key: cord-0892258-ixew079m
authors: Osman, Wessam; Al Fahdi, Fatma; Khamis, Faryal; Salmi, Issa Al; Khalili, Huda Al; Gokhale, Antara
title: Serum Calcium and Vitamin D levels correlation with severity of COVID-19 in hospitalized patients in Royal Hospital, Oman
date: 2021-04-20
journal: Int J Infect Dis
DOI: 10.1016/j.ijid.2021.04.050
sha: efd40890788785b4864601a94c1393fea21171a1
doc_id: 892258
cord_uid: ixew079m

Introduction There have been studies revealing hypocalcemia in severe covid-19 and low vitamin D levels that warranted further studies. Objective Our study investigates the correlation between calcium levels at presentation as a primary endpoint, and pre-existing calcium levels as a secondary endpoint, to severity of disease presentation and progression. Method Observational cohort study in adults admitted with COVID-19 from March till September 2020. Multiple clinical scales, laboratory parameters, were used to correlate corrected calcium and vitamin D associations with risk factors and outcomes. Results Around 445 patients were included in the study. Hypocalcemic patients had more abnormal laboratory parameters and longer hospitalization duration. Hypocalcemia was in 60-75% of all age groups (p-value 0.053), for which 77.97% were ICU admissions (p-value 0.001) and 67.02% were diabetic (p-value 0.347). There were non-significant correlations between Vitamin D and almost all the parameters except for chronic respiratory diseases with P value of 0.024. Conclusion It can be concluded that hypocalcemia is a significant and reliable marker of disease severity and progression regardless of underlying comorbidities. Meanwhile, Vitamin D levels fail to reflect correlation with severity of COVID-19 infections.

Coronaviruses (CoVs) are enveloped single-stranded RNA viruses with a highly diverse nature. Over the past 2 decades, two novel CoVs severe acute respiratory syndrome CoV (SARS-CoV) and Middle East respiratory syndrome CoV (MERS-CoV) have recently emerged to cause severe human disease. They were found to causes multiple symptomatic effects in respiratory, enteric, hepatic, and neurological systems in humans and animals. The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a newly emerging zoonotic corona virus that was discovered in Wuhan, China in December 2019 and has been identified as Coronavirus Disease 2019 . The virus's main manifestations on patients include fever, cough and shortness of breath that can progress rapidly in some cases to severe pneumonia, acute respiratory distress syndrome (ARDS) and septic shock. (Khamis et al., 2020) The World Health Organization (WHO) has declared coronavirus disease 2019 (COVID-19) as a public health emergency of grave international concern.(KEŠEĽOVÁ et al., 2020) Although the clinical picture of SARS, MERS, and COVID-19 seems to be similar, differences were noted since early reports at the beginning of the pandemic. Hence a proper characterization of the full pathophysiology of this disease, clinical progression, possible sequalae, are critical to battle the detrimental effects of this disease.

The National Health Commission of China has issued a series of diagnosis and treatment recommendations and suggested classifying the disease into four grades: mild, moderate, severe and critical.(Health Commission of PRC, 2019) Recent studies have reported the clinical characteristics and prognosis of COVID-19 with varied severity. (Guan et al., 2020; Huang et al., 2020; Wang et al., 2020; Xu et al., 2020; Zhu et al., 2020) The underlying pathophysiology of this virus leading to disease progression and organs dysfunction remain to be further explored.

In our country, as of November 1 st 2020, we had 115,734 confirmed cases, 105,700 recoveries and 1246 deaths reported. Worldwide cases stood at a staggering 46,632,558 cases, with 17,715,649 recoveries and 1,201,927 deaths (as per COVID-19 Dashboard by the Center for Systems Science and Engineering at Johns Hopkins University). This unopposed progression of disease has hampered the best healthcare systems around the world, with unparalleled rapidity and calamitous economic effects. So far evidence for definitive treatment regimens have proved anecdotal at best. (Dong et al., 2020) Due to the high mortality and the lack of effective treatments in critical patients, early identification of possible parameters that may predict clinical progression would be crucial to stratify care required for patients accordingly. (Guan et al., 2020; Maclaren et al., 2020) This is particularly of significance to identify patients that are most likely to require early intensivist setting care . (Lipsitch et al., 2020) Huang et al reported that patients admitted into intensive care unit (ICU) had more severe clinical symptoms and more abnormal serum parameters. (Huang et al., 2020) Throughout this pandemic, there has been a panoply of biochemical parameters suggested in literature that have been found to be inextricably linked to clinical progression of patients in different populations. J o u r n a l P r e -p r o o f There have been a few published studies regarding the significance of calcium levels as a predictor of severity of disease, amongst other biomarkers like D-Dimer, CRP, and Ferritin. Millet and Whittaker, 2018; Sun et al., 2020) This study aims to investigate this link in our population of COVID patients as well, namely corrected calcium to the progression of our patients through a particular set of parameters biochemical and clinical. As for vitamin D levels, there have been a few studies that investigated the levels of vitamin D with previous disease outbreaks like H1N1 and SARS. (Grant et al., 2020) The secondary endpoint in our study is to prove that the correlation of clinical progression of our COVID cases is not linked to Vitamin D levels as compared to corrected calcium levels.

This is an observational cohort study that was conducted at the Royal Hospital, which is a tertiary health-care hospital in Muscat. The data was extracted from our hospital's COVID registry established for all patients since the start of the pandemic in our population. The registry includes multiple parameters such as epidemiological characteristics, clinical symptoms, comorbid diseases and laboratory parameters. The laboratory parameters included corrected calcium and vitamin D levels. Our lab uses the Siemens Atellica chemistry system (CH930), Germany. Hypocalcemia for this study was defined a corrected calcium levels below 2.1mmol/L, and low vitamin D level <30nmol/L. This study included all patients >15 years of age admitted to the hospital with COVID-19 infection.

The assessment of clinical condition on admission and progression during hospital stay was measured using several clinical tools. The WHO Ordinal scale of clinical improvement was used on admission and discharge (Please refer to Table 1 ). (World Health Organization, 2020) This scale ranks patients in meaningful categories but does not differentiate between underlying causes. (Siegerink and Rohmann, 2018) The CURB-65 score was also used for admission evaluation. (Lim et al., 2003) Data analysis was performed using STATA statistical software version 13.0, USA. The significance level was set at α = .05, and all tests were 2-tailed. The original scores of the 4 measurement tools that are not normally distributed and so are presented as medians with interquartile ranges (IQRs). The ranked data, which were derived from the counts of each level of diagnosis Calcium levels and Vitamin D are presented as numbers and percentages. The nonparametric Mann-Whitney U test was used for continuous variables, multivariable logistic regression analysis was used to different variable against our calcium and vitamin D levels, and the associations between risk factors and outcomes was presented as odds ratios (ORs) and 95% CIs. A p-value of <0.05 was considered statistically significant in our analyses.

A total of 445 hospitalized patients were included in the study. Baseline characteristics of our patient characteristics are summarized in Table 2 .

Multiple clinical scales and laboratory values were used to classify patient's severity on admission and discharge. Ordinal Scale on admission showed 66.4% scaled (0-4) and 33.6% J o u r n a l P r e -p r o o f were (5-8). On the contrary, the scale on discharge showed 82.6% scaled (0-4) and 17.1% were (5-8).

As per the CURB 65 score on admission 37.4% were (0), 33.3% were (1),20% were

(2),6.9% were (3),2.1% were (4) and only 0.5% were (5). Males were noted predominantly more severe than female as detailed in the table.

As per the laboratory severity markers on admission, low total WBC count (<2.2 x10*9) was seen in 2.9%, particularly lymphopenia present in 68.5%. Hypocalcemia (<2.1 mm/L) as predicted was seen in 68.8% (Males 65.6% & Females 34.4%), CRP was >10mg/L in 92.5%, ferritin >708 mcg/L in 55.6%, Troponin was >49 pg/mL in 53%, ALT was >49 iU/L in 37.9%, Ddimer was >0.5 mg/L FEU in 76.9% and LDH >246 iU/L in 88.3%. Vitamin D was only deficient in 5% of our population as expected, with a mean of 66.6 and range 17-128 nmol/L Diabetes and hypertension were the most frequent co-morbidities accounting ( Diabetes Mellitus was seen in 67.02% of hypocalcemia group and 71.36% were non-diabetic (P value=0.347). The P value was 0.024 for chronic respiratory diseases as 54.55% had hypocalcemia. Chronic liver diseases and chronic kidney diseases had a non-significant P value of 0.717 and 0.262 respectively.

Leukocytosis >10x109 combined with normal WCC (2.2-10x109) showed a P value of 0.096. Normal lymphocytes (1.2-4) and lymphopenia (<1.2) showed a P value of 0.079 in correlation to hypocalcemia, as 71.69% with normal Lymphocytes and 71.69% of lymphopenic patients had hypocalcemia. CRP was raised in 71.54% of hypocalcemia group representing a J o u r n a l P r e -p r o o f significant P value of 0.001. High ferritin showed a P value of 0.019, correlating significantly with hypocalcemia. Other markers like ALT and LDH showed a P value of 0.073 and <0.001, respectively. No significant correlation with Vitamin D was found (P value=0.144).

Oxygen requirements were statistically significant with a P-value of 0.006, with 77.95% of hypocalcemia patients needing >15L/min of O2. The need for NIV in hypocalcaemic patients was 84.81% (P-value=0.001). ARDS and intubation had P values of 0.012 and <0.001 respectively.

Surprisingly, sepsis and death had non-significant correlation as P value was 0.0838 for sepsis and 0.72 for death.

The meta-analysis showed non-significant correlations between Vitamin D and almost all the parameters except for chronic respiratory diseases with P value of 0.024 and CRP P value 0.014 and ALT where P value was 0.029, while the Two Sample T Test of calcium and vitamin D against other parameters are displayed in Table 5 .

This study showed that 33% of patients in this cohort had ordinal scale of 5-8 on admission whereas it was 17% at discharge. The CURB score of 2 and above was present in one third of patients and was more severe in males. Diabetes and hypertension were main comorbidities occurring in 50% of participants. Hypocalcemia (<2.1 mm/L) was present in more than two thirds of the participants mainly in males. Hypocalcemia was associated with chronic respiratory diseases. Patients with hypocalcemia had worse ordinal scale, lymphopenia, CRP, J o u r n a l P r e -p r o o f LDH, longer hospital stay, ICU admission, ARDS and intubation and higher oxygen requirements.

Hypocalcemia showed no significant association with death, however, there was a trend towards a statistical significance association with sepsis (P=0.08).

A high incidence of hypocalcemia was observed in critically ill patients admitted in hospitals in Wuhan Covid-19 during the beginning of the epidemic. It was therefore hypothesized that low serum calcium levels were associated with the severity of disease and prognosis in COVID-19. Hypocalcemia was detected in 60% of patients at hospital admission and in 70% during hospitalization in a large group of SARS patients in North America. (Booth et al., 2003) Data of patients with Ebola virus infection from United States and European hospitals reported a similar incidence of hypocalcemia. (Uyeki et al., 2016) Several studies have investigated the clinical and laboratory characteristics of COVID-19 patients, including inflammatory and organ injury biomarkers . (Harries and Takarinda, 2020) Many cases of COVID-19 at presentation have been reported to have hypocalcemia. (Bossoni et al., 2020) However, no particular population data were available on calcium levels in COVID-19. (Puig-Domingo et al., 2020) Several studies revealed a correlation between hypocalcemia, higher mortality and poor clinical outcome in hospitalized critically ill patients. (Akirov et al., 2017; Cheungpasitporn et al., 2018) Calcium plays a crucial role in viral fusion of various enveloped viruses such as SARS-CoV, MERS-CoV, and Ebolavirus. It is known to directly interact with the fusion peptides of these viruses to promote their replication. (Booth et al., 2003; Nathan et al., 2020; Straus et al., 2019) In patients with SARS CoV and Ebolavirus, hypocalcemia was a very frequent laboratory finding.

Despite its regularity, the underlying pathophysiology, clinical relevance and prognostic significance were not fully investigated. (Nathan et al., 2020; Straus et al., 2019) Calcium may prove as a useful biochemical marker of disease severity. Since measuring calcium is a simple blood test that can be initiated upon presentation in emergency visit for most patients, it may prove a quick indicator for the clinician to discern severity of the case.

Whether this hypocalcemia represents the pathophysiology of COVID-19, a dysregulation of calcium homeostasis, or perhaps linked to vitamin D levels as well is yet to be investigated.

Since a high incidence of hypocalcemia in COVID-19 patients may predict severity of illness and the need for hospitalization, we suggest that calcium should always be assessed at initial hospital evaluation. Hypocalcemia may have a negative impact on cardiac function and may be even lethal when severe and acute. Monitoring and maintaining adequate calcium levels in all hospitalized patients with COVID-19 is recommended. (Holick, 2007) As a scientific postulation for the mechanism behind hypocalcemia in COVID-19

infections, it was found that these patients have elevated levels of unbound fatty acids and unsaturated fatty acids. The latter can bind to calcium with a favorable (-20KJ/mol) enthalpy and cause acute significant hypocalcemia. They can also induce cytokine storm and multiorgan system failure. In severe disease, hypoalbuminemia can also be induced by unsaturated fatty acids. This may affect corrected albumin measurements like in our study. It was also found that if calcium is rapidly corrected, it may bind and neutralize these unsaturated fatty acids early in the disease, thereby preventing mitochondrial injury, and the subsequent widespread cellular injury, organ failure and sepsis that follows. Despite the logic underlying this explanation, it may be counterintuitive to ascertain that a single mechanism can explain such a widespread J o u r n a l P r e -p r o o f phenomenon in a very heterogenous population with different degrees of severities, ethnic backgrounds and manifestations worldwide. Singh et al., 2020) As for Vitamin D, its metabolism and actions are well studied . (Holick, 2007) Vitamin D3 is produced in the skin through the action of UVB radiation forming 7-dehydrocholesterol, which is then followed by a thermal reaction. Vitamin D3 is then converted to 25(OH)D in the liver and then to the hormonal metabolite 1,25(OH)2D (calcitriol) in the kidneys. Most of the activity of vitamin D comes from calcitriol entering the vitamin D receptor in the nucleus. It is a DNA binding protein that interacts directly with regulatory sequences near target genes. It recruits chromatin active complexes that participate genetically and epigenetically in modifying transcriptional output. (Pike and Christakos, 2017) Calcitriol then helps to regulate serum calcium concentrations, by a negative feedback loop with parathyroid hormone (PTH). (Holick, 2007) There have been several reviews that considered the various possibilities by which vitamin D may reduce the risk of viral infections and death. (Abhimanyu and Coussens, 2017; Beard et al., 2011; Gombart et al., 2020; Greiller and Martineau, 2015; Gruber-Bzura, 2018; Hewison, 2012; Lang and Aspinall, 2017; Rondanelli et al., 2018; Wei and Christakos, 2015) One review considered the role of vitamin D in reducing the risk of common cold, attempted to group these mechanisms into three categories: cellular natural immunity, adaptive immunity and physical barrier. (Rondanelli et al., 2018) Vitamin D maintains tight junctions, gap junctions, and adherens junctions (e.g. by E-cadherin) across cellular structure. (Schwalfenberg, 2011) Several articles have discussed the increase in infections by viruses and other organisms caused J o u r n a l P r e -p r o o f due to the disruption of these junction integrities. (Chen et al., 2020; Kast et al., 2017; Rossi et al., 2020) Vitamin D enhances cellular innate immunity partly through the induction of antimicrobial peptides; including 1,25-dihdroxyvitamin D defensins, human cathelicidin, and LL-37 cathelicidin-derived antimicrobial peptide. (Adams et al., 2009; Laaksi, 2012; Liu et al., 2006) Cathelicidins demonstrate direct antimicrobial activities against an array of micro-organisms; Vitamin D supplementation also promotes expression of genes associated with antioxidation (glutathione reductase and glutamate-cysteine ligase modifier subunit). (Lei et al., 2017) Increased glutathione production spares the use of ascorbic acid (vitamin C), which is J o u r n a l P r e -p r o o f known for its antimicrobial activities, and has been proposed as supplementation to decrease effects of COVID-19. (Bâldea, 2020; Colunga Biancatelli et al., 2020; Mousavi et al., 2019) While vitamin D levels vary widely in the northern hemisphere of the planet, our regions fare differently and for different reasons. Studies in Oman have reported high prevalence of vitamin D deficiency (87.5%) in healthy Omanis. (Abiaka et al., 2013) The lack of exposure to sunlight is one of the main causes for vitamin D deficiency. (El-Hajj Fuleihan, 2009; Guan et al., 2020; Kast et al., 2017) Given that most of our admissions were expatriate from mostly Asian nationalities, our study population presented a wide range of ethnic backgrounds to incorporate any inherent differences in vitamin D levels. The link between calcium levels and vitamin D level may be of significance if it can be proven as with other studies done worldwide. (Meltzer et al., 2020) As our data suggests, particularly from the multivariate analysis, hypocalcemia is a very reliable marker for disease progression as a predictor and as part of the overall symptomatology of the disease. The worse the hypocalcemia, the more severe the clinical progression of the patients with complications. Although death as an outcome in our univariate analysis had p-value 0.684, and in multivariate analysis had a p-value of 0.72, this may be due to other interfering factors including differences in therapeutic regimens which were not mentioned in our study.

There was a slight limitation in the corrected calcium measurements in our laboratory.

Our laboratory uses the Siemens Atellica chemistry system (CH930), Germany. Its method is based on CPC method (O-Cresolphthalein complexone), a colorimetric method. there was a change in albumin method which is an element in the equation for corrected calcium. The J o u r n a l P r e -p r o o f change was from Bromocresol green (BCG) to Bromocresol purple (BCP). Both methods are colorimetric or dye binding methods. They are both known to overestimate albumin concentration in hospitalized patients as the dye is reacting with acute phase reactants in these patients. This overestimation of albumin results in underestimation of calcium especially at albumin levels over 40 g/L. However, this effect is at a lesser extent in BCP compared to BCG.

Through our observation of non COVID cases, it seems that the margin of error was negligible, as non COVID cases presented with majority normal corrected calcium values.

In conclusion, we highly recommend the use of corrected calcium levels as a predictor of possible clinical progression, and initial stratifying parameters for further need of intensive care. Also given the possible theory behind its mechanism, rapid correction is advised to prevent further injury at the cellular level and stifle further provocation of the disease. As for Vitamin D levels, it represented no particular significance in our population to recommend correction or otherwise.

We declare that this manuscript is original, has not been published before and is not currently being considered for publication elsewhere.

We know of no conflicts of interest associated with this publication, and there has been no financial support for this work that could have influenced its outcome. As First Author, I confirm that the manuscript has been read and approved for submission by all the named authors.

Ethical approval has been granted by our hospital's Research Committee in July 2020. The current "Instructions to Authors" has been read by all authors and hereby ensure compliance with those instructions and acceptance of the conditions posed. J o u r n a l P r e -p r o o f 

The role of UV radiation and Vitamin D in the seasonality and outcomes of infectious disease

Vitamin D status and anthropometric indices of an Omani study population

Vitamin D-Directed Rheostatic Regulation of Monocyte Antibacterial Responses

Cathelicidin impact on inflammatory cells

Calcium levels on admission and before discharge are associated with mortality risk in hospitalized patients

What Can We Learn from the Time Evolution of COVID-19 Epidemic in Slovenia? ArXiv 2020

Antiviral activity and increased host defense against influenza infection elicited by the human cathelicidin LL-37

Vitamin D and the anti-viral state

Clinical Features and Short-term Outcomes of 144 Patients with SARS in the Greater Toronto Area

Severe hypocalcemia in a thyroidectomized woman with Covid-19 infection

Mechanisms underlying the effect of vitamin D on the immune system

Vitamin D and 1,25(OH)2D regulation of T cells

The Epidemiological Characteristics of an Outbreak of 2019 Novel Coronavirus Diseases (COVID-19) -China

Epidemiological features and time-series analysis of influenza incidence in urban and rural areas of Shenyang

Impact of admission serum calcium levels on mortality in hospitalized patients

The antiviral properties of vitamin C

An interactive web-based dashboard to track COVID-19 in real time

Vitamin D deficiency in the Middle East and its health consequences for children and adults

Hypocalcemia: the quest for the cause of a major biochemical feature of COVID-19

Hypocalcemia is highly prevalent and predicts hospitalization in patients with COVID-19

A review of micronutrients and the immune system-working in harmony to reduce the risk of infection

Evidence that vitamin d supplementation could reduce risk of influenza and covid-19 infections and deaths

Modulation of the immune response to respiratory viruses by vitamin D

Influence of drugs on vitamin D and calcium metabolism

Vitamin D and influenza-Prevention or therapy?

Clinical characteristics of 2019 novel coronavirus infection in China

Faculty Opinions recommendation of Presenting Characteristics, Comorbidities, and Outcomes Among 5700 Patients Hospitalized With COVID-19 in the New York City Area. Fac Opin -Post-Publication Peer Rev Biomed Lit

Health Commission of PRC N. Chinese guidelines for diagnosis and treatment of malignant lymphoma 2018 (English version)

The role of cathelicidin and defensins in pulmonary inflammatory diseases

An update on vitamin D and human immunity

Medical progress: Vitamin D deficiency

Clinical features of patients infected with

1,25-Dihydroxyvitamin D 3 and IL-2 Combine to Inhibit T Cell Production of Inflammatory Cytokines and Promote Development of Regulatory T Cells Expressing CTLA-4 and FoxP3

Respiratory syncytial virus infection influences tight junction integrity

The impact of the covid-19 outbreak on aviation

Clinical characteristics and outcomes of the first 63 adult patients hospitalized with COVID-19: An experience from Oman

Vitamin D and respiratory infection in adults

Vitamin D Status and the Host Resistance to Infections: What It Is Currently (Not) Understood

Mechanisms of action of vitamin D as supplemental therapy for Pneumocystis pneumonia

Suppresses the in Vitro Proliferation and Im1Unoqabulin Production By Normal Human Peripheral Blood Cells

Defining community acquired pneumonia severity on presentation to hospital: An international derivation and validation study

Defining the Epidemiology of Covid-19 -Studies Needed

Toll-like receptor triggering of a vitamin D-mediated human antimicrobial response

Preparing for the Most Critically Ill Patients with COVID-19: The Potential Role of Extracorporeal Membrane Oxygenation

Aging decreases the capacity of human skin to produce vitamin D3

Effect of high doses of vitamin D supplementation on dengue virus replication, Toll-like receptor expression, and cytokine profiles on dendritic cells

Association of Vitamin D Status and Other Clinical Characteristics With COVID-19 Test Results

Physiological and molecular triggers for SARS-CoV membrane fusion and entry into host cells

Immunomodulatory and antimicrobial effects of vitamin C

Calcium Ions Directly Interact with the Ebola Virus Fusion Peptide to Promote Structure-Function Changes That Enhance Infection

Biology and Mechanisms of Action of the Vitamin D Hormone

COVID-19 and endocrine diseases. A statement from the

Self-Care for Common Colds: The Pivotal Role of Vitamin D, Vitamin C, Zinc, and Echinacea in Three Main Immune Interactive Clusters (Physical Barriers, Innate and Adaptive Immunity) Involved during an Episode of Common Colds -Practical Advice on Dosages

Viral strategies predisposing to respiratory bacterial superinfections

A review of the critical role of vitamin D in the functioning of the immune system and the clinical implications of vitamin D deficiency

Impact of your results: Beyond the relative risk

Mechanistic basis and therapeutic relevance of hypocalcemia during severe COVID-19 infection

Ca2+ ions promote fusion of Middle East Respiratory Syndrome coronavirus with host cells and increase infectivity

Serum calcium as a biomarker of clinical severity and prognosis in patients with coronavirus disease 2019: a retrospective crosssectional study 2020

Clinical Management of Ebola Virus Disease in the United States and Europe

Alacsony D-vitamin-szint a Semmelweis Egyetem betegei körében: a központi laboratóriumban egy év alatt meghatározott D-vitaminszintek retrospektív értékelése

Clinical Characteristics of 138 Hospitalized Patients with 2019 Novel Coronavirus-Infected Pneumonia in Wuhan, China

Mechanisms underlying the regulation of innate and adaptive immunity by vitamin D

World Health Organization. WHO R&D Blueprint novel Coronavirus COVID-19 Therapeutic Trial Heal Organ

Clinical findings in a group of patients infected with the 2019 novel coronavirus (SARS-Cov-2) outside of Wuhan, China: Retrospective case series

Vitamin D Alleviates Rotavirus Infection through a Microrna-155-5p Mediated Regulation of the TBK1/IRF3 Signaling Pathway In Vivo and In Vitro

A Novel Coronavirus from Patients with Pneumonia in China