Inverse Correlation between 25-OH Vitamin D Levels and Severity of Viral Respiratory Illness in Infants

Citation: Fairchok M, Schofield C, Chen W, Pugh M, Bigg H, et al. (2017) Inverse Correlation between 25-OH Vitamin D Levels and Severity of Viral Respiratory Illness in Infants. J Infect Dis Epidemiol 3:030. doi.org/10.23937/2474-3658/1510030 Received: January 13, 2017; Accepted: April 14, 2017; Published: April 17, 2017 Copyright: © 2017 Fairchok M, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


Introduction
The prevalence of 25-OH vitamin D insufficiency (level of < 30 ng/mL) in persons aged > 1 year in the United States is 31% [1].Growing evidence suggests that in addition to calcium homeostasis, vitamin D also plays key roles in innate and adaptive immunity and thus vitamin D deficiency has been hypothesized to be a culprit in the risk and severity of Acute respiratory infection (ARI) in both adults and children.Vitamin D receptors are widely expressed amongst cells of the innate and adaptive immune system, including dendritic cells and macrophages.When stimulated, these cells produce • Page 2 of 7• Fairchok et al.J Infect Dis Epidemiol 2017, 3:030 mL were considered Vitamin D insufficient (VDI).Levels ≥ 30 ng/mL were considered to be Vitamin D sufficient (VDS).Although there is a debate about the cutoff level for vitamin D sufficiency, we chose to use the 30 ng/mL cutoff based on the work of Bahn, et al. who determined that a level of at least 32 ng/ml is needed for activation of the immune response [10].
Both the serum sample and the nasal swab from each subject were sent at room temperature to Laboratories Northwest, co-located on campus, for 25-OH vitamin D assay of the serum, and detection of viral pathogen.The 25-OH vitamin D levels were obtained using the Abbott Architect Chemoluminescent Microparticle Immunoassay (Abbott Laboratories, Abbott Park, IL).Viral pathogens were detected with the eSensor® Respiratory Viral Panel (Genmark Diagnostics, Carlsbad, CA), a multiplex Polymerase chain reaction (PCR) based assay that tests for Influenza viruses A and B, Adenovirus, Human parainfluenza viruses 1-3, Human rhinovirus, Respiratory syncytial virus (RSV) A and B, and Human metapneumovirus.If phlebotomy was unsuccessful, the subject was removed from the study.

Outcome measures
Primary outcome measures included the use of intravenous fluids and antibiotics, need for intensive care and oxygen supplementation, maximum amount of oxygen flow needed, and the number of days of fever, of oxygen requirement, of intensive care, and of hospitalization.

Statistical analysis
First, we examined bivariable associations between demographics and potential risk factors by 25-OH Vitamin D level (VDI vs. VDS).We performed chi-square tests (exact test, if appropriate) for dichotomous and categorical variables and Wilcoxon rank-sum tests for continuous variables.We then performed multivariable logistic regression to examine factors associated with vitamin D levels, including variables that were p ≤ 0.05 in the bivariable analysis.Next, we examined differences in clinical severity by categorical 25-OH vitamin D levels (VDI vs. VDS) using chi-square tests (exact test, if appropriate) for categorical outcomes and Wilcoxon rank-sum test for continuous outcomes.We adjusted p-values in the bivariable analysis to control for multiple comparisons using False discovery rate (FDR) [11].We performed multivariable analysis for clinical outcomes that were significantly different across 25-OH Vitamin D categories with adjustment of potential confounders: ordinal logistic regression for ordinal outcomes (i.e.volume level of oxygen use), and linear regression for duration of hospitalization and oxygen supplementation.We fit the linear models with log transformed duration data; however, for intuitive understanding, we presented the exponentiated values of point estimates and 95% Confidence intervals (CIs) in the linear models.Tests with p Human cathelicidin protein (HCAP-18), which augments the microbial killing capacity of phagocytes and activates anaphylactoid factors that promote neutrophil and monocyte migration to the site of infection [2].In addition to its immunologic activating effects, vitamin D also suppresses the release of potentially damaging pro-inflammatory cytokines and chemokines, to include IL-1β, IL-6, IL-8, and TNF-α [3].
Conversion of vitamin D to the active form in humans begins with vitamin D3 production in the skin upon exposure to sunlight.Given the effects of vitamin D on immunity, it is plausible that the seasonality of ARI may be driven in part by periods of active vitamin D insufficiency during the winter and early spring months attributable to lower rates of skin conversion to D3.Although several studies have now shown an increased incidence of ARI in subjects with low 25-OH vitamin D levels [4][5][6][7][8][9], very little is known regarding 25-OH vitamin D levels and their relationship to the severity of ARI.We therefore investigated the correlation between 25-OH vitamin D levels and severity of viral respiratory tract infections in a prospective cohort of otherwise healthy children hospitalized at a community based children's hospital in Tacoma, WA.We hypothesized that children with lower 25-OH vitamin D levels would have more severe disease.

Patients and Methods
From February 14, 2014 to May 11, 2015, we recruited otherwise healthy children < 2 years of age with a febrile presumed viral, lower respiratory illness admitted to the Mary Bridge Children's Hospital (MBCH).MBCH is a tertiary care children's hospital with 82 beds located in Tacoma, WA.Inclusion criteria included a fever and at least one of the following symptoms: cough, congestion, increased work of breathing, or wheezing.Exclusion criteria included chronic medical illness; primary diagnosis of a bacterial infection; and history of asthma, immunodeficiency or prematurity.Respiratory infections were categorized as bronchiolitis if the subjects had increased work of breathing or wheezing in addition to the cough and congestion.All treatment decisions were made by the inpatient pediatric hospitalist attendings, independent of this study.All subjects' parents or guardians provided signed informed consent prior to enrollment.The study was approved by the Infectious Disease Institutional Review Board of the Uniformed Services University of the Health Sciences (IDCRP-045), and by the Multicare Institutional Review Board.
At enrollment, demographics, medical history, clinical symptoms, vital signs and physical exam findings were recorded by clinical research personnel using a standard clinical record form, and a nasopharyngeal swab (Nylon-flocked, Copan Diagnostics, Corona, CA) for respiratory viral detection was collected.A serum sample of approximately 3 mL was obtained for 25-OH vitamin D analysis.25-OH vitamin D levels of < 30 ng/ ≤ 0.05 (2-sided) were considered statistically significant.Analyses were performed using SAS 9.3 (SAS Institute, Inc., NC).

Results
We approached 158 eligible subjects for participation in the study.Subjects were only enrolled during the viral respiratory season.Fifty (32%) declined, with 90% citing the need for a blood sample.Of the 108 subjects enrolled, 14 (12.9%) were administratively withdrawn due to inability to obtain a blood specimen and 4 (0.2%) declined collection of nasal swab, leaving 90 subjects who completed the study.There were no demographic differences among those who declined, withdrew or completed the study.Twenty-two (24%) patients required intensive care during their stay.Mean age of enrollees was 8 months, with 43% female.Seventy-one percent were Caucasian, 6% Pacific Islander, 4% African-American, 4% Native American, and 15% more than one race.
Associations between 25-OH vitamin D categories and clinical severity are shown in Table 3.We noted that the proportion of intravenous fluid supplementation, intensive care treatment, artificial ventilation, and antibiotic treatment were all markedly higher in subjects with VDI when compared to VDS subjects, but the differences were not statistically significant after accounting for multiple comparison.Among those who received oxygen support (N = 68), VDI subjects used significantly higher level oxygen support compared to VDS subjects Note: a Based on chi-square for categorical variables (exact test if appropriate) or Wilcoxon rank-sum test for continuous variables; b Median (Med.) and interquartile range (IQR) for continuous variables; c both parents are white; d Missing group is not included in statistical testing.Pathophysiological evidence has been mounting that a sufficient level of 25-OH vitamin D is needed to protect against severe RSV and other viral respiratory tract infections.According to Bahn, et al. the activation of HCAP-18, which is critical in the immune response to respiratory viruses, requires a minimum 25-OH vitamin D level of 32 ng/mL [10].Randolph, et al. found that children hospitalized with RSV bronchiolitis were more likely to carry the GC1s haplotype, which is associated with higher levels of 25-OH vitamin D binding protein, and consequently, less freely available 25-OH vitamin D [12].Additionally, Hansdottir, et al. found that human (p = 0.020).The difference remained significant in multivariable ordinal logistic regression: VDI subjects were more likely to use higher levels of oxygen support (OR = 4.58; 95% CI 1.58-13.23;data not shown) adjusting for breastfeeding, age and RSV detection.
Figure 2 displays further evidence of more severe disease in VDI subjects.We found that VDI subjects had significantly longer median days of hospitalization (5 versus 3 days, p = 0.030), and of oxygen support for subjects requiring oxygen (4 versus 1 days, p = 0.001) when compared to VDS subjects.However, the median duration of parent-reported fever was similar between the two groups (2 versus 2 days, p = 0.830).
Multivariable log-transformed linear regression analysis verified that VDI patients had longer duration of hospitalization (1.4 days longer, 95% CI: 1.0, 1.9 days) and oxygen use (2.2 days longer, 95% CI: 1.4, 3.5 days) compared to VDS subjects with adjustment of breastfeeding, age and RSV detection (data not shown).

Discussion
We found that infants with 25-OH vitamin D levels < 30 ng/mL suffered from more severe viral respiratory infections according to several objective measures when compared to those infants with sufficient levels, even when adjusting for age, breastfeeding and RSV status.We had a high level of VDI in our population, but we did not find any risk factors associated with VDI other than breastfeeding and younger age, suggesting that the most important predictor of neonatal vitamin D status in our population was likely related to maternal vitamin D stores.disease burden [17].Conversely, two other studies of vitamin D prophylaxis failed to show any effect on the rate of ARI: no reduction in upper respiratory tract infection was observed among U.S. adults aged 45-75 years who were given vitamin D3 supplementation [18]; similarly vitamin D3 supplementation among adults in New Zealand had no impact on the incidence nor severity of respiratory infections [19].
Few studies have addressed whether vitamin D insufficiency is correlated with an increased severity of symptoms with ARI.One prospective study of 28 Japanese children hospitalized with lower respiratory tract infections did show a significant correlation between VDI and use of supplementary oxygen and artificial ventilation [20].A study of 300 German adults with community acquired pneumonia showed that a modestly more severe course of illness was associated with low levels of 25-OH vitamin D [21].By contrast, no such association was found in a large study investigating 25-OH vitamin D levels and the duration of respiratory tract infections among elderly Norwegians [22].
Recently, Beigelman, et al. reported a study with a similar design to ours, investigating the relationship of vitamin D levels and severity of RSV bronchiolitis amongst hospitalized infants, but with opposite results [23].Of note is that the 25-OH vitamin D assay used in their study was different than our assay, which may also affect the validity of comparing results of our studies.They found no differences in severity as measured by duration of hospitalization, lowest oxygen saturation level and bronchiolitis severity score in their population related to vitamin D status.However, there are several important differences in our populations-the mean age of their subjects was lower, at 4 versus 8 months; they had a larger percentage of African American participants (42% versus 4%); and most notably a very low number of subjects had vitamin D insufficiency compared to airway epithelial cells infected with RSV and exposed to 25-OH vitamin D resulted in a decreased inflammatory response with minimal impact on the capacity for viral clearance [13].
From a clinical perspective, increased rates of viral respiratory tract infections have been shown in both infants and children with insufficient levels of 25-OH vitamin D [4][5][6][7][8][9].Several studies, all outside the U.S., have shown that newborns with lower cord blood levels of 25-OH vitamin D are at increased risk for acute nasopharyngitis [4], lower respiratory tract infections [5,7,9], and RSV bronchiolitis [6].Another study of pregnant Norwegian women detected a decreased risk of lower respiratory tract infections during the first 3 years of life in those infants with higher maternal midpregnancy 25-OH vitamin D levels [14].A prospective study of 743 Canadian children ages 3-15 years also found a correlation between lower serum 25-OH vitamin D levels and rates of laboratory-confirmed viral respiratory tract infection [8].A limitation of such studies has been the difficulty in proving causal association, particularly when potentially confounding factors such as younger age and season have also been associated with both increased rates of viral respiratory tract infections, and vitamin D insufficiency.
More definitive evidence of VDI and the risk of viral respiratory infection stems from prospective studies of vitamin D prophylaxis [15][16][17].Grant, et al. demonstrated a reduction in primary care visits for ARI among New Zealand infants randomized to receive prophylaxis with high dose vitamin D, and whose moms were also prophylaxed during pregnancy [15].Goodall, et al. also found a reduction in laboratory confirmed upper respiratory tract viral infections and viral loads in Canadian adults given vitamin D3 [16].In another study, Swedish adults with frequent respiratory infections given vitamin D3 prophylaxis also had a reduction in our subjects (median level of 36.8 ng/mL versus 24.5 ng/mL).The relationship of vitamin D to bronchiolitis severity might vary according to age.Racial differences in vitamin D metabolism may also exist.Finally, given the critical threshold of 30 ng/mL needed to trigger the immunologic effect of 25-OH vitamin D, [10] Beigelman's study may not have had enough subjects with low vitamin D levels to discern an effect on severity.
Our study provides novel information in that it is one of very few to date investigating whether lower levels 25-OH vitamin D levels correlate with the severity of viral respiratory infections.Although our numbers were small, we were able to demonstrate higher severity of the infection among these infants, even after controlling for viral etiology, age and breastfeeding status.Specifically, infants with VDI had significantly longer durations of hospitalization and oxygen use, and also required a higher level of oxygen support.These findings augment those of the Japanese study [20] and provide support for the importance of maintaining 25-OH vitamin D levels ≥ 30 ng/mL to not only prevent respiratory infections in children, but lessen their severity.
Previous work has revealed that 25-OH vitamin D levels in newborns directly correlates with maternal levels during pregnancy [24], and that supplementation of women during pregnancy with higher dose 25-OH vitamin D may result in less VDI in newborns, as well as in decreased rates of asthma in childhood [25].Our findings further highlight the importance of addressing the issue of maternal VDI during pregnancy.Higher supplementation beyond prenatal vitamins may be warranted, particularly as there was no difference in levels associated with those infants who had been prescribed supplemental vitamin D compared to those without.Potentially there was poor compliance with administering the vitamin D to the infants.
Our study had several limitations.A larger study might have allowed a number of sub-group analyses to further tease out severity impact.25-OH vitamin D levels were measured at the time of clinical presentation/ enrollment, and the correlation and impact of levels over the duration of illness could not be determined.Lastly, our population was confined to otherwise healthy subjects in the Pacific Northwest, and so our findings may not be applicable to other populations in other regions of the US.

Conclusion
Our findings suggest that achieving sufficient levels of 25-OH vitamin D in healthy infants and toddlers might be an effective intervention to reduce severe viral respiratory infections.Further interventional studies in either infants or pregnant females would be helpful in assessing this strategy to prevent viral respiratory infections that are associated with severe clinical outcomes in infants and children.

Figure 2 :
Figure 2: Duration of receiving medical support among ARI pediatric cases who needed such treatment shown by vitamin D level as box-and-whisker plots.Note: The boxes represented the median (thick line) and 25 th and 75 th percentile (box end) of duration in each group.The whisks indicate 1.5 times of interquartile range below 25 th percentile and above 75 th percentile.Difference of duration between vitamin D groups were tested using Wilcoxon rank sum test.P-value was adjusted for multiple comparison.

Table 1 :
Association between demographics, risk factor and level of vitamin D among 90 children hospitalized.

Table 2 :
Multivariable analysis of potential confounders, including age, RSV detection and breastfeeding status, and 25-OH Vitamin D sufficiency among 90 children hospitalized for viral respiratory tract infections.

Table 3 :
Association between vitamin D level and clinical severity outcomes.
Note: a Based on chi-square for categorical variables (exact test if appropriate) or Wilcoxon rank-sum test for continuous variables.False discovery rate was presented for multiple comparison adjusted p-values; b Not Done group is not included in statistical testing.• Page 5 of 7• Fairchok et al.J Infect Dis Epidemiol 2017, 3:030