Lack of association between vitamin D insufficiency and clinical outcomes of patients with COVID-19 infection

Background A protective effect of vitamin D against COVID-19 infection is under investigation. We aimed to analyze the effect of vitamin D sufficiency on the clinical outcomes of patients infected with COVID-19. Methods In this cross-sectional study we analyzed the vitamin D levels of COVID-19 patients who were admitted to Razi Hospital (an infectious disease referral center in Mazandaran province in northern Iran) from February to March 2020. Overall, a cutoff point of 30 ng/mL was used for the definition of vitamin D sufficiency. Results One hundred fifty-three patients were analyzed in this study who had laboratory documentation of a 25(OH) D level at the time of hospitalization. The vitamin D levels of the patients were 27.19 ± 20.17 ng/mL. In total, 62.7% (n = 96) of the patients had a 25(OH) D level of less than 30 ng/mL and 37.25% (n = 57) had a 25(OH) D level of more than 30 ng/mL. In total, 49% (n = 75) of the patients suffered from at least one underlying disease. The univariate and multivariable regression showed that vitamin D sufficiency was not associated with a statistically significant lower risk of adverse clinical outcomes of COVID-19 such as duration of hospitalization and severity of infection (P > 0.05). Conclusions Sufficient vitamin D levels were not found to be protective against adverse clinical outcomes in patients infected with COVID-19. Chronic disorders in COVID-19 patients were found to have greater relevance than vitamin D levels in determining the adverse outcomes of the infection. Further studies are needed to determine the role of vitamin D level in predicting the outcomes of COVID-19 infection.


Introduction
Coronavirus 2019 or COVID-19, a respiratory infectious disease, has led to a pandemic of pneumonia-related illness [1][2][3]. The clinical features of this disease vary from asymptomatic or mild (in over 80%) to severe cases leading to acute respiratory syndrome and respiratory failure, requiring hospitalization in the intensive care unit, sepsis, septic shock and death [4][5][6]. The rapidly increasing number of infected individuals in highly critical states requiring intensive resources is a huge public health challenge worldwide [7]. Unfortunately, no suitable antiviral treatment has been found for this disease so far, and all therapeutics used are based on hypotheses that do not have sufficient evidence to support their use. Given the high economic importance of the COVID-19 pandemic, it is necessary to find methods that reduce the risk of infection and mortality at a low cost [8]. The status of the immune system is affected by multiple factors that may contribute to the risk of morbidity and mortality related to viral infections such as COVID-19. There are several vitamins and essential elements that have been shown to be necessary for a robust immune system response [9,10]. Recent studies have highlighted a crucial supportive role for vitamin D in immune system functions, particularly in balancing the inflammatory response to viral infection [9,11,12]. Vitamin D is a fat-soluble vitamin that plays a key role in modulating both innate and adaptive immune responses. Previous studies have also shown that adequate levels of vitamin D have been shown to reduce the risk of viral respiratory infections and the length of hospital stay [1,13,14]. On the other hand, there is no accurate information about the COVID-19 virus in this field and the role of vitamin D supplementation in reducing the risk of infection is still under evaluation [15][16][17]. There is a high prevalence of vitamin D deficiency among Iranians based on several studies [18,19]. Mazandaran province is located in the north of Iran, along the southern coast of the Caspian Sea with a moderate climate. In this study, we investigated the effect of vitamin D sufficiency on the clinical outcomes of patients with COVID-19 infection hospitalized in Razi hospital, which is a referral center of COVID-19 patients in Mazandaran province.

Study design, participants and data source
This was a cross-sectional, retrospective study analyzing vitamin D levels of COVID-19 patients > 18 years old who were admitted to Razi Hospital, an infectious disease referral center in Mazandaran province, located in the north of Iran, from February to March 2020. The census method was used for sampling. The exclusion criteria was vitamin D intake in the past month prior to the diagnosis of COVID-19. The study was approved by the Ethics Committee of Mazandaran University of Medical Science, Ramsar international branch (IR.MAZUMS.RI-B.REC.1399.019). Hospital medical records were analyzed from the inpatient database of Razi teaching hospital affiliated with Mazandaran University of Medical Sciences.

Definitions and data collection
The definitive diagnosis of COVID-19 infection was based on the positive results of real-time reverse transcriptase-PCR (RT-PCR) assay for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) using nasopharyngeal swab.
The vitamin D levels were measured by a high performance liquid chromatography (HPLC) method at the time of admission to the hospital. Based on the Endocrine Society's Practice Guidelines on Vitamin D, overall, a cutoff point of 30 ng/mL was used for the definition of vitamin D sufficiency [20].
Definitions of COVID-19 severity were based on the World Health Organization COVID-19 clinical management guideline: 1-Mild stage (definitive COVID-19 patients without evidence of viral pneumonia or hypoxia); 2-Moderate disease (clinical signs of pneumonia [fever, cough, dyspnea, tachypnea] but no signs of severe pneumonia, including SpO2 ≥ 90% on room air not requiring supplemental oxygen); 3-Severe stage (with clinical signs of pneumonia [fever, cough, dyspnea, tachypnea) plus one of the following: respiratory rate > 30 breaths/ min; severe respiratory distress; or SpO2 < 90% on room air; 4-Critical (Acute respiratory distress syndrome (ARDS), sepsis/septic shock, acute thrombosis) or other conditions that would normally require the provision of life sustaining therapies such as mechanical ventilation (invasive or non-invasive) or vasopressor therapy [21]. Severe and critical categories were grouped together as "severe" in our study.
Data such as demographic information (age, sex, body mass index [BMI], and living area), medical history, comorbidities, and diagnostic data of the patients, including vitamin D levels and the outcomes were extracted from medical files. Age was categorized into three classifications (50>, 50-65, and > 65 years old) [22]. The following outcomes were measured: duration of hospitalization, lung involvement, ICU admission, invasive and non-invasive mechanical ventilator use, severity of disease, and in-hospital mortality.

Statistical analysis
Statistical analysis was performed using the Stata software version 14.0. Differences between groups (patients with 25(OH) D level ≥ 30 and patients with 25(OH) D level ≤ 30) were determined by the Chi-square test or Fisher's exact test. Ordinal logistic regression was used to determine the association between variables and severity of diseases. Univariate linear regression was performed to evaluate the association between vitamin D status, demographic characteristics and existing comorbidities with adverse outcomes of COVID-19. Variables with a P-value less than 0.2 according to a univariate analysis and vitamin D status were included in the multivariable analysis. The descriptive values below 5% (P-value< 0.05) were considered statistically significant.  Tables 1 and 2.
The average hospitalization stay of patients were 6.3 ± 4.12 days. Bilateral lung involvement was seen in 17.64% of patients. In total, 6.53% of patients were admitted to the ICU. Invasive mechanical ventilator was utilized for 1.96% of patients while 9.08% of patients were under non-invasive ventilation. Based on severity of disease, 41.7% of patients were categorized into the mild form of COVID-19 disease, while 41.2 and 11.8% experienced the moderate and severe / critical forms of COVID-19,

Univariate and multivariable regression analysis for adverse outcome of COVID-19
There were few patients with some adverse outcomes of COVID-19 such as ICU admission (10/153), mortality (5/153), and invasive ventilation (3/153). Therefore, the univariate and multivariable linear regression was performed for hospitalization duration. The univariate and multivariable linear regression results showed that there was no significant association between vitamin D status and duration of hospitalization as well as the demographic variables and chronic disorders (Table 3).

Discussion
The association between vitamin D status and seasonal respiratory infections has been proven in several studies [12,23,24]. Optimizing vitamin D status could improve the immune response and has been suggested as possibly protective in COVID-19 infection [10,25,26]. The COVID-19 outbreak began during the winter and a common feature of the inhabitants of all countries north of the 42nd parallel is a vitamin D insufficiency [27]. These facts resulted in the concept of using vitamin D for the prevention of COVID-19 infection or using vitamin D as an intervention strategy in COVID-19 patients [3,23]. In our study, only 37. negative controls [28,29]. Consistent with our study findings, the study by Hastie et al. did not support a potential link between vitamin D concentrations and risk of COVID-19 infection [25,28]. Older age and comorbidities are linked to an insufficient vitamin D supply [30,31]. It is notable that 64.05% of our patients were greater than 50 years old.  [25,33,34]. In addition, demographic data reviewed from several studies highlighted the severity of COVID-19 was more significant in male compared to female patients [35,36]. Moreover, delays in hospitalization and less access to health facilities can be factors involved in more severe forms of the disease among patients residing in rural areas. Several studies have reported that a higher prevalence of vitamin D deficiency was observed in patients with worse COVID-19 outcomes [13,14,17,37]. In our study, there was no evidence to suggest that assessment of vitamin D can serve as an indicator of the outcomes of COVID-19 infection. In line with our findings, Hastie et al. reported that the measurement of vitamin D would not be useful to evaluate the risk of COVID-19 in clinical practice [25].
There are some limitations in this research due to its cross-sectional retrospective design. The first drawback is that we included only patients who had documented vitamin D levels. The other drawback of our study is its small sample size, as well as some confounding factors, such as smoking and socioeconomic status were not recorded for most patients and could have an admissible impact on the COVID-19 severity.

Conclusion
Despite the fact that the number of patients with an insufficient vitamin D level was greater in our study, and approximately 44% of patients fell into the moderate and severe/critical categories, some requiring invasive/non-invasive ventilation, ICU admission, longer hospitalization, and death, there was no statically significant evidence that insufficient vitamin D levels might play a role in adverse outcomes of COVID-19 infection.
Chronic disorders in COVID-19 patients likely have greater relevance than vitamin D levels in determining the adverse outcomes of the infection. Therefore, vitamin D supplementation may not be helpful in the prevention or treatment of COVID-19. As there is still no effective pharmaceutical treatment for COVID-19, general vaccination of the community, as well as adherence to health protocols, are the main tools to reduce morbidity and mortality caused by this disease. Larger prospective studies are needed however to support or refute our study findings.