Correlation of Vitamin D deficiency with chest X-rays severity scores and different inflammatory markers in severe and critical COVID-19 patients

Summary Objective To determine the relationship between Vitamin D deficiency with Chest X-Rays severity score and Different Inflammatory Markers in Severe and Critical COVID-19 Patients. Design A cross-sectional study Setting The study was conducted in COVID-19 isolation units at Mardan Medical Complex Teaching Hospital (MMCTH) and Bacha Khan Medical College, Pakistan Participants 206 patients who tested positive for COVID-19 by PCR were included in the final analysis. Data Collection/Intervention We collected demographic, comorbidity, laboratory, and clinical outcome data from the electronic records of admitted, deceased, or discharged patients. Main outcome measure Frequency of symptoms, comorbidities, mortality and morbidity, chest x-ray severity scores, different inflammatory markers in Vitamin D deficient Covid-19 patients Results 128(62.14%) were severe and 78(37.5%) were critical COVID-19 patients. The whole cohort had 82(39.80%) males and 124(60.20%) females, with a median age of 55 IQR (50-73). Study participants' median Vitamin D level was 14.01ng/ml, with a minimum of 7.5ng/ml and a maximum of 70.8ng/ml. 67/206 patients died, with a fatality ratio of 32.5%. 54/67(80.59%) suffered from one or more comorbid conditions. Conclusion Low Vitamin D levels were linked to a higher risk of death, higher x-ray severity scores, and different inflammatory markers. Vitamin D levels greater than 30ng/ml for older patients and greater than 40ng/ml in older patients with comorbidities were associated with reduced severity and mortality in patients with COVID-19. Funding None declared


INTRODUCTION
Vitamin D boosts the innate immune response, lowering the risk of infection from SARS-CoV-2 exposure. Vitamin D is also involved in regulating the adaptive immune system and inflammation. Infected people with sufficient levels are less likely to develop hyperinflammatory (severe) COVID-19 (the cytokine or bradykinin "storm"). 1 Vitamin D suppresses viral multiplication by releasing defensin and cathelicidin proteins from macrophages and monocytes. 2,3 To eliminate respiratory pathogens, Vitamin D induces apoptosis and autophagy in the affected epithelium. 4 Some COVID-19 patients with severe symptoms had low T-lymphocyte counts. 5 Vitamin D supplementation boosts T-lymphocyte levels 6 , supporting the idea that it could help cure COVID-19 or reduce the severity and progression to mortality.
One of the pandemic's most serious issues is severe COVID-19 progression in some patients. Thrombotic events and cytokine storms have been linked to severe COVID-19 infection. These events cause fatalities. [7][8] Vitamin D reduces cytokine storm risk and controls thrombotic pathways. 9,10 Vitamin D deficiency has been directly linked to increased COVID-19 severity and mortality. 11,12 In recent years, Vitamin D deficiency has increased the risk of multiple illnesses, including systemic infection. 13 Since Vitamin D plays an immunomodulation role 14 , production of antiviral peptides to enhance innate immunity 3 and bolstering mucosal defenses, Vitamin D deficiency can impact immunological activities. In clinical investigations, acute respiratory illnesses, such as influenza ep-SARS-CoV-2 is rapidly spreading and poses an unpredictable risk to human health worldwide. 20 Acute respiratory disease syndrome (ARDS) 21 appears to be caused by SARS-CoV-2 predominantly through the immune evasion mechanism during infection, followed by hyper response and cytokine storm in some individuals. 22 Angiotensin-converting enzyme 2 allows SARS-CoV-2 to infiltrate alveolar and intestinal epithelial cells at the cellular level. 24 When the renin-angiotensin system is out of whack, it might cause an influx of cytokines, which can eventually cause lethal acute respiratory distress syndrome. 23 As a result of these findings, the impact of Vitamin D deficiency on COVID-19 infection and outcomes has gathered substantial attention. Studies on the association between Vitamin D deficiency and COVID-19 mortality are scarce, and most current research focuses on COVID-19 infection, severity, and therapy. So, the literature on this topic calls for more clinical investigations. 24,25,26 It is crucial to learn why COVID-19 is so different in terms of mortality and severity worldwide. Better nutrition's potential to boost immunity is a significant consideration. Vitamin D and other nutrients play essential roles in immune system function. However, Vitamin D's potential function in reducing COVID-19 infection and mortality is little understood. Therefore, this study aimed to determine if there is a correlation between Vitamin D deficiency with chest x-ray severity scores and inflammatory marker levels in severely and critically ill COVID-19 patients.

METHODS
This cross-sectional study was conducted in the COVID-19 isolation units of the Mardan Medical Complex (MMC) in Khyber Pakhtunkhwa, Pakistan, from Novem-ber9, 2021, to March21, 2022. The sample was raised using the convenience sampling technique. We collected demographic, comorbidity, laboratory, and clinical outcome data from the electronic records and the charts of deceased or discharged admitted patients. COVID-19 patients in this study were split into two groups: Severe and Critical.
Respiratory distress equivalent to 30 breaths per minute, oxygen saturation <93% at room air, arterial oxygen partial pressure (PaO2), or FiO2 of 300mmHg corresponding to 0.133kPa) were considered "severe" in COVID-19 patients. As determined by an independent medical practitioner, if a patient needed mechanical ventilation, was in septic shock, or required to be admitted to an intensive care unit, they were considered "critical" (ICU). 27

Inclusion and exclusion criteria
Adult patients over the age of 18 years who tested positive for COVID-19 by rRT-PCR and were classified as severe or critical COVID-19 patients were included in the final analysis after providing written informed consent within the first 24 hours of admission. Patients who tested negative on rRT-PCR for COVID-19, patients below the age of 18 years, patients referred to another medical facility during hospitalization, patients hospitalized more than once, and patients whose Vitamin D test results were found missing were excluded.

Inflammatory markers and Vitamin D measurements
HORIBA ABX micros es60 hematology analyzer, Roach Cobas e411 Immunoassay Analyzer, and an Automated Chemistry Analyzer Architect c4000 abbot were used to measure hemoglobin, total leukocytes count, lymphocytes count, neutrophils count, platelets count, D-Dimer, C-Reactive Protein, Ferritin, Lactate Dehydrogenase, and Vitamin D. Many guidelines define Vitamin D deficiency as serum Vitamin D levels <20ng/mL. The Mayo Clinic's definition of Vitamin D insufficiency (<30ng/ml) was used in this study. 28

Ethical considerations
Patients were enrolled in this study after authorization by Institutional Review Boards Reference No 192/BKMC, Dated 01/11/2021, of Mardan Medical Complex and Bacha Khan Medical College Pakistan. Participants gave their fully informed consent and were free to opt-in or out of the study at any point.
Participants were required to sign the box showing their informed consent, confirming that they had read the research purpose, understood it, and wanted to participate. To acquire a clear image of the study, uneducated participants were instructed in their native tongue about its significance. All participants received assurances of confidentiality, and all collected data were treated with confidentiality.

Statistical analysis
Data are presented as appropriate, as the median and interquartile range (IQR), or as frequencies and percentages. To explore the connection between categorical factors such as gender, age, comorbidities, symptoms, and chest radiography, the Pearson Chi-Square Test and Fisher Exact Test were utilized. The Kruskal Wallis One-Way Analysis of Variance (ANOVA) was used to test for inter-group differences in severe patients, critical patients, and Vitamin D deficiency. The 95% confidence interval was used for all statistical tests (95%CI). The Pearson Correlation Method was used to investigate the correlation between Vitamin D Deficiency with Chest Xray severity scores and different inflammatory Markers. Relationships between Vitamin D Deficiency with Chest X-ray severity scores and different inflammatory Markers were studied using scatter plots. SPSS version 26 was used for all statistical analyses. P-value <0.05 was considered statistically significant.

Inflammatory markers of Severe and Critical COVID-19 patients
In COVID-19 patients, the inflammatory marker values in the severe group with Vitamin D levels <30ng/ml were considerably higher than those in the severe group with Vitamin D levels >30ng/ml, showing a significant difference between the two groups. In the critical group, the inflammatory markers were much greater than in the severe group of vitamin D levels <30ng/ml vs >30ng/ml. When comparing severely ill and critically ill COVID-19 patients, however, both groups of patients revealed statistically significant evidence that Vitamin D levels >30ng/ml reduced the severity of inflammatory markers in COVID-19 patients. (Table 3

Chest X-Ray Severity Scores of Severe and Critical COVID-19 patients
The total Chest X-ray severity scores were 128(62.14%) in the severe group and 78(37.86%) in the critical group. The number of Chest X-ray severity scores was lowest in critical COVID-19 patients, with 12(15.38%) in >30ng/ml Vitamin D group compared to severe COVID-19 patients, 30(23.43%) had the lowest >30ng/ml Vitamin D group. In critical COVID-19 patients, the number of patients increased by 66(84.61%) when <30ng/ml vitamin levels were employed compared to severe COVID-19 patients when <30ng/ml vitamin levels were used, 98(76.56%) patients had Vitamin D levels <30ng/ml. Thus, Vitamin D levels of >30ng/ml reduced the chest Xray severity scores in patients diagnosed with COVID-19. (Table 3,4)

Correlation between Vitamin D deficiency with inflammatory marker and chest X-ray Severity Scores
A substantial positive and robust negative association was found in the Pearson correlation and scatter plot between Vitamin D and chest X-ray severity score and inflammatory markers. Vitamin D had a high positive correlation with hemoglobin and lymphocytes in severe and critical patients but a negative correlation with total leukocyte count, neutrophils, Platelets, C-reactive protein, Lactate dehydrogenase, D-dimer, ferritin, and chest Xray Rale score. (Table 5

DISCUSSION
In this study, patients with Vitamin D deficiency, particularly severe and critical patients, as well as older age and with multiple comorbidities, had a higher risk of death from COVID-19 infection. Because higher Vitamin D levels were found to have a strong negative relationship with worse chest x-ray severity scores and excess inflammation as measured by inflammatory markers, adequate Vitamin D levels may protect the immune system, particularly in the elderly. In addition, Vitamin D seemed to mute the disadvantages of having multiple comorbidities in severe and critical COVID-19 patients. Our findings suggest that Vitamin D deficiency may increase the incidence of COVID-19 severity and its progression to mortality.
This study's favourable results concerning Vitamin D sufficiency being somewhat protective in COVID-19 are consistent with earlier findings. Receiving some sun is probably required to recuperate from Vitamin D insufficiency. Total sun hours from March to April were 333.4 in 2019 and 349.4 in 2020. The difference in sunlight was only 4.8%, which is insignificant; however, Vitamin D supplementation and dietary habits are likely to be key issues. 30 The role of Vitamin D during the COVID-19 pandemic in reducing infection risk, disease severity, and death was fiercely disputed. 31 Just as India has a high rate of Vitamin D deficiency, 32 52.91% of COVID-19 patients in Pakistan had Vitamin D levels of <20ng/ml in our study. The proportion was 79.11% when a <30ng/mL limit was employed to evaluate Vitamin D deficiency levels.
Kernan, K. et al. found that ferritin is a significant immune dysregulation mediator through its direct immunosuppressive and pro-inflammatory activities, particularly in severe hyperferritinemia. An essential host defensive mechanism during infection is elevated ferritin levels, which remove defence immune cell function. Additionally, it could be protective by reducing free radical generation and regulating immunomodulation. 33 In a study from Spain, Vitamin D insufficiency was found to dramatically boost ferritin levels, which was likewise observed in our study. 34 An Indian study compared previously published data on average Vitamin D levels with death reports from multiple states and concluded that Death rates might be more significant in Vitamin D deficient areas. However, this study had substantial limitations because individual Vitamin D levels were not tested, and most of the historical data used was heterogeneous and limited. 32 Our findings are consistent with other hospital-based studies, which found that low Vitamin D levels were linked to severe/critical COVID-19 disease, 35,36 greater rates of ICU admission, 37 and higher levels of inflammatory markers, 38 and death. 33 An interesting finding of this study 39 was that Vitamin D insufficiency is associated with increased elderly morbidity.
In this study's Severe and Critically COVID-19 patients, there appears to be a relationship between Vitamin D deficiency and high D-dimer levels. Elevated D-dimer levels signal the activation of pro-inflammatory cytokine cascades (and downregulation of the anti-inflammatory cytokine cascade). When D-dimer levels are high, the risk of death increases. 40 The cytokine storm observed in COVID-19 infection may be due to Vitamin D deficiency-induced dysregulation of innate and adaptive immunity. Vitamin D has an impact on both bacterial and viral innate immune responses. It works by inhibiting pro-inflammatory cytokines includes such as interleukin (IL)-1, interleukin (IL)-6, interleukin (IL)-8, interleukin (IL)-12, and tumor necrosis factor-alpha TNF-α. 41 Vitamin D stimulates the development of T regulatory cells while inhibiting the transition of naive T cells into pro-inflammatory Th17 cells. 42,43 Vitamin D has anti-inflammatory effects in human alveolar epithelial cells and helps wound healing. 44 Vitamin D also helps maintain the endothelium's health, and a lack of it causes vascular permeability and leakage. [45][46] Men are more vulnerable to ACE2 receptor dysregulation and, presumably, higher COVID-19 morbidity due to Vitamin D deficiency since it increases the X-chromosome-associated "Renin-Angiotensin" System (RAS) activity. 47 Vitamin D and CRP had a -0.879 correlation value, which was significant in inflammatory disorders (p=0.001). 48 taking COVID-19 into account as an inflammatory condition. Our study found a correlation coefficient of -0.795 with a p-value=0.000 for severe patients and a correlation coefficient of -0.656 for critical patients. Multiple connections between lymphocyte immunological markers and Vitamin D insufficiency were discovered in individuals with CRVD, CAD, T2DM, and hypertension. 49 Our study found associations between lymphocyte inflammatory markers and Vitamin D deficiency, such as COVID-19 patients who showed higher percentages of severe and critical cases and were more likely to die.
Older COVID-19 patients with comorbidities were more likely to indicate low immune function, with higher rates of comorbidities including diabetes, hypertension, asthma, COPD, hepatitis B, hepatitis C, and cancer. This was supported by the proportion of patients with a decreased number of lymphocytes and an increase in inflammatory markers in older COVID-19 patients being significantly higher than that in non-comorbid COVID-19 patients. Pimental et al. found that neutrophil counts increased in the low Vitamin D group compared to the normal Vitamin D group 50 and our findings were comparable.
Campi et al. found that Vitamin D levels were significantly lower in patients admitted to ICU with COVID-19, as were LDH and platelet levels. 51 Our investigation found that Vitamin D deficiency was also associated with lower LDH and platelet levels in COVID-19 patients. In addition, this study discovered a positive relationship between Vitamin D deficiency and hemoglobin level, consistent with previous studies demonstrating that Vitamin D deficiency with low hemoglobin level and rachitic symptoms were associated with increased severity of acute lower respiratory tract infections. 52 Waheed, S. et al. found that Chest X-ray at presentation showed bilateral ground-glass appearance in Vitamin Ddeficient COVID-19 patients. After three days of Vitamin D supplementation, it showed improvement of chest X-ray ground-glass appearance. 53 In another study, Breslin É. et al. found that low Vitamin D levels were associated with a higher risk of infiltrates on chest X-rays. 54 in our investigation, a significant negative correlation between chest X-ray severity scores and Vitamin D deficiency in severe and critical COVID-19 patients, were discovered.
The conclusions of the current study have several limitations. First, the investigation was done in a single centre (Mardan, Pakistan) multi-centre study would provide more strong conclusions. Second, From November 2021 to April 2022, less sun exposure, a direct source of Vitamin D, may aggravate the illness. Despite this limitation, winter seasonal variation demonstrates that this study population was significantly less exposed to sunlight. In addition, because this study only included individuals with low sun exposure, these findings may not apply to those with high sun exposure. Third, only quantitative indicators were measured, with no regard for the time between infection and admission. This information was unavailable at the time of admission. The time between infection and admission is a significant independent predictor of infection risk. In COVID-19 patients, timely hospitalization has a significant beneficial effect on reducing severity progression to mortality.
Fourth, when evaluating inflammatory symptoms, individuals with asymptomatic, mild, or moderate presentations were advised to stay at home and were not included in the current study. Our findings show that Vitamin D deficiency is not common in these patients, so the majority of patients admitted to the hospital were in a severe and critical stage of the disease, were deficient in Vitamin D, and linked to an increased incidence of COVID-19 hospitalization. Fifth, most of our patients were elderly, an independent risk factor for COVID-19. It is suggested that countries in which elderly people live among the general population should impose more severe preventive mitigation measures than countries in which elderly people are kept apart from the general population. This is due to the fact that the disease spreads more quickly and severely among elderly patients with Vitamin D deficiency than it does among younger patients.

CONCLUSION
Vitamin D modulates the immune system in addition to improving the ability of the innate immune system to combat COVID-19 infection. Vitamin D is also involved in the regulation of the adaptive immune system and inflammation. Vitamin D deficiency was correlated with an increase in several inflammatory markers and chest x-ray severity scores. In Severe and Critical COVID-19, Vitamin D levels greater than 30ng/ml in older age and greater than 40ng/ml in older patients with comorbidities were associated with reduced severity and mortality. Randomised controlled trials and large population research should be conducted to validate these preliminary findings.