data Sources Ovid MEDLINE (1946 to 24 of April 2020) and PubMed (2020 to 17 of September 2020). Eligibility Criteria for Selecting Studies A search using the search terms: [(cholecalciferol or ergocalciferol or vitamin D2 or vitamin D3 or vitamin D or 25OHD) and (SARS-CoV-2 or coronavirus or COVID or betacoronavirus or MERS-CoV or SARS-CoV or respiratory infection or acute respiratory distress syndrome or ARDS)]m.p. was conducted on the 24/04/2020 (Search A) and 17/09/2020 (Search B). Results 91 studies were identified as being concerned with Acute Respiratory Infection (ARI)/Acute Respiratory Distress Syndrome (ARDS) and vitamin D, and 25 publications specifically explored the role of vitamin D deficiency in the development and progression of SARS-CoV-2/COVID-19 related ARDS. Search “A” identified three main themes of indirect evidence supporting such an association. Consistent epidemiological evidence exists linking low vitamin D levels to increased risk and severity of respiratory tract infections. We also report on plausible biological processes supporting such an association; and present weaker evidence supporting the benefit of vitamin D supplementation in reducing the risk and severity of ARIs. Uncertainty remains about what constitutes an appropriate dosing regimen in relation to reducing risk/severity of ARI/ARDS. More recent evidence (Search B) provided new insights into some direct links between vitamin D and COVID-19; with a number of cohort and ecological studies supporting an association with PCR-positivity for SARS-CoV-2 and vitamin D deficiency. The exact efficacy of the vitamin D supplementation for prevention of, or as an adjunct treatment for COVID-19 remains to be determined; but a number of randomized control trials (RCTs) currently underway are actively investigating these potential benefits. Conclusion Our rapid review of literature supports the need for observational studies with COVID-19 infected populations to measure and assess vitamin D levels in relation to risk/severity and outcomes; alongside RCTs designed to evaluate the efficacy of supplementation both in preventive and therapeutic contexts. The overlap in the vitamin D associated biological pathways with the dysregulation reported to drive COVID-19 outcomes warrants further investigation.


INTRODUCTION
Vitamin D deficiency affects almost 50% of the population worldwide (Holick, 2007). Its high prevalence is a particularly important public health issue because hypovitaminosis D is an independent risk factor for total mortality in the general population (Melamed et al., 2008). The potential consequences of vitamin D deficiency are wide ranging. It plays a role in the development and progression of chronic diseases such as cardiovascular disease, bone health, autoimmunity, type-2 diabetes, cancer and depression. The less well recognized yet important role of vitamin D includes its effect in modulating the innate and adaptive immune systems, and deficiency of vitamin D is associated with an increased susceptibility to respiratory infections (Dancer et al., 2015;McCartney and Byrne, 2020).
As the pandemic of a novel betacoronavirus known as severe acute respiratory syndrome corona virus 2 (SARS-CoV-2) or 2019 novel coronavirus disease  continues to advance globally, studies on risk factors for intensive care unit admission and mortality are emerging. Early studies from China and Italy report that older age, male sex, smoking status, existing comorbidities or a compromised immune system increase the risk of developing severe and even fatal respiratory disease associated with COVID-19 . Mortality rates from COVID-19 vary significantly between countries. Countries that lie below 35 degrees North have relatively low mortality rates (e.g., 14.6% of patients in northern latitudes to 0.6% of patients in southern latitudes) . Although this discrepancy has been suggested to be a consequence of the high prevalence of elderly populations in European countries (Bureau, 2020); 35 degrees North is also the latitude above which people do not receive sufficient sunlight to retain adequate vitamin D levels during winter, suggesting a possible role for vitamin D in determining outcomes from COVID-19 (Lips et al., 2019). United Kingdom and United States data from critically ill patients with COVID-19 indicate that people from Black, Asian and minority ethnic (BAME) backgrounds are at Abbreviations: 25(OH)D, 25-hydroxy vitamin D; ALRI, acute lower respiratory infection; ARI, acute respiratory infection; ARDS, acute respiratory distress syndrome; ALI, acute lung injury; ACEI ACEII, angiotensin-converting enzymes I and II; BAME, black, asian and minority ethnic backgrounds; RCTs, Randomized Clinical Trials; RAS, renin-angiotensin system; RTI, respiratory tract infection; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2. a higher risk of developing more severe symptoms than white people (Centre. ICNAaR., 2020). There are also serious concerns regarding the disproportionate impact of COVID-19 on BAME healthcare professionals (Siddique, 2020). Ethnicity is a complex epidemiological entity made up of genetic, social, cultural and behavioral patterns but a growing body of literature indicates that, worldwide, immigrants experience health deterioration after their arrival into their adopted country, and moreover, they have lower vitamin D compared to the native-born population (Holick, 2010;Edwards et al., 2014;Granlund et al., 2016;Martin et al., 2016). Given the prevalence of vitamin D deficiency in BAME populations (Holick, 2007), studies evaluating its role in promoting COVID-19 disease severity are currently underway.
The current scoping review specifically focuses on the potential effects of vitamin D on the risk and severity of COVID-19 associated respiratory dysfunction. We hypothesize that deficiency of vitamin D may be associated with an increased morbidity/mortality of COVID-19 respiratory infection.

METHODS
Following the PRISMA extension for scoping reviews guidelines (Tricco et al., 2018), a initial search was conducted in Ovid MEDLINE, a bibliographic database including references to journal articles from 1946 to date, with the following search terms: [(cholecalciferol or ergocalciferol or vitamin D2 or vitamin D3 or vitamin D or 25OHD) and (SARS-CoV-2 or corona virus or COVID or betacoronavirus or MERS-CoV or SARS-CoV or respiratory infection or acute respiratory distress syndrome or ARDS)].mp. [mp = title, abstract, original title, name of substance word, subject heading word, floating sub-heading word, keyword heading word, organism supplementary concept word, protocol supplementary concept word, rare disease supplementary concept word, unique identifier, synonyms] on the 24 of April 2020 (Search A). Given the rapid turnaround of COVID-19 related literature published following the first peak of the SARS-CoV-2 pandemic, we conduced a second search in PubMed, a bibliographic database that comprises more than 30 million citations for biomedical literature from MEDLINE, life science journals, and online books, with the following search terms: [(cholecalciferol or ergocalciferol or vitamin D2 or vitamin D3 or vitamin D or 25OHD) and (SARS-CoV-2 or corona virus or COVID or betacoronavirus or MERS-CoV or SARS-CoV or respiratory infection or acute respiratory distress syndrome or ARDS)] on the 17 of September 2020 (Search B). There is no published protocol associated with this scoping review.
The inclusion criteria for both searches considered both pediatric and adult publications. Moreover, no restrictions were placed on publication type, with all systematic reviews, narrative reviews, meta-analyses, original research articles (experimental, observational and clinical trials), commentaries, letters, and editorials identified in the MEDLINE search being considered for this review. However, articles concerned with respiratory chronic diseases, such as asthma, and other non-respiratory related pathologies were excluded. Non-English publications, duplicate studies, errata, trial protocols without data and studies not related to human viruses/infections were also excluded. Further exclusions were applied in the second search with only publications with full text available, in human models and published in 2020 included. Studies describing protocols, experimental studies and publications reviewed in the first search were further excluded. Moreover preprints were not included in this review.
Publications were initially screened by title and abstract by three independent reviewers, with potentially relevant studies undergoing a full text review. Following the inclusion and exclusion criteria described above, some studies were further excluded. The included studies were then summarized and classified depending on the topic. Potential bias in some of the observational and interventional studies was identified based on the study design. No formal assessment of quality of selected papers or effect measurement was undertaken given the breadth of publication types being considered and given the scope of the current review.

RESULTS
A total of 128 studies were identified from the first search "A." Full text review was undertaken on 115 articles which were assessed as potentially suitable for inclusion after title and abstract screening. After further exclusion of papers not concerned with COVID-19, ARI/ARDS and vitamin D, the final number of publications included was 91 (Table 1). Figure 1 presents a PRISMA diagram of the review strategy. Reviewed papers on search consisted of 7 systematic reviews with meta-analyses, 13 narrative reviews, 16 randomized trials, 33 observational studies and 5 experimental studies. The remaining (Quesada-Gomez et al., 2020) publications were letters, editorials, commentaries and position statements (Supplementary Table S1). Search "B" identified 112 publications of which 101 were suitable for full inclusion after exclusion of duplicates. After further exclusion for non-Covid related, not relevant and only protocols, 59 publications were reviewed (Supplementary Table S1). Twenty five (8 observational, 4 ecological, 1 interventional non-RCT, 4 Systematic review/meta-analysis/review and 8 editorial) new studies specifically explored the role of vitamin D deficiency in the progression of ARDS in COVID-19 infection (Arnold, 2020;Aygun, 2020;Brenner et al., 2020;Chakhtoura et al., 2020;D'Avolio et al., 2020;Ebadi and Montano-Loza, 2020;Faul et al., 2020;Haj Bloukh et al., 2020;Hastie et al., 2020;Ilie et al., 2020;Laird et al., 2020;Martineau and Forouhi, 2020;Meltzer et al., 2020;Ohaegbulam et al., 2020;Panagiotou et al., 2020;Quesada-Gomez et al., 2020; Raisi-Estabragh et al.,  2020 ;Silberstein, 2020;Singh et al., 2020;Slominski et al., 2020;Sun et al., 2020;Townsend et al., 2020;Tramontana et al., 2020;Whittemore, 2020) (Tables 2, 3). Figure 2 presents a PRISMA diagram of the review search B. Review of search "A" studies found no direct evidence of any association between vitamin D and risk or severity of COVID-19; however, our literature review identified three main themes of indirect evidence supporting an association for vitamin D and SARS-CoV-2 exposure: (1) an association of vitamin D deficiency with increased incidence and severity of respiratory infections (including 12 research studies exploring ARDS/ARI, Table 4

Biological Mechanisms of Interaction of Vitamin D and Clinico-Pathological Features of SARS-CoV-2 Infection
The pathogenic mechanisms of COVID-19 disease have not been completely elucidated yet. However, a number of dysregulated pathways are being increasingly recognized as directly related to COVID-19 morbidity and mortality (Jakovac, 2020;Misra et al., 2020;Panarese and Shahini, 2020). These include the role of renin-angiotensin pathways in viral entry into alveolar cells and subsequent acute lung injury, dysregulated immune responses and activation of coagulation pathways (Quesada-Gomez et al., 2020). Interestingly, our literature review identified a number of studies/reviews (n = 24) providing some insight into the mechanistic explanations for the observed epidemiological relationship between vitamin D deficiency and increased risk/severity of respiratory infections (Ginde et al., 2009;Laaksi, 2012;Pfeffer and Hawrylowicz, 2012;He et al., 2013;Parekh et al., 2013;Lambert et al., 2014;Dancer et al., 2015;Greiller and Martineau, 2015;Vargas Buonfiglio et al., 2017;Xu et al., 2017;Jolliffe et al., 2018;Miraglia Del Giudice et al., 2018;Iyer and Bansal, 2019;Aygun, 2020;Brenner et al., 2020;Carter et al., 2020;Jakovac, 2020;Kakodkar et al., 2020;Maes et al., 2020;McCartney and Byrne, 2020;Misra et al., 2020;Molloy and Murphy, 2020;Panarese and Shahini, 2020;Quesada-Gomez et al., 2020;Tian and Rong, 2020;Zheng et al., 2020). Studies and narrative reviews suggest that entry of SARS-CoV-2 infection into alveolar epithelial cells, triggered by binding of the virus surface spike (S)-protein to the angiotensin converting enzyme 2 (ACE2) receptor, may lead to dysregulation of the renin-angiotensin system (RAS) resulting in acute lung injury due to toxic overaccumulation of angiotensin II (Ang II) in alveolar cells (Pfeffer and Hawrylowicz, 2012;Arnold, 2020;Carter et al., 2020;Jakovac, 2020;Kakodkar et al., 2020;Misra et al., 2020;Panarese and Shahini, 2020;Tian and Rong, 2020). Evidence suggests that vitamin D deficiency causes over-activation of the pulmonary RAS, providing a possible explanation for the observed inverse relationship between blood pressure and serum 25(OH)D levels (Parekh et al., 2013;Xu et al., 2017). Vitamin D may exert protective effects on acute lung injury, at least in part, by regulating the balance between the expression of members of the RAS (Xu et al., 2017). It is therefore plausible that vitamin D deficiency may exacerbate dysregulation of the pulmonary RAS induced by SARS-CoV-2 infection.
ACE2 receptor required for virus entry, is expressed by endothelial cells and its density in each tissue may correlate with the severity of the disease in that tissue (Jia et al., 2005;Gheblawi et al., 2020;Perico et al., 2020;Xu et al., 2020). Endothelial dysfunction is a common feature of the clinical manifestations observed in COVID-19 (e.g., high blood pressure, thrombosis, kidney disease, pulmonary embolism, Kawasaki vasculitic disease, cerebrovascular and neurological disorders) (Sardu et al., 2020b). A systematic and comprehensive evaluation of literature by Sardu et al. (2020b) report that in the hospitalized patients with COVID-19 disease the worsening disease severity, including ICU admissions, could be played by endothelial dysfunction that is more enhanced in patients with co-morbidities. Study by. Guan et al. reported that 23.7% 23.7% of hypertensive patients had disease severity (vs. 13.4% of normotensive subjects), and that 35.8% (vs. 13.7%) reached the composite endpoint of ICU admission, mechanical ventilation and death (Guan et al., 2020). Paradoxically, many of the therapies for pre-existing cardiovascular diseases can also increase the ACE2 levels resulting in a negative outcome regard to infectivity and outcome of COVID−19. However, given the high incidence of cardiac injury and adverse outcomes in hypertensive patients during the COVID-19 pandemic, experts do not recommend that hypertensive patients should discontinue and/or switch therapies during COVID-19 infection. Interestingly, vitamin D treatment has been shown to inhibit ACE2 expression in the kidneys (Lin et al., 2016;Ali et al., 2018), and thereby could prevent COVID-19 entry into the cell in patients with pre-existing co-morbidities. Additionally, Zhou et al. recently showed a significant association between levels of D-dimer > 1 µg/mL and COVID-related death [OR (95 %CI):18.42 (2.64-128.55)], with coagulation dysfunction associated with fatal ARDS in COVID-19 patients (Sardu et al., 2020c;Zhou et al., 2020). Vitamin D and its associated metabolites are implicated in the regulation of thrombosis-related pathways by modulation of genes such as those related to angiogenesis . It has been proposed that this is likely to be the mechanism of action for the increased risk of thrombotic events associated with a vitamin D deficient state (Tian and Rong, 2020). These studies strongly support investigation Positive association The number of cases/country is affected by the number of tests performed and also by the different measures taken by each country to prevent the spread of infection, and the difference in the number of infected patients in the population will also mean different levels of exposure for the population. Mortality might be a better marker of the number of cases in the population although even that can be influenced by the variations in the approach or management of the disease.

No association
Research studies addressing vitamin D and severity of ARI/ARDS.
Frontiers in Physiology | www.frontiersin.org of vitamin D replacement in patients with pre-existing comorbidities. Genetic studies have found a polymorphism in the vitamin D receptor (VDR) gene associated with upper respiratory tract infections in children and adults (Jolliffe et al., 2018). Dysregulated immune responses associated with excessive and prolonged cytokine/chemokine responses known as a 'cytokine storm' , seem to be directly related to worse outcomes in COVID-19 (Carter et al., 2020;Slominski et al., 2020;Tian and Rong, 2020). All cells of the immune system have been shown to express the VDR, including T cells, and literature suggests that it is now well accepted that 1,25(OH)2D and vitamin D are important immune system regulators (Laaksi, 2012;Parekh et al., 2013;Greiller and Martineau, 2015;Miraglia Del Giudice et al., 2018;Iyer and Bansal, 2019;Maes et al., 2020;Misra et al., 2020;Tian and Rong, 2020). Vitamin D has been linked to innate immunity through identification of the cationic antimicrobial peptide cathelicidin as a vitamin D target gene and VDR upregulation in monocytes. Previous studies have proposed cathelicidin activation as a potential mechanism driving vitamin D protective effects on respiratory tract infections (Laaksi, 2012;Lambert et al., 2014;He et al., 2016;Vargas Buonfiglio et al., 2017;Molloy and Murphy, 2020), e.g., in tuberculosis (Ginde et al., 2009;Molloy and Murphy, 2020). A cross-sectional analysis in a mostly African-American population showed that lung function decrements associated with low cathelicidin were greatest in individuals with low vitamin D levels (Lambert et al., 2014). Additionally, vitamin D is involved in downregulating the production of pro-inflammatory cytokines [tumor necrosis factor α (TNF α), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-12 (IL-12), and interferon-gamma (IFN-γ)] which contribute to the cytokine storm (Pfeffer and Hawrylowicz, 2012;He et al., 2013;Barnett et al., 2014;Zhang et al., 2019;Aygun, 2020;Maes et al., 2020;Orrù et al., 2020;Tian and Rong, 2020). Downregulation of IFNγ and IL-6 inflammatory responses have been reported as being negative prognostic indicators in critically ill ventilated patients including those with COVID-19 (McCartney and Byrne, 2020). Other vitamin D functions such as the musculoskeletal properties can be also potentially beneficial in the context of COVID-19 (Tramontana et al., 2020). While the data are far from conclusive in attributing a role for vitamin D in influencing the risk and severity of COVID-19, collectively these studies provide a strong rationale for more pre-clinical and clinical research for further exploration.

Vitamin D Supplementation in Relation to Acute Respiratory Tract Infections
Over the years numerous randomized controlled trials (RCTs) have evaluated whether vitamin D supplementation can decrease the risk of acute respiratory tract infections (ARTI) and its severity. Such studies have yielded conflicting results. Pediatric RCTs exploring the association of vitamin D supplementation with reduction of ARIs have mainly reported no association (Ginde et al., 2009;Camargo et al., 2012;Maxwell et al., 2012;Goldring et al., 2013;Grant et al., 2015Grant et al., , 2016Xiao et al., 2015;Omand et al., 2017;Somnath et al., 2017;Das et al., 2018;Lee et al., 2018;Hauger et al., 2019;Loeb et al., 2019). A large cohort study assessing the effects on serum vitamin D levels following oral vitamin D supplementation in children (0-5 years) found no association between either vitamin D levels or supplementation on health-service utilization (HSU) for upper respiratory tract infections (URTIs) (Omand et al., 2017). Similarly, a meta-analysis of 7 RCTs presented a non-significant association between vitamin D supplementation and reduced risk of ARIs in children [RR (95%CI): 0.79 (0.55-1.13)] (Xiao et al., 2015). Pre-natal and infant studies have also found no effect (Goldring et al., 2013;Grant et al., 2016). The relevance of these studies to adults, however, is unclear.
Several studies undertaken in adults show inconsistent results especially in the elderly population (Murdoch et al., 2012;He et al., 2013;Jolliffe et al., 2013;Dubnov-Raz et al., 2015;Martineau et al., 2015Martineau et al., , 2017Martineau et al., , 2019Vargas Buonfiglio et al., 2017;Aloia et al., 2019;Camargo et al., 2019;Kakodkar et al., 2020). Whilst a systematic review of 39 studies including observational studies and RCTs reported inconsistent results for vitamin D supplementation for prevention of ARI (Jolliffe et al., 2013). Moreover, a meta-analysis in 2017 of individual data from 10,933 study subjects in 25 RCTs, showed an 12% overall protective effect of oral vitamin D3 supplementation against bacterial and viral ARTI [OR(95%CI): 0.88 (0.81-0.96)]. Benefit was greater in those receiving daily or weekly vitamin D (P for interaction = 0.05) and in those with serum 25(OH)D levels < 25 nmol/L [adjusted OR (95%CI): 0.30(0.17 -0.53)] (Martineau et al., 2017(Martineau et al., , 2019. A UK study examined the effect of 14 weeks of vitamin D3 supplementation (5000 IU/d) during winter in male athletes, on antimicrobial proteins in saliva. The authors observed increases over time in saliva secretory immunoglobulin A (SIgA) and the antimicrobial peptide cathelicidin in the vitamin D3 group (both P = 0.03), suggesting that these changes may increase resistance to respiratory infections (He et al., 2013).
A number of RCTs studies in mechanically ventilated, critically ill patients with high dose supplementation (250,000-500,000 IU vitamin D) have proven to be safe and observed association with decreased of hospital length stay and increase of hemoglobin serum levels (Perron and Lee, 2013;Han et al., 2016;Putzu et al., 2017;Smith et al., 2018;Ebadi and Montano-Loza, 2020). Additionally, it is important to mention the diversity observed among the RCTs with respect to supplementation regimen (monthly/weekly/daily), the intake dose (1000-5000 IU/d, 14000/w, 12000-200000 IU/m, or 300000 IU single dose) and the duration of the intervention (1 year -> 3 years) make it difficult to have clear crossstudy comparisons. Moreover, baseline vitamin D levels were not assessed in many studies, and many cases vitamin D intake and respiratory events were self-reported. However, based on these studies although vitamin D supplementation can be concluded to be preventive for ARIs in adults, uncertainty around therapeutic benefit/dosing required remains.

DIRECT EVIDENCE FOR VITAMIN D and SARS-CoV-2 INTERACTION
A more up-to-date search (Search B) identified a number of studies providing a more direct link or evidence for an association of vitamin D and COVID-19. These are summarized below.
Vitamin D Deficiency and COVID-19 Incidence/Disease Severity Human requirement for vitamin D, assessed by measuring 25(OH)D levels in blood plasma or serum, is achieved primarily through the synthesis of this pre-hormone in the skin during exposure to ultraviolet B (UVB) radiation, with minor contribution from diet, year round. This process is negatively influenced by skin pigmentation. A level of 20-50 nanograms/milliliter is considered adequate for healthy people. A level less than 12 ng/mL indicates vitamin D deficiency (Holick, 2007). Vitamin D deficiency have reported to be associated with PCR-positivity for SARS-CoV-2 individuals in two different cohorts. D'Avolio et al. observed significantly lower 25(OH)D levels (p = 0.004) in PCR-positive patients for SARS-CoV-2 (median value 11.1 ng/mL) compared with negative patients (24.6 ng/mL) in a cohort Study in Switzerland suggesting a potential role of the vitamin D in the COVID-19 infection (D'Avolio et al., 2020). Similar results were observed in a healthy workers cohort in Chicago where the vitamin D deficient group had a higher risk for COVID-19 positivity compared with the control group (RR = 1.77; 1.12-2.81) (Meltzer et al., 2020). This positive association was also observed in a large US cohort study (n = 191,779) where SARS-CoV-2 positivity was strongly and inversely associated with circulating 25(OH)D levels after adjusting for latitudes, races/ethnicities, both sexes, and age ranges (Kaufman et al., 2020). Moreover, Faul et al. (2020) presented a small cohort of patients with PCR-positive SARS-CoV-2 related pneumonia, where a baseline serum 25OHD level less than 30 nmol.l-1 was associated with a hazard ratio (HR) for intubation of 3.19 (95 percent confidence interval, 1.05-9.7), (p = 0.03) suggesting that low 25OHD level, contributes to severe disease and progression to ARDS in some patients infected with SARS-CoV-2 (Faul et al., 2020). In line with this results, low serum 25[OH]D levels in patients hospitalized with COVID-19 was associated with greater disease severity but not with mortality in a United Kingdom COVID-19 patients cohort (Panagiotou et al., 2020). Serum calcium levels are well recognized to be significantly positively correlated with vitamin D levels; and higher incidences of organ injury, septic shock, and higher 28day mortality have been observed in COVID-19 patients with lower calcium levels (Sun et al., 2020). Collectively these results provide a strong rationale for further exploration of the potential role of vitamin D and its supplementation where deficient in COVID-19 infection; as well as its role as a predictive biomarker for COVID-19 severity (Weir et al., 2020).
Study by Whittemore (2020) reported a statistically significant correlation between lower COVID-19 death rates and a country's latitude, suggesting a correlation between sunlight exposure and reduced mortality. In support of this, two other ecological studies have explored the correlation between mean vitamin D levels and incidence and COVID-19 related mortality across selected European countries (Ilie et al., 2020;Singh et al., 2020). Both studies found a strong inverse correlation between mean vitamin D level in various European countries and COVID-19 mortality (Ilie et al., 2020;Singh et al., 2020) or COVID-19 incidence (Ilie et al., 2020) which was also supported by the results of the systematic review by Laird et al. (2020). However, ecological approaches are at risk of potential confounding effects given the variation in measurements of a dependent variable such as number of detected COVID-19 cases; which for example may be significantly different from the number of true cases primarily due to differences in local screening policies (Garg et al., 2020;Gostic et al., 2020;Maruotti et al., 2020). Additionally, none of the abovementioned studies report the duration of time between the identification of first cases in a country and the reported death rates are unknown. It is possible that death rates in a country where cases began to appear recently will appear lower than those in countries fighting the virus for a longer time. For example, Portugal (39 nmol/L) and Sweden (73.5 nmol/L) were reported as having the lowest or highest mean serum vitamin D levels for the populations respectively. However, the number of fatalities as of 16/10/2020 in Sweden are higher than Portugal, contradicting the conclusions drawn by the authors. Interestingly, two cohort studies exploring the potential association of vitamin D deficiency and the high incidence of COVID-19 in BAME individuals have not reported positive correlations between the two (Hastie et al., 2020;Raisi-Estabragh et al., 2020). Raisi-Estabragh et al. (2020) specifically explore whether cardiometabolic exposures, including vitamin D, socioeconomic, lifestyle and behavioral exposures could explain the differential patterns of COVID-19 incidence observed in male and BAME patient populations. The authors could not conclude that sex and ethnicity differential pattern of COVID-19 were explained adequately by variations in cardiometabolic factors, 25(OH)-vitamin D levels or socio-economic factors, however, a sensitivity analysis specifically exploring the interaction of BAME ethnicity and vitamin D levels was not conducted. Hastie et al. (2020) found no association between plasma 25(OH)D concentrations and COVID-19 infection in the UK biobank (Faul et al., 2020) nor in the BAME population after adjusting for potential confounders. However, the results need to be interpreted with caution due to the over adjustment of the analyses including mediators of disease and the potential error in the outcome assessment and vitamin D levels Roy et al., 2020). Both studies found significantly lower levels of vitamin D in the COVID-19 positive population and both studies assessed baseline vitamin D levels (samples taken between 2006 and 2010). Some authors have suggested that the interaction between BAME ethnicity, worse COVID-19 outcomes and vitamin D deficiency is mediated by other factors such as obesity and socioeconomic factors (Chakhtoura et al., 2020;Muscogiuri et al., 2020;Orrù et al., 2020;Sardu et al., 2020a;Townsend et al., 2020). These hypotheses need to be further investigated in observational studies.
We can conclude here that there is a potential benefit of vitamin D supplementation for prevention and treatment of COVID-19 patients, however the true association between mean plasma 25(OH)D levels and COVID-19 outcomes remains unclear and needs to be further explored with welldesigned RCTs.

Vitamin D Supplementation for COVID-19 Prevention/Treatment
Considering that adverse events related to vitamin D toxicity are rare (Kimball et al., 2017;McCartney and Byrne, 2020;Slominski et al., 2020), experts have since recommended vitamin D supplementation of 10,000 IU/d of vitamin D3 for a few weeks to rapidly raise 25(OH)D levels, followed by 5000 IU/d, with the goal of raising 25(OH)D levels to >40-60 ng/mL (100-150 nmol/L) for populations at risk of influenza and/or COVID-19 (Aloia and Li-Ng, 2007;Martineau et al., 2009Martineau et al., , 2019Urashima et al., 2010;Alexander et al., 2020;Chakhtoura et al., 2020;Siuka et al., 2020;Tramontana et al., 2020;van der Meulen, 2020). For treatment of COVID-19 patients, higher vitamin D3 doses were recommended by the authors (Ebadi and Montano-Loza, 2020;Liu et al., 2020;Slominski et al., 2020). A recent case series publication on 4 COVID-19 patients with vitamin D deficiency reported that all 4 patients treated with cholecalciferol 1000 IU/d or ergocalciferol 50,000 IU/d had a good outcome and were discharged within 14 days; with biomarker changes from day 0-6 observed (Ohaegbulam et al., 2020). Caccialanza et al. (2020) have also recently published a protocol implementation for early nutritional supplementation of non-critically ill patients hospitalized with COVID-19 in Italy, whereby cholecalciferol is promptly prescribed according to blood tests results [50,000 IU/wk. if 25(OH)D < 20 ng/mL; 25,000 IU/wk. if 25(OH)D 20 to <30 ng/mL]. Beigmohammadi et al. (2020) RCT for patients with COVID-19 admitted to intensive care will supplement the intervention arm with a number of vitamins including a single dose of Vitamin D 600,000 IU to assess severity and mortality rate. Quesada-Gomez et al. (2020) suggested that the stimulation of VDR in patients with SARS-CoV-2 infection, may reduce ARDS due to VDR's capacity as a negative regulator of renin-angiotensin system in alveolar cells and recommended oral calciferol supplementation as a therapy approach. However, according to our literature review the exact efficacy of the above-mentioned measures for prevention of, or as an adjuvant treatment for COVID-19 remains to be determined.
The results of 9 ongoing RCTs (26 registered) on vitamin D are early awaited as they will help to elucidate the role of vitamin D in COVID-19 infection and its potential benefit in prevention and treatment of COVID-19 disease (Dos Santos, 2020). These include: 4 RCTs exploring survival rate on COVID-19 patients [NCT04334005 (single dose of 25,000 UI of vitamin D), ZnD3-CoVici (NCT0435149) (2000 IU per day for 2 months), CoVitTrial (NCT04344041) (single dose of cholecalciferol 400,000 IU compared to a single dose of 50,000 IU), COVIDIOL (NCT04366908)] [Calcifediol or not (calcifediol in soft capsules: 0.532 mg on the day of admission and 0.266 mg on day 3 and 7 and then weekly until discharge or ICU admission)]; 2 studying association with COVID symptoms and hospitalization [LEAD COVID-19 (50,000 IU of vitamin D, weekly for 2 weeks), HELPCOVID-19 (NCT04335084) (No dose available)] and 1 study exploring the potential preventive effect of calcifediol in healthy population [(NCT04386850) (25 mcg of 25OHD3 once daily at bedtime for 2 months and the control group will receive placebo daily for 2 months)] (Quesada-Gomez et al., 2020). Moreover, Martineau (2020) have launched a RCT (COVIDENCE UK) which aim to recruit 12000 individuals to investigate how diet and lifestyle factors might influence transmission of SARS-CoV-2, severity of COVID-19 symptoms, speed of recovery, and any long-term effects.

CONCLUSION
The spread of novel SARS-CoV-2 virus, resulting in COVID-19 has attracted renewed interest in vitamin D; being dubbed the 'magic-bullet' or 'cure' for COVID-19. The aim of this publication was to assess the current evidence between vitamin D deficiency and increased morbidity/mortality with COVID-19 respiratory dysfunction. Lack of standardization of the clinical cut-offs for diagnosing hypovitaminosis D, the heterogeneity in supplementation regimes and duration, and the many self-reported measures of exposures and outcomes in RCTs make robust metanalyses difficult. Collectively, our scoping review highlights that the epidemiological studies exploring an association between circulating levels of 25[OH]D (the biomarker of vitamin D status) and incidence and severity of COVID-19 show a positive association but are mainly observational studies and limited in number. However, two ecological studies have observed inverse correlations between vitamin D status and incidence and mortality of COVID-19 (Ilie et al., 2020;Singh et al., 2020), also supported by other studies including a systematic review (D'Avolio et al., 2020;Kaufman et al., 2020;Laird et al., 2020;Meltzer et al., 2020;Panagiotou et al., 2020). Supplementation in a small case study of COVID-19 patients had a positive outcome , supporting previous observations in ICU mechanically ventilated patients (Perron and Lee, 2013;Han et al., 2016;Smith et al., 2018). Based on the above described studies vitamin D supplementation can be concluded to be preventive for ARIs in adults, but uncertainty around therapeutic benefit/dosing required remains.
In conclusion, the evidence presented here supports further observational and multicenter well-designed randomized clinical studies on the role of vitamin D in preventing and/or treating COVID-19 infections. The overlap in the vitamin D associated biological pathways with the dysregulation reported to drive COVID-19 outcomes warrants further investigation, as does the role that vitamin D levels at time of presentation of COVID-19 may have as predictive biomarkers of disease severity. It is important for future work that implications for at-risk populations (BAME, elderly, pregnant, immunocompromised, obese) are taken into consideration when defining the vitamin D dose regimens and duration. Moreover, clinical trials focused on inpatients presenting with COVID-19 are needed to provide a better understanding of the potential of vitamin D as an adjunct treatment.

AUTHOR'S NOTE
AS and SI affirm that the manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned (and, if relevant, registered) have been explained.

AUTHOR CONTRIBUTIONS
AS and SI conceived the study idea. AS, JK, and KB performed the literature search. AS, KB, and JK acquired the data and performed the credibility assessments. AS, KB, and SI developed the core criteria used in this study. AS wrote the first draft of the manuscript. AS, KB, DJ, MV, and SI critically revised the manuscript. AS and SI had full access to all of the data in the study, and took responsibility for the integrity of the data and the accuracy of the data analysis. AS and SI were the guarantors. AS attested that all listed authors meet authorship criteria and that no others meeting the criteria have been omitted. All the authors contributed to the article and approved the submitted version.