Effect of Vitamin D supplementation to reduce respiratory infections in children and adolescents in Vietnam: A randomized controlled trial

Background It is uncertain whether vitamin D can reduce respiratory infection. Objective To determine whether vitamin D supplementation reduces influenza and other upper viral respiratory tract infections. Methods A total of 1300 healthy children and adolescents between the ages of 3 and 17 years were randomized to vitamin D (14 000 U weekly) or placebo for 8 months in Vietnam. The primary outcome was reverse transcriptase (RT)‐PCR–confirmed influenza infection, and the coprimary outcome was multiplex PCR–confirmed non‐influenza respiratory viruses. Participants, caregivers, and those assessing outcomes were blinded to group assignment. Results A total of 650 children and adolescents were randomly assigned to vitamin D and 650 to placebo. The mean baseline serum 25‐hydroxyvitamin D levels were 65.7 nmol/L and 65.2 nmol/L in the intervention and placebo groups, respectively, with an increase to 91.8 nmol/L in the vitamin D group and no increase, 64.5 nmol/L, in the placebo group. All 1300 participants randomized contributed to the analysis. We observed RT‐PCR–confirmed influenza A or B occurred in 50 children (7.7%) in the vitamin D group and in 43 (6.6%) in the placebo group (hazard ratio [HR]: 1.18, 95% CI: 0.79‐1.78). RT‐PCR–confirmed non‐influenza respiratory virus infection occurred in 146 (22.5%) in the vitamin D group and in 185 (28.5%) in the placebo group (hazard ratio [HR]: 0.76, 95% CI: 0.61‐0.94). When considering all respiratory viruses, including influenza, the effect of vitamin D in reducing infection was significant, HR: 0.81, 95% CI: 0.66‐0.99. Conclusion Vitamin D supplementation did not reduce the incidence of influenza but moderately reduced non‐influenza respiratory viral infection.


| INTRODUC TI ON
Influenza and other respiratory viruses account for substantial morbidity in children. [1][2][3][4][5] The prime strategy for preventing respiratory infection is vaccination against influenza. Protection against influenza with vaccines is approximately 60% effective, but may be lower, particularly when there is a mismatch between antigens in the vaccine and the circulating influenza strains. 6 In the absence of available vaccines against other respiratory viruses, strategies in addition to influenza vaccination may be of clinical benefit to children.
It has been proposed that vitamin D, ingested as cholecalciferol (vitamin D) or ergocalciferol (vitamin D 2 ), can reduce viral respiratory infection, possibly by stimulating expression of antimicrobial peptides, such as the defensin retrocyclin-2. 7,8 Observational studies in children have demonstrated an association between vitamin D levels and respiratory infection, but have been inconsistent. 9-14 A recent systematic review and meta-analysis of individual participant data of vitamin D clinical trials of children and adults reported a reduced risk of acute respiratory infection (odds ratio 0.88, 95% CI: 0.81-0.96). 15 Important limitations were that definitions of respiratory infection in children varied considerably (eg, including pneumonia, 16 otitis media, 17 and exacerbation of asthma 18,19 ) and the vast majority of trials (23 of 25) did not include any laboratory confirmation of respiratory infection. 15,20 Conducting randomized controlled trials of vitamin D supplementation to prevent influenza and other respiratory infections can be challenging in settings where vitamin D deficiency may not be prevalent and uptake of influenza vaccination is relatively high. 21,22 In contrast, in most low-and middle-income countries, such as Vietnam, children are not routinely vaccinated against influenza and vitamin D deficiency in children has been reported to be more prevalent. 23 We conducted a randomized trial of vitamin D in children in Vietnam to assess its effectiveness in reducing laboratory-confirmed influenza and non-influenza viral respiratory tract infections. We hypothesized that vitamin D would reduce both laboratory-confirmed influenza and non-influenza respiratory viral infection compared to placebo.

| Study design
A placebo-blinded randomized controlled trial. The study was conducted in two phases. In the first phase, we enrolled participants

| Participants
We enrolled children and adolescents between the ages of 3 and 17 years in Thanh Liem District of Vietnam. Thanh Liem is a rural district of Ha Nam Province, part of the Red River Delta region of Vietnam, located 50 km south of Hanoi. Children born prematurely at gestational age <32 weeks, children with any chronic illness (except asthma), children with impaired vitamin D metabolism (eg, antiseizure medications), and children with a sibling participating in the study (to reduce clustering effects) were excluded. For all participants aged 7-17 years, an assent form was obtained. Signed parental consent was required for all participants.

| Randomization and blinding
Participants were assigned at random to one of the two study groups (vitamin D or placebo), in a 1:1 ratio. Computer-generated randomization was performed by an external research organization, with treatment assignments made in random permuted blocks of 4. The placebo liquid, fractionated coconut oil, was identical in appearance and taste to the vitamin D liquid. All study medication was packaged in identical-appearing bottles. The appropriate bottle of study medication, which was stored at the commune health centers at the study sites, was labeled with the participant identification number and that individual received the contents of the pre-labeled bottle with the participant identification. Participants were randomized in sequential order as they were enrolled. All participants and their parents, research field staff, study investigators, and research staff at McMaster University and at National Institute of Hygiene and Epidemiology were blinded.

| Procedures
Children and adolescents randomized to vitamin D received 7 drops

| Outcomes
The primary outcome of this study was reverse transcriptase (RT)-PCR-confirmed influenza infection using multiplex PCR for influenza A and influenza B (using modified CDC primers for matrix A gene). The coprimary outcome, non-influenza respiratory viral infection, was measured using a separate multiplex PCR for parainfluenza 1, 2, 3, metapneumovirus, RSV, entero-rhinovirus, and adenovirus.
Secondary outcomes included the following: influenza-like illness, defined as cough and fever of ≥38.0°C, receipt of antibiotics, use of over the counter medication for respiratory symptoms, pharmacy visits, private medical clinic visits, and medical center or hospital visits. These outcomes were measured through parental report with confirmation through review of commune health center records when possible.
Research staff monitored for signs and symptoms of toxicity using checklists for symptoms. The major potential toxicity was hypercalcemia which could present as kidney stones. Data on toxicity were recorded monthly.

| Statistical analysis
We calculated our sample size based on the results of the first phase of the study. We had enrolled and randomized 400 participants and followed them over 12 months. split between the two primary outcomes, differences with P < 0.025 for 2-tailed tests were considered significant for these outcomes.
For all other outcomes, differences with P < 0.05 were considered significant. Statistical analyses were conducted using sas version 9.2 (SAS Institute, Cary, NC) and R version 3.2.

| Patient involvement
No patients were involved in the design of the research question, study design, or outcome measures. Because of patient preference, we did use oropharyngeal as opposed to nasopharyngeal swabs. A summary of the vitamin D levels was disseminated to participants.

| Participants
There were 1641 children and adolescents assessed for eligibility in two  Table 1.
The mean age of participants was 8.5 years (standard deviation [SD] 4.0 years); 52.2% were female; and both figures were similar between the two study groups (Table 1)

| Outcomes
We observed RT-PCR-confirmed influenza A or B in 50 children (7.7%) in the vitamin D group and in 43 children (6.6%) in the placebo group (  (Table 3). There were a total of 177 (27.2%) influenza and non-influenza respiratory viruses in the vitamin D group and 209 (32.2%) in the placebo group.
We found no significant difference between vitamin D and placebo groups for RT-PCR-confirmed influenza, hazard ratio [HR]: 1.18, 95% CI: 0.79-1.77 (Figure 2). We found that vitamin D significantly reduced non-influenza respiratory viral infection, HR: 0.76, 95% CI: 0.61-0.94 (P = 0.011). Although the attack rates differed between the two stages of the study, the relationship between the two groups was similar (Table 2). When considering all respiratory viruses, including influenza, the effect of vitamin D in reducing infection was significant, HR: 0.81, 95% CI: 0.66-0.99.
When we compared the vitamin D group to the placebo group for secondary outcomes, we found the following: 50 (7.7%) vs 62 CI: −6.2%-4.4%), and 338 (52.0%) vs 361 (55.5%) that used over the counter medications for respiratory infection (absolute difference −3.5%, 95% CI: −9.0%-1.9%). No significant difference in these outcomes between groups was found. Only two participants in each group visited a private clinic, and only one in the vitamin D group and two in the placebo visited a hospital or medical center for respiratory symptoms. Only one serious adverse event was reported, which was not related to the study, a hospitalization for a scheduled tonsillectomy.
We conducted a post hoc subgroup analysis limited to participants who at baseline had 25OHD levels less than 50 nmol/L. In this cohort, there were only 14 cases of influenza, and the hazard ratio was 2.5 (5% CI: 0.78-7.9), P = 0.12. When we conducted the analysis using other respiratory viruses as the outcome, there were 46 events, HR: 0.92, 95% CI: 0.52-1.66, P = 0.8.

| D ISCUSS I ON
We

| Strengths and limitations of the study
Strengths of the study were that it was a randomized placebocontrolled trial with laboratory-confirmed respiratory viral infection as the main outcome and measurement of vitamin both at baseline and at follow-up.
One possible limitation was the use of oropharyngeal specimens instead of nasopharyngeal specimens. This was based on adherence to testing, and we reasoned that an increase in the number of swabs would make up for any reduction in sensitivity. The attack rate for all respiratory viruses in our study was 35%, which is comparable to other studies that have used RT-PCR to detect infection in children. 24

| Comparison with other studies
Two previous trials of vitamin D in children reported either an effect of vitamin D on all respiratory viruses or no effect on all influenza. 28,29 These studies had limitations such as lack of RT-PCR testing, 28,29 survey-reported symptom outcomes at the end of the study, 28 and the lack of criteria for ascertaining outcomes 29 that limit inferences and comparisons to our trial. A randomized trial of highdose versus standard-dose vitamin D supplementation in Canadian young children did not demonstrate a reduction of overall wintertime upper respiratory tract infections. 30 Our study was conducted in a middle-income country where children and adolescents are not routinely vaccinated against influenza. Vitamin D levels, while not as low as anticipated, may still be lower in this population than in many settings in developed countries. Therefore, our results may apply to children and adolescents in other low-and middle-income countries.

| CON CLUS ION
Our results show that vitamin D supplementation does not reduce influenza but can reduce non-influenza respiratory infections in children and adolescents aged 3-17 years in a low-and middleincome country. Our findings imply that vitamin D supplementation can play a moderate role in reducing illness caused by respiratory viruses. ML, BW, PS, and EP were responsible for the analysis and interpretation of data. ML produced a first draft of the manuscript, and all authors provided intellectual input. ML is the guarantor.

CO PY R I G HT
The Corresponding Author has the right to grant on behalf of all authors and does grant on behalf of all authors, a worldwide licence to the Publishers and its licensees in perpetuity, in all forms, formats, and media (whether known now or created in the future), to (a) pub- (f) license any third party to do any or all of the above.

E TH I C A L A PPROVA L
The study was approved by the research ethics board of McMaster University and that of the Ministry of Health in Vietnam.

DATA S H A R I N G
Patient-level data and statistical code are available upon request from the corresponding author at loebm@mcmaster.ca.

TR A N S PA R EN C Y
Lead author (ML) affirms that the manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned (and, if relevant, registered) have been explained.