The association between vitamin D deficiency and the clinical outcomes of hospitalized COVID-19 patients

Background Vitamin D deficiency is an emerging public health problem that affects more than one billion people worldwide. Vitamin D has been shown to be effective in preventing and reducing the severity of viral respiratory diseases, including influenza. However, the role of vitamin D in COVID-19 infection remains controversial. This study aimed to analyze the association of vitamin D deficiency on the clinical outcome of hospitalized COVID-19 patients. Methods A prospective cohort study was conducted among hospitalized COVID-19 patients at two COVID-19 referral hospitals in Indonesia from October 2021 until February 2022. Results The median serum 25(OH)D level in 191 hospitalized COVID-19 patients was 13.6 [IQR=10.98] ng/mL. The serum 25(OH)D levels were significantly lower among COVID-19 patients with vitamin D deficiency who had cardiovascular disease (p-value=0.04), the use of a ventilator (p-value=0.004), more severe COVID-19 cases (p-value=0.047), and mortality (p-value=0.002). Furthermore, serum 25(OH)D levels were significantly different between patients with mild and severe COVID-19 cases (p-value=0.019). Serum 25(OH)D levels in moderate and severe COVID-19 cases were significantly different (p-value=0.031). Lower serum 25(OH)D levels were significantly associated with an increased number of comorbidities (p-value=0.03), the severity of COVID-19 (p-value=0.002), and the use of mechanical ventilation (p-value=0.032). Mortality was found in 7.3% of patients with deficient vitamin D levels. However, patients with either sufficient or insufficient vitamin D levels did not develop mortality. Conclusions COVID-19 patients with vitamin D deficiency were significantly associated with having cardiovascular disease, mortality, more severe COVID-19 cases, and the used of mechanical ventilation. Lower serum 25(OH)D levels were associated with an increased number of comorbidities, COVID-19 severity, and the use of mechanical-ventilation. Thus, we suggest hospitalized COVID-19 patients to reach a sufficient vitamin D status to improve the clinical outcome of the disease.

who had cardiovascular disease (p-value=0.04), the use of a ventilator (p-value=0.004),more severe COVID-19 cases (p-value=0.047), and mortality (p-value=0.002).Furthermore, serum 25(OH)D levels were significantly different between patients with mild and severe COVID-19 cases (p-value=0.019).Serum 25(OH)D levels in moderate and severe COVID-19 cases were significantly different (p-value=0.031).Lower serum 25(OH)D levels were significantly associated with an increased number of comorbidities (p-value=0.03), the severity of COVID-19 (p-value=0.002), and the use of mechanical ventilation (p-value=0.032).Mortality was found in 7.3% of patients with deficient vitamin D levels.However, patients with either sufficient or insufficient vitamin D levels did not develop mortality.

Conclusions
COVID-19 patients with vitamin D deficiency were significantly associated with having cardiovascular disease, mortality, more severe COVID-19 cases, and the used of mechanical ventilation.Lower serum 25(OH)D levels were associated with an increased number of comorbidities, COVID-19 severity, and the use of mechanicalventilation.Thus, we suggest hospitalized COVID-19 patients to reach a sufficient vitamin D status to improve the clinical outcome of the disease.

Introduction
Coronavirus Disease-2019 (COVID- 19) is a rapidly spreading pandemic disease caused by Severe Acute Respiratory Syndrome Corona-Virus-2 (SARS-CoV-2), the seventh coronavirus that infect humans.2][3][4][5] There is a lack of evidence-based data about the risk factors for the infection, as well as the most effective treatments.Current hospital-based management is focused on the excessive inflammatory response and respiratory support due to the fact that targeted antiviral therapies have not been widely accessible. 6tamin D is a versatile steroid hormone that plays multiple roles in the body, including the regulation of bone and calcium metabolism. 7,8It also supports the innate and adaptive immune systems against respiratory viruses. 7,9It controls the innate immune system by stimulating the synthesis of antimicrobial peptides such as IL-37, cathelicidins, and defensivins. 1,10,114][15][16][17] Vitamin D deficiency was widespread across Southeast Asian countries, despite extensive exposure to sunlight. 18Based on current evidence, vitamin D helps prevent and mitigate the severity of viral respiratory diseases, such as influenza. 4,7,19,20However, the role of vitamin D in COVID-19 infection remains unclear. 4,7rthermore, this is the first study that analyzes the International Severe Acute Respiratory and Emerging Infections Consortium Coronavirus Clinical Characterisation Consortium (ISARIC-4C) score in a group of patients with vitamin D deficiency.The ISARIC 4C mortality score provides an approach for evaluating the risk of mortality upon admission by utilizing demographic and physiological parameters.This scoring system is derived from a comprehensive population cohort study conducted at the national level in the United Kingdom. 21Understanding the clinical course of COVID-19 is crucial until a viable vaccination becomes widely accessible, due to the lack of specific therapies and the tremendous health and economic impact of the pandemic. 1,22In this situation, deficient vitamin D is a modifiable risk factor due to its safety and affordability. 1,23,24The aim of this study was to assess the association of vitamin D deficiency on the clinical outcome of hospitalized COVID-19 patients.Thus, a comprehensive understanding can be obtained as a promising strategy for evaluating the prognosis and treatment for COVID-19 patients.

Study design
This study was a cross-sectional study conducted at two COVID-19 referral hospitals in Jakarta, Indonesia (National Emergency Hospital Wisma Atlet Kemayoran and Dr. Cipto Mangunkusumo General Hospital), from October 2021 until February 2022.The included subjects were COVID-19 positive (confirmed by reverse transcription-polymerase chain reaction [RT-PCR]) and admitted to the hospital; aged 18 years and older.The exclusion criteria were COVID-19 patients with clinically asymptomatic and severely affected COVID-19 patients who arrived using mechanical ventilation prior to admission.This study specifically involved subjects registered with mild, moderate, or severe disease according to WHO interim guidance and Indonesian government policy at admission. 25The STROBE guidelines (Strengthening the Reporting of Observational Studies in Epidemiology) were followed for this study.

REVISED Amendments from Version 3
The reviewer did raise a very important concern which will improve our manuscript.
-The title was updated.
-We have included additional information in the discussion section.
-We have included several factors that are considered to be limitations of our study.
-We added more references to enhance the quality of our paper.
Any further responses from the reviewers can be found at the end of the article

Data collection
The SARS-CoV-2 infection was confirmed through positive RT-PCR obtained from nasal and oropharyngeal swabs collected. 26The examination was carried out in the Biosafety Level 3-facility (BSL-3) with Biological Safety Cabinet Class II (BSC-II).
During the admission, each patient had 3-5 mL of blood collected in an acid citrate dextrose tube from a cuffed venous sample.The samples were transported to the laboratory in a cold chain for the measurement of vitamin D. Vitamin D status was evaluated by measuring serum 25(OH) D or 25-hydroxyvitamin D levels.The results were gathered using Roche Diagnostics' Cobas e411, a competitive electrochemiluminescent protein binding assay.

Statistical analysis
Statistical Package for the Social Sciences (SPSS) version 27 for Macintosh was used to analyze the data that was collected.The serum 25(OH) D levels between two subgroups were analyzed with either Mann-Whitney U test for 2 subgroups and Kruskal-Wallis test for more than 2 subgroups.

Ethical approval
Ethical approval for this study was granted by Ethics Committee of the Faculty of Medicine, Universitas Indonesia (ethical approval number: KET533/UN2.F1/ETIK/PPM.00.01/2021) and by the Ethics Committee of Wisma Atlet Hospital Jakarta (029/KERSDCWA/2021).The Declaration of Helsinki was implemented during this study.

Results
This cross-sectional study included 191 subjects.Before being enrolled, each participant signed a written consent form.The characteristics of the included subjects can be observed in Table 1.From the 191 subjects, 54.5% were female.comorbidities, the severity of COVID-19, and the use of mechanical ventilation.Among 191 patients, mortality was found in 7.3% of patients with deficient vitamin D levels.However, subjects with either sufficient or insufficient vitamin D levels did not develop mortality.

Discussion
1][32] Whereas the skin's absorption of sunlight is established as the primary source of vitamin D, other variables, including age, comorbidities, and skin pigmentation, may alter the vitamin D level. 15,32Based on the skin's sensitivity to ultraviolet (UV) light, the majority of Indonesians have either Fitzpatrick skin phototype IV (with medium to dark brown) or phototype V (dark brown).A lower vitamin D is associated with darker skin pigmentation due to the higher melanin present in darker skin. 18,32,33Other factors, including haze, altitude, and air pollution, also alter the ultraviolet B radiation. 32,34e beneficial effect of vitamin D to reduce the severity of respiratory tract infection remains controversial. 4,76][37] The current study investigated the association of vitamin D deficiency to the clinical outcome of hospitalized patients at two COVID-19 referral hospitals in Indonesia.We found that compared to insufficient and sufficient, those with deficient vitamin D status had more number of comorbidities (Table 2).In COVID-19 patients with deficient vitamin D were significantly associated with cardiovascular disease (Table 3).Our findings was supported by de la Guía-Galipienso et al., that revealed vitamin D deficiency may play a critical role in the initiation of inflammation, myocardial calcification, and endothelial dysfunction, which are risk factors for cardiovascular disease. 27,380][41][42] Left ventricular hypertrophy, vascular dysfunction, and arterial stiffness have been associated with vitamin D deficiency.A deficiency of the vitamin D receptor causes an increase in left ventricular mass and elevated levels of atrial natriuretic peptide, as well as cardiac metalloproteases and disturbances in homeostasis.Furthermore, the development of fibrotic extracellular matrix induces left ventricular dilation. 27,43,44tamin D has been shown to have a number of beneficial effects on the cardiovascular system, including natriuretic peptide secretion, inhibition of the renin-angiotensin-aldosterone system (RAAS), anti-hypertrophic effects, and inhibition of cardiomyocyte proliferation. 39,45,46Calcitriol and its analogues activate VDR, which directly suppresses angiotensin I expression and local angiotensin II synthesis in myocardial, kidney tissue, and renal arteries. 39,47Studies have revealed that vitamin D enhances the anti-hypertensive effects of angiotensin 1-7 by inducing the production of angiotensin-converting enzyme 2 (ACE2). 39,48,49MiR-106b-5p, which acts on juxtaglomerular cells to boost renin synthesis, has been shown to be directly influenced by VDR-deficient immune cells. 39,49reover, vitamin D affects the progression of HF through modulating the production of metalloproteinases.Evidence strongly suggests that vitamin D has an anti-inflammatory effect by preventing nuclear factor kappa B (NF-κB) and promoting the production of IL-10, which have a significant role in the progression of CVD. 39,50,51Vitamin D deficiency induces arterial stiffness and endothelial dysfunction in blood vessels, which in turn leads to enhanced inflammation, endothelial cell malfunction, and atherogenesis. 27,52e severity of COVID-19 were significantly associated with the lower serum 25(OH) D levels (Tables 2, 3 and Figure 1).Vitamin D is an immunomodulatory hormone with antibacterial and anti-inflammatory properties, and it plays a crucial role in the immune system.Vitamin D has been reported to exert its effects against COVID-19 by limiting the viral transmission, diminishing viral replication, and optimizing viral clearance. 27,534][55][56][57] According to the research of Daneshkhah et al., a lack of vitamin D raises C-reaction protein (CRP) levels, which in turn elevates the risk of a cytokine storm. 27,580][61] Thus, increasing vitamin D levels to adequate levels may help to prevent COVID-19 infection and complications. 27,53,54,59,62thermore, Sabico et al. that conducted a multi-center randomized clinical trial in Middle East, a region with high prevalence of vitamin D deficiency, revealed that a daily oral supplementation of 5000 IU vitamin D3 for 2 weeks reduced the recovery time for gustatory sensory loss and cough among patients with mild to moderate COVID-19 symptoms and sub-optimal vitamin D status. 63 the best of our knowledge, this is the first study that analyse the ISARIC-4C score in a group of patients with vitamin D deficiency.We found that serum 25(OH) D levels had no significant association with ISARIC-4C Score (Tables 2, 3).
In contrast with study by Wellbelove et al. that concluded the ISARIC-4C mortality score is good predictors for 30-day mortality in COVID-19 (AUROC of 0.74-0.88). 64The ISARIC-4C consortium established the ISARIC-4C Mortality Score to predict the mortality of hospitalized COVID-19 patients.Multicentre cohort study was conducted among 74,944 participants at 260 different hospitals.However, the ISARIC-4C has been internally validated but not externally validated.Hence, further study is warranted to fully understand the potential of ISARIC-4C as a prognostic tool to classify patients into specific management groups. 6,29,65rum 25(OH) D levels were significantly lower among subjects that used the ventilator (Tables 2, 3).Among all patients, mortality was found in 7.3% of patients with deficient vitamin D levels.However, patients with either sufficient or insufficient vitamin D levels did not develop mortality (Table 2).Serum 25(OH) D levels in vitamin D deficiency subjects were significantly lower in the COVID-19 patients with mortality status (Table 3).Our findings were consistent with the cohort study by Angelidi et al. and the single-center retrospective study by Alguwaihes et al., which discovered that lower 25(OH) D levels were associated with increased mechanical ventilation needs and mortality risk among hospitalized patients. 66,67ior studies have revealed that vitamin D deficiency has been correlated to a 58% increased risk of acute respiratory infection, prolonged mechanical ventilation, and a 10-fold increase in mortality risk. 66,68,69 contrast, a single-center prospective study conducted in the Indian subcontinent, a region with a high prevalence of deficient vitamin D, demonstrated no statistically significant difference in the median length of stay (LOS) between patients with sufficient vitamin D and deficient vitamin D (p-value = 0.176).The LOS for patients with deficient vitamin D was 12 days (95% CI: 10, 12 days), and the LOS for patients with sufficient vitamin D was 11 days (95% CI: 10, 13 days).They also showed that deficient vitamin D (defined as 25(OH) D < 30 ng/mL) in patients with COVID-19 was not associated with the length of hospital stay, the need for mechanical ventilation, or the mortality rate.70 These different results could be explained by different cut-offs to define deficient vitamin D. In our study, a serum 25(OH) D level of less than 20 ng/mL (50 nmol/L) was considered deficient according to the Endocrine Society Clinical Practice Guidelines.27 On the other hand, most hospitalized patients with COVID-19 have numerous comorbidities, and this population tends to have lower vitamin D levels.Another cohort study by Luigi et al., which excluded the effect of reverse causality and the concomitant comorbidities, demonstrated that vitamin D levels upon admission to the hospital can be used to prospectively predict worse outcomes for both severe and non-severe COVID-19.71 Thus, vitamin D levels in such a setting should be interpreted with caution.70 As a steroid hormone, vitamin D interacts with the vitamin D receptor located in the nucleus of cells to have physiologic effects.17,66 The interaction of 25(OH) D with other steroid hormone receptors may have physiological effects similar to glucocorticoids.66,72 Although the underlying mechanisms of vitamin D's protection against severe COVID-19 are unknown, it is established that vitamin D reduces the production of proinflammatory cytokines such as Th1, TNF-α, interferon-β, IL-6, and promotes the production of anti-inflammatory responses such as T regulatory cells and Th2.55,66,[73][74][75] There are several explanations for vitamin D's beneficial effects on critically ill patients.Initially, critically ill patients who are given vitamin D supplements will have their plasma vitamin D concentrations restored.Furthermore, vitamin D regulates the synthesis of immune system effector molecules such as β-defensin and cathelicidin, which are both antimicrobial peptide.55,66,76,77 Cathelicidin enhances the production of anti-inflammatory cytokines while decreasing the synthesis of pro-inflammatory cytokines.As a result, vitamin D deficiency could increase the risk of sepsis and inflammation in severely ill patients by diminishing the immune response and modulatory effects on innate immunity.[78][79][80][81][82][83] Furthermore, several COVID-19 pandemic studies contained some substantial biases and were not representative of the real-world conditions of the COVID-19 pandemic.84,85 In regions where authorities implemented home isolation and social distancing measures, individuals with mild to moderate cases of COVID-19 were generally not hospitalized.84,86 In contrast, in urban areas experiencing a high prevalence of COVID-19 cases and facing constraint intensive care resources, particularly mechanical ventilation, the majority of the hospital admissions were primarily for the individuals with severe or critical cases of COVID-19.84,[87][88][89] Hospitalized people from a single center or a few centers were unlikely to represent the distribution of COVID-19 cases in an area.Also, there were evident selection biases in why and where people were hospitalized in these studies.84,90,91 Multiple studies have reported the presence of censoring among subjects who were still hospitalized leading to potential biases in the interpretation of the findings.84,87,92 On the contrary, we were able to identify every COVID-19 patient in our area, all of whom were admitted to the hospital.We also conducted a comprehensive follow-up for all the patients.The characteristics of our subjects were more similar to they who were exposed to the SARS-CoV-2 infection in a non-epidemic setting.As the result, our study provided a significant potential for widespread applicability and reflected the real-world conditions of the COVID-19 pandemic.84,[93][94][95] The strength of this study lies in the fact that it is the first study to analyze the ISARIC-4C Score in COVID-19 patients with deficient vitamin D. The majority of this study's data were collected during the Omicron variation's development and can be utilized to make comparisons to the Delta variant or any other variants.However, this study has several limitations that should be considered to improve the further research.First, this study did not include a healthy control group as a reference population. Seond, after patients were discharged, serum 25(OH) D levels were not measured.Third, the observational design and small sample size could potentially miss an important finding in this present study.Fourth, we did not collect any information on the use of vitamin D supplementation.Fifth, it was not possible to determine the impact of disease on vitamin D levels due to reverse causality.Furthermore, considering the established associations between vitamin D levels and comorbidities, and relationship between comorbidities and COVID-19 outcomes, it is difficult not to consider the possibility that vitamin D may become an epiphenomenon typically present in those with a more severe disease.
Hence, despite these limitations, our study clearly showed that lower vitamin D levels at admission represent a strong and reliable factor predicting worse outcomes.We demonstrates that hospitalized COVID-19 patients with vitamin D deficiency had a higher risk of using mechanical ventilation and mortality from respiratory failure and other complications.Additionally, a prior meta-analysis revealed that people with a deficient vitamin D level had an increased risk of SARS-CoV-2 infection and COVID-19-related hospitalization.][98]

Conclusion
We found that lower serum 25(OH) D levels were associated with an increased number of comorbidities, COVID-19 severity, and the use of mechanical ventilation.COVID-19 patients with vitamin D deficiency status were significantly associated with having cardiovascular disease, mortality, more severe COVID-19 cases, and the used of high-flow nasal canule (HFNC) or ventilator.This study doesn't diminish the significance of the continuing vaccine effort against the health-economic burden of SARS-CoV-2 infection.As a result, we strongly suggest achieving sufficient vitamin D status, which may serve as an important adjuvant strategy to improve clinical outcomes before vaccines become widely available.COVID-19 outcomes.VD deficiency has been associated with many diseases.However, its supplementation appears to lack conclusions.It will be helpful to show that VD supplementation will help outcomes of COVID-19.Without it, the value of this study is limited.

Is the work clearly and accurately presented and does it cite the current literature? Yes
Is the study design appropriate and is the work technically sound?Partly

Are sufficient details of methods and analysis provided to allow replication by others? Partly
If applicable, is the statistical analysis and its interpretation appropriate?I cannot comment.A qualified statistician is required.

Are all the source data underlying the results available to ensure full reproducibility? Yes
Are the conclusions drawn adequately supported by the results?Partly

Is the work clearly and accurately presented and does it cite the current literature? Partly
Is the study design appropriate and is the work technically sound?Partly

If applicable, is the statistical analysis and its interpretation appropriate? Partly
Are all the source data underlying the results available to ensure full reproducibility?Partly

Are the conclusions drawn adequately supported by the results? Partly
Competing Interests: No competing interests were disclosed.
I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.

1.
The biggest issue in design is as to why were patients with mild COVID 19 illness included and hospitalized.No guidelines recommend hospital admission of mild cases of COVID 19 and inclusion of these cases involves a serious issue in design which would clearly affect the outcomes of the patients and the results of the study.Inclusion and the very hospitalization of such cases is as such flawed and seriously affects the results.

2.
The details of the ISARIC-$C Mortality score should normally be a part of the Methods section.While the authors mention this as the first usage in COVID-19 studies, they have not unfortunately described this in the appropriate section of the manuscript. 3.
An important study from India is missing from the 'Discussion' section.The study is also from an area where we expect abundance of sunlight too.In this study ( Such discrepant studies need to be part of the discussion of a study like the one that authors have conducted and the possible reasons for such dichotomy proposed.

4.
Medicine, Faculty of Medicine, Universitas Indonesia -Dr.Cipto Mangunkusumo General Hospital The manuscript provides an analysis of vitamin D levels in 189 patients with COVID-19 in Indonesia and correlates the vitamin D levels with the outcomes of illness using ISARIC-4C mortality score among others.While the concept of the study is good in a tropical country with high sunlight exposure, there are some issues in design that make me uncomfortable for the interpretation of the results.

1.
The study has been put forth as a 'cohort' study.However it obviously is not a longitudinal one and hence needs to be classified as an observational cross sectional study.

Response:
The reviewer did raise a very important concern which will improve our manuscript.We agree with the reviewer's constructive comments that our study is an observational cross sectional study.

2.
The biggest issue in design is as to why were patients with mild COVID 19 illness included and hospitalized.No guidelines recommend hospital admission of mild cases of COVID 19 and inclusion of these cases involves a serious issue in design which would clearly affect the outcomes of the patients and the results of the study.Inclusion and the very hospitalization of such cases is as such flawed and seriously affects the results.

Response:
We appreciate your constructive suggestions.We have provided additional explanations regarding the inclusion criteria in both the methods and discussion sections.
During the initial phase of the pandemic, Indonesia encountered a challenging situation as it emerged as the first and enduring epicenter.The incidence of positive cases and deaths has experienced a significant surge, leading to the highest numbers in the Southeast Asian region.The Indonesian government has established a national policy that emphasizes emergency hospitals, which are intended to isolate mild cases and stop community transmission.This Indonesian government strategy has been proven to approach disease containment successfully in Wuhan, China, which was reflected by a significantly reduced COVID-19 mortality rate.Thus, this study includes patients who have been registered with mild, moderate, or severe disease in accordance with the policy established by the Indonesian government. 82rthermore, several COVID-19 pandemic studies contained some substantial biases and were not representative of the real-world conditions of the COVID-19 pandemic. 84,85In regions where authorities implemented home isolation and social distancing measures, individuals with mild to moderate cases of COVID-19 were generally not hospitalized. 84,86In contrast, in urban areas experiencing a high prevalence of COVID-19 cases and facing constraint intensive care resources, particularly mechanical ventilation, the majority of the hospital admissions were primarily for the individuals with severe or critical cases of COVID-19 .84,87,88,89Hospitalized people from a single center or a few centers were unlikely to represent the distribution of COVID-19 cases in an area.Also, there were evident selection biases in why and where people were hospitalized in these studies. 84,90,91Multiple studies have reported the presence of censoring among subjects who were still hospitalized leading to potential biases in the interpretation of the findings. 84,87,92On the contrary, we were able to identify every COVID-19 patient in our area, all of whom were admitted to the hospital.We also conducted a comprehensive follow-up for all the patients.The characteristics of our subjects were more similar to they who were exposed to the SARS-CoV-2 infection in a non-epidemic setting.As the result, our study provided a significant potential for widespread applicability and reflected the real-world conditions of the COVID-19 pandemic. 84,93,94,95xpertise to confirm that it is of an acceptable scientific standard.

Shaun Sabico
Department of Biochemistry College of Science, King Saud University, Riyadh, Riyadh Province, Saudi Arabia The present prospective study by Rachman and colleagues analyzed the impact of vitamin D deficiency among covid-19 patients and found that those who had more comorbidities and had more severe manifestations of covid-19 were more likely to be vitamin D deficient.Although the data itself is not new, this is one of the very few studies coming from Southeast Asia where vitamin D deficiency is not expected to be as pronounced in other regions.The use of ISARIC-4C mortality score is also an added novelty.Despite this fact, their findings largely echo previous observations.I have several comments: Major: Please add more details how covid-19 was diagnosed with reference.Was it through nasopharyngeal swab?And where was vitamin D measured?Was it in a BSL3 facility? 1.
The statistics could have been expanded to control for confounders such as age and BMI.These were not explicitly mentioned in the data analysis.Furthermore, with the number of variables measured, the p-value should have been Bonferroni adjusted unless the authors prove this wasn't necessary.

2.
Lastly, the study design appears to be cross-sectional and not prospective.The observational design and small sample size have limited the findings to at best, suggestive.The limitation section could have been expanded taking into consideration the points raised.

3.
Minor Several studies that support your findings from the Middle East where deficiency is very pronounced are suggested to be added including clinical trials on vitamin D and covid-19.Similar findings from different regions and ethnic groups reinforce the role of vitamin D in severity of covid-19.
1. Thank you for your construction feedback.We have added more details on the methods.The SARS-CoV-2 infection was confirmed through positive RT-PCR obtained from nasal and oropharyngeal swabs collected. 77The examination was carried out in the Biosafety Level 3facility (BSL-3) with Biological Safety Cabinet Class II (BSC-II).

References
During the admission, each patient had 3-5 mL of blood collected in an acid citrate dextrose tube from a cuffed venous sample.The samples were transported to the laboratory in a cold chain for the measurement of vitamin D. The statistics could have been expanded to control for confounders such as age and BMI.These were not explicitly mentioned in the data analysis.Furthermore, with the number of variables measured, the p-value should have been Bonferroni adjusted unless the authors prove this wasn't necessary. 1.
The reviewer did raise a very important concern which will improve our manuscript.We strongly agree that the reviewer's suggestion should be considered for further researches regarding this issue.The confounders such as age and BMI could not be controlled due to the minimum sample size of the subgroups and the fact that the number of samples is unequally distributed, which has not fulfilled the criteria to conduct the analysis.Thus, we have raised the point that the small sample size has become our limitation.We plan to improve the design and participants selection in the next study.
Furthermore, we have added Figure 1 (a bivariate analysis that has been adjusted with the Bonferoni correction).Lastly, the study design appears to be cross-sectional and not prospective.The observational design and small sample size have limited the findings to at best, suggestive.The limitation section could have been expanded taking into consideration the points raised. 1.
We have added the points of observational design and small sample size as limitations of our study.Additionally, we want to verified that our study's design is cohort prospective.Even if the risk variables (vitamin D levels) are measured upon admission, the outcome is followed and observed until the patient leaves the hospital, becomes worse, requires a ventilator, or passes away.The person had not used a ventilator when first recruited.

Minor
Several studies that support your findings from the Middle East where deficiency is very pronounced are suggested to be added including clinical trials on vitamin D and covid-19.Similar findings from different regions and ethnic groups reinforce the role of vitamin D in severity of covid-19.Please don't be hesitant to contact us if you have any concerns or suggestions about the revised manuscript.If you have any suggestions, we would greatly appreciate hearing them.Thanks for your attention.We eagerly await your response.
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Figure 1
Figure 1 presented the bivariate analysis performed on COVID-19 patients who were vitamin D deficiency.Serum 25(OH) D levels were significantly different between patients with mild and severe COVID-19 cases (p-value < 0.001).Serum 25(OH) D levels in mild and moderate COVID-19 cases were also significantly different (p-value 0.002).

Figure 1 .
Figure 1.The bivariate analysis of serum 25(OH) D levels based on COVID-19 severity among deficient vitamin D subjects after adjusted with Bonferroni correction.Analyzed using Mann-Whitney U test.Significant at p-value < 0.05.The green double-arrow denotes statistically significant difference.The red double-arrow denotes nonstatistically significant difference.
Underlying data Figshare: Impact of Vitamin D Deficiency in Relation to the Clinical Outcomes of Hospitalized COVID-19 Patients, DOI: https://doi.org/10.6084/m9.figshare.22145768.v2. 99This project contains the following data: -The data here is only for research paper validation of corresponding author Andhika Rachman entitled: "Impact of Vitamin D Deficiency in Relation to the Clinical Outcomes of Hospitalized COVID-19 Patients".The raw data consists of subjects characteristics and the levels of serum 25-hydroxy-vitamin D of hospitalized COVID-19 patients.Data are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).

Figure 1
presents the bivariate analysis performed on COVID-19 patients with vitamin D deficiency.Serum 25(OH)D levels were significantly different between patients with mild and severe COVID-19 cases (p-value < 0.001).Serum 25 (OH) D levels in mild and moderate COVID-19 cases were also significantly different (p-value 0.002).

1 .
Thank you for your advice.We have improved our paper by citing the multi-center randomized clinical trial in the Middle East that conducted by Sabico et al.We have also cited the studies carried out by Al-Daghri et al. and Alguwaihes et al.

Table 2 .
The categorized levels of serum 25(OH) D levels based on the influencing factors.

Table 3
showed that serum 25(OH) D levels were significantly lower among COVID-19 patients with vitamin D deficiency who had cardiovascular disease, the use of a ventilator, more severe COVID-19 cases, and mortality.Mortality was found in 9.59% of COVID-19 patients with vitamin D deficiency.

Table 3 .
The effect of vitamin D deficiency among each subgroup.

Table 3 .
Continued Abbreviations: BMI, body mass index; DM type II, diabetes mellitus type II; HFNC, High-flow nasal canule.Bold value denotes statistical significance.a Analyzed using Mann-Whitney U test.b Analyzed using ANOVA test.c Analyzed using Kruskal-Wallis test.d Analyzed using t-test.
This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Is the work clearly and accurately presented and does it cite the current literature? Partly Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Partly If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? No source data required Are the conclusions drawn adequately supported by the results? Yes Competing Interests: No
1. Sabico S, Enani MA, Sheshah E, Aljohani NJ, et al.: Effects of a 2-Week 5000 IU versus 1000 IU Vitamin D3 Supplementation on Recovery of Symptoms in Patients with Mild to Moderate Covid-19: A Randomized Clinical Trial.Nutrients.2021; 13 (7).PubMed Abstract | Publisher Full Text 2. Al-Daghri NM, Amer OE, Alotaibi NH, Aldisi DA, et al.: Vitamin D status of Arab Gulf residents screened for SARS-CoV-2 and its association with COVID-19 infection: a multi-centre case-control study.J Transl Med.2021; 19 (1): 166 PubMed Abstract | Publisher Full Text 3. Alguwaihes AM, Sabico S, Hasanato R, Al-Sofiani ME, et al.: Severe vitamin D deficiency is not related to SARS-CoV-2 infection but may increase mortality risk in hospitalized adults: a retrospective case-control study in an Arab Gulf country.Aging Clin Exp Res.2021; 33 (5): 1415-1422 PubMed Abstract | Publisher Full Text competing interests were disclosed.Reviewer Expertise: Nutrition, medical sciences, vitamin D, metabolism I

confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
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