Discussion
In summary, statistically significant differences in SARS-CoV-2 seropositivity were found within the entire cohort, and several subgroups across the four VD categories. The subgroups included both sex subgroups; age >50 years subgroup; BAME subgroup; BMI<30 kg/m² subgroup and both comorbidity subgroups. There were no statistically significant differences between the seropositivity rates in the paired subgroups in any VD category. All curves displayed varying U-shaped curves, with ethnicity showing the most variation between subgroups: BAME subjects showed marked increase in seropositivity as VD level moved into the deficient areas, whereas the white cohort showed less variation in seropositivity across the VD continuum.
Seropositivity rate was significantly higher in the severely deficient category in comparison with any of the other VD categories within both the total population and the male subgroup. After a reduction in seropositivity rate between a VD level of 30 nmol/L and ~80 nmol/L, there was an unexpected increase in seropositivity rate beyond VD ~80 nmol/L. This resulted in a U-shaped curve that was reflected within all the subgroups.
A significantly larger proportion of the BAME population were severely VD deficient relative to the total white population. This finding was consistent across the total population, but also both the SARS-CoV-2-positive and SARS-CoV-2-negative BAME groups individually. These results add to the literature that individuals of darker skin are more likely to be VD deficient because of a greater melanin content, reducing the availability of ultraviolet B (UVB) rays for VD3 synthesis.7 This evidence is also consistent with a recent study in the UK which found a significantly higher proportion of VD deficiency among newborns within the BAME ethnic group.13 Regardless of whether VDD is a cause or consequence (or both) of COVID-19, further investigation into VD supplementation in those of BAME ethnic group is warranted.
The U-shaped curves observed in this study challenge the current understanding of the relationship between VD and COVID-19, in which susceptibility is assumed to reduce with increasing VD levels.14 No published studies regarding COVID-19 have reproduced such results, that is, those demonstrating increasing seropositivity at both ends of the VD spectrum. This U-shaped curve, however, has appeared in the wider literature regarding VD. For example, a large sample (n=24 094) study by Amrein et al (2014) investigated the relationship between hospital admission VD and mortality.15 Interestingly, after a reduction in mortality with increasing VD levels, 90-day mortality rate began to increase beyond ~125 nmol/L levels, with an independent predictor of mortality beyond 150 nmol/L. In contrast, a review in 2016 identified all the studies (at the time) that investigated VD level against multiple outcomes within a U-shaped distribution: they concluded that the results were unlikely to be valid due to the lack of consideration of vulnerable individuals taking VD supplementation.16 There has been increased interest during the course of the pandemic of the role of VD supplementation to protect from COVID-19,17 with evidence from meta-analysis that supplementation reduces risk of acute respiratory tract infection.18 However, in our study, only eight individuals were on VD supplementation at the time of enrolment. Only two individuals had VD levels over 80 nmol/L from a total of 51 subjects and therefore would have minimal contribution to the observed U-shaped effect.
The only studies where the U-shaped curves were possibly significant were associated with allergy, due to a changing balance in the Th1/Th2 axis.16 Due to U-shaped curves being identified in several studies against disease risk, further investigation is required to understand this phenomenon. One common explanation is whether the cause of increased disease risk passed a certain VD is due to the individual being on supplementation due to being a clinically vulnerable individual. However, our cohort were healthcare staff with few comorbidities. One possible mechanism of the U-shape curve could be due to induction of fibroblast growth factor-23 at higher levels of 25(OH)D (>100 nmol/L 25(OH)D) and the consequent inhibition of 1-hydroxylase in immune cells.19
There has been interest in the role of VD binding protein (DBP) in relation to COVID-19 infection. This study did not measure DBP; polymorphisms in the DBP gene have been shown to be associated with severity of COVID-19 infection.20 DBP is the main serum binder of 25(OH)D and has an indirect role in the activation of innate immune cells. DBP levels have been shown to drop in patients with ARDS,4 which may indirectly lead to increase in free 25(OH)D which may enhance the availability of 25(OH)D to immune cells. Furthermore, single nucleotide polymorphisms contribute a relatively small proportion of overall VD availability.21 Therefore, the role of DBP in the setting of COVID-19 remains unclear.
Limitations
There are a number of limitations to this study. The aggregation of multiple ethnicities into a singular ‘BAME’ subgroup provided a challenge, and due to the study population did not allow further subcategorisation of ethnicity for which there may be further susceptibilities.6 The study was conducted during the first wave of the COVID-19 pandemic and therefore predated mass testing for COVID-19 and PCR testing was only being conducted within hospitalised patients. Individuals were recruited based on displayed symptoms of COVID-19 and isolation. A limitation is that we have looked at seropositivity, rather than infection, though the used assay has a very high sensitivity for PCR proven disease. Other members of staff isolating for symptoms suggestive of COVID-19 who were not antibody positive may have had other respiratory tract infection or alternative diagnosis. Inclusion bias is another limitation raised consistently; individuals were recruited based on displayed symptoms of COVID-19 and isolation. This potentially increased the risk of selecting individuals that were more susceptible to COVID-19, regardless of their VD levels. Furthermore, healthcare workers had a higher risk of COVID-19 infection, particularly those who were patient facing, and so the population is not easily generalisable outside of a healthcare population. The amount of time the subjects may have been infected was not considered, and so VD levels versus severity of disease (alongside confounding influences) will affect the ability to interpret the results. As the median duration from symptom onset to testing was 48 days, this would have allowed sufficient time to develop an antibody response following infection. Another issue which is raised consistently in similar literature is seasonal variability in VD;22 however the participants were recruited within a tight timeframe in May, which should reduce effect of seasonal variation. There could be other confounding factors such as nutritional intake and use of fortified foods which were not assessed in the study and are another limitation. The U-shaped curves were derived by grouping the samples into four VD bins, principally because there were limited numbers at each end of the VD spectrum.