Randomized Controlled Trial of Vitamin D Supplementation on Improvement of Pneumonic Children at Tertiary Pediatric Hospital in Egypt
Phase: PHASE1
Conditions: Child, Hospitalized, Vitamin D Deficiency, Pneumonia
Sponsor: Cairo University

1 
 
Cover page 
Study Protocol with statistical analysis plan  (SAP) 
Title: Randomized Controlled Trial of Vitamin D supplementation on Improvement of 
Pneumonic Children at Tertiary Pediatric Hospital in Egypt 
Date of the Document: 24-July-2019   
REC approval number: D-12-2019 
 

2 
 
Introduction: 
Globally, pneumonia is considered one of the prominent causes of children’s morbidity and 
mortality, particularly in developing countries, causing 29% of the under-Five mortality(1).
 
According to the World health organization estimates, 66% of deaths due to pneumonia occur in 
infancy, where 90% occur in developing countries(2).
 
In Egypt, according to UNICEF 2018, Acute Respiratory Tract Infection (ARTIs)was estimated to 
account for 11% and 19% of the under-five and post-neonatal mortalities respectively(3). 
Despite the well-recognized role of vitamin D in metabolism and homeostasis in the general 
population, there is now growing interest in its probable association with pneumonia(4). 
Basically, 25(OH) D and 1,25 (OH)2D are surrogate markers of the active form of vitamin D in the 
body. Recently, it has been highlighted that Vit D(1,25(OH)2D ) has a crucial role in host defenses, 
inflammation, and immunity. Furthermore, Vit D (1,25(OH)2D ) promotes the expression of 
cathelecidin and B-defensin (antimicrobial peptides) that are broadly expressed in the body and playa 
vital role in innate immunity as a result of their chemotactic action and toxin neutralization(5). 
Due to its short half-life (12-36 hours) compared to 25(OH)D (3 weeks), 1,25(OH)2D is proposed to 
provide a more  valid measure for better prediction of the vitamin D role in reducing the severity and 
improving the prognosis of patients with (ARTIs)(6). 
Globally, about 30% to 90% of Under-5 children experience vitamin D deficiency. This could vary 
among children, according to the socioeconomic, environmental and behavioral circumstances. 
Studies evaluating the association of 1,25 (OH)2D  deficiency and the severity of respiratory tract 
infection, are rare and showed controversial findings(7,8). 
However, an Indian systematic review polled the results of 12 studies, with 2279 participants, 
highlighted the significant correlation between vitamin D deficiency and incidence and severity of 
ALRIs(9). 
A prospective cohort study conducted in Yemen in 2009, examined the ability of deficient levels of 
vit.D to predict the outcomes of severe pneumonia.  The study documented the significant association 
between vitamin D deficiency with neutropenia and hypoxia in patients with severe pneumonia, thus 
predicting poor prognosis(10). 
In Egypt 2010, a case-control study conducted on children aged 2 to 5 years to examine the impact of 
vitamin D deficiency on the susceptibility of pneumonia. The study illustrated that Vitamin D 
deficiency is associated with a higher incidence and more severe pneumonia(11). 
Hashemian H., et al 2017, advocated providing children(particularly suffering from pneumonia) with 
adequate amounts of vitamin D supplements(12). 
Nevertheless, few studies have been conducted to evaluate the impact of vitamin D supplementation 
on the outcome of pneumonic infants. 

3 
 
Thus, we urge to conduct a randomized controlled trial (RCT) in Abou ElReesh tertiary Pediatric 
hospital, to evaluate the effects of vitamin D3 supplementation to children with pneumonia. We 
postulated that supplementation of 100 000 IU of vitamin D3 (Cholecalciferol) will reduce the 
duration of illness in those children and improve their outcome. 
Subjects and Methods: 
Study design and study setting 
The current study is a randomized, double-blinded placebo-controlled trial (RCT) that was conducted 
in Cairo University Abou ElReesh Children hospital, which is a university-affiliated teaching hospital 
in Egypt, to evaluate the effect of vitamin D3 supplementation on the outcome of treatment of 
children with pneumonia. Pneumonia cases were recruited from two of the hospital general pediatric 
departments and the four pediatric intensive care units (PICUs) of the hospital. 
Sample Size and study population  
To detect a 20% difference in the mean duration of pneumonia between the vitamin D arm and 
control arm [5 days vs. 6 days (SD 2)] allowing Type I error of 5% and Type II error of 10%, 86 per 
group (172 total) is the minimum number to be recruited [13]. After allowing for 10% for 
nonresponse or loss for follow-up and 25% for exclusion of children diagnosed with sufficient or 
toxic level of vitamin D after vitamin D testing (Box 1) according to prevalence of vitamin 
deficiency in a pediatric hospital of eastern India [14], 233 was the minimum number to start with.   
Mild and moderate pneumonia cases were recruited from two of the hospital general pediatric 
departments chosen by simple random sample from a total of six hospital departments while severe 
pneumonia cases were recruited from the four PICUs of the hospital. 
All children (259 children) between 1 month and 12 years of age admitted to the selected 
departments during the period from 9th September 2019 to 15th December 2019 and diagnosed 
clinically with pneumonia according to World Health Organization criteria of severity [15] (Box 2) 
were screened for the inclusion criteria. Children who had clinical signs of rickets (2 children), severe 
illnesses (meningitis, heart or renal disorders, measles, severe malnutrition, endocrine dysfunction, 
hypercalcemia, hyperthyroidism, and suspected tuberculosis) (4 children) or were known to have 
received high-dose vitamin D treatment in the past 3 months (3 children) were excluded from the 
study. Children with sufficient (52 children) or toxic (7 children) levels of vitamin D were also 
excluded. 
Thus, 191 child (93 in the intervention arm and 98 in the control arm) who had pneumonia with 
insufficient or deficient level of vitamin D and their parents consented to participate were actually 
enrolled in the study, completed the baseline and the outcome assessments and continued the daily 
clinical follow up till their discharge (improvement or death) from the hospital.  

4 
 
Box 1: Level of vitamin D [16]
Sufficient vitamin D (25(OH)D:  ≥ 30 ng/mL), 
Insufficient vitamin D (25(OH)D or at risk of deficiency : 10-29 ng/mL), and  
Deficient vitamin D (25(OH)D:  <10 ng/ mL). 
1 
Box 2: Diagnosis of Pneumonia [15]
Pneumonia: (i) Age-specific tachypnoea (>60 ⁄ min if <2 months; >50 ⁄ min if 2–11 months; >40 if 
12–24 months) and (ii) absence of wheeze (with or without fever). 
Fever: Axillary temperature >37.50 _C (age 1 week– 3 months) or >38.0 _C (2–23 months). 
axillary temperature was measured using a standard mercury thermometers 
Respiratory rate was measured twice for one full minute while the child is quiet using stopwatches 
taking the average count. 
Mild pneumonia: Minimally increased work of breathing, no hypoxemia, able to tolerate PO 
Moderate pneumonia: Hypoxemia, inability to tolerate PO, moderately increased work of 
breathing (grunting, retracting, tachypnea). 
Severe Pneumonia: Significantly increased work of breathing, altered mental state, concern for 
respiratory failure sepsis, failure to maintain oxygen saturation with Fi02 of 50%, need for positive 
pressure ventilation. Oxygen saturation was measured using a pulse oximeter with a probe on a finger 
or toe, in room air. 
Improvement (discharge criteria): (Meets all) tolerating PO, not hypoxemic (> 90%), mildly 
increase or normal work of breathing [17]. 
Vitamin D testing 
Two milliliters of venous blood was collected on plain tubes, left for 10 min to clot and then 
centrifuged at 3000 rpm for 5 min; the separated serum samples were stored at -20 °C till the time of 
assay and used for detection of concentrations of serum 25OH Vitamin D Total ( 25OH Vitamin D2 
and D3) by ELISA using (DIAsource 25OH Vitamin D Total ELISA Kit, Catalogue No. KAP 1971) 
supplied by DIA source Immunoassays S.A. (Rue Du Bosquet, 2 B-1348 Louvain-la Neuve, Belgium 
), according to the manufacturer’s instructions and based on the principle of solid-phase Enzyme-
Linked Immunosorbent Assay performed on microtiter plates. The amount of substrate turnover is 
determined colourimetrically by measuring the absorbance, which is inversely proportional to the 
total 25OH Vitamin D (D2 and D3) concentration. A calibration curve is plotted and the total 25OH 
Vitamin D (D2 and D3) concentrations of the samples are determined by dose interpolation from the 
calibration curve. 
Random allocation of study groups  

5 
 
The children were individually randomized into intervention or control groups using a random 
number sequence elicited in an Excel spreadsheet with no limitations. The allocation was further 
hidden by using closed dark envelopes. A biostatistician and an office secretary who were not 
members of the investigating team did independently the randomization, repackaging, sequencing, 
and allocation concealment. None of the study staff and participants’ parents was aware of the drug 
or placebo being dispensed. The codes were revealed only at the time of final data analysis.   
The children were followed clinically on a daily base by the three study pediatricians to assess the 
resolution or deterioration of signs and symptoms of pneumonia till being discharged from the 
hospital whether due to recovery or death. The routine treatment of inpatient children is free of 
charge in Abou ElReesh Children's hospital, while vitamin D was paid by the researchers to be given 
free of charge to the children in the intervention arm during the study period.  
Baseline assessment  
Data was collected for each participant regarding socio-demographic variables (age, sex, feeding 
practices), skin color, nature and duration of presenting symptoms. All children were examined for 
vital signs (temperature, heart rate, respiratory rate, blood pressure, oxygen saturation), pallor, 
cyanosis, nasal flaring, grunt, and mental status. Pa O2/FIO2 was measured and serum creatinine, C 
reactive protein, platelet count and serum bilirubin laboratory tests were done. SOFA score was 
assessed for severe pneumonia cases.  
Intervention  
All children were treated with antibiotics according to WHO classification and treatment of 
childhood pneumonia at health facilities 2012 [18],  at enrollment after obtaining consent from 
parents and completing the baseline assessment, children were given a single injection of one ml of 
100.000 IU of vitamin D3 (Cholecalciferol), vitamin D3 obtained from 2 ml vials containing 200,000 
IU each (Devarol- S- 200.000 I.U. produced by Memphis for Pharmaceutical and Chemical 
Industries) and stored in manufacturer’s recommended conditions in a dry, cool environment for 1–
16 weeks (depending on the date of recruitment)  or placebo which is 1 ml saline injection. Syringes 
were labeled with a unique ID number and given by the blinded doctors choosing the next syringe 
with a randomization code. (only office secretary aware of randomization codes).  
Hospital follow-up 
 Children were monitored with recording data every eight hours for respiratory rate, chest indrawing, 
oxygen saturation, auscultation findings, fever, feeding, cyanosis, and mental status. The child was 
discharged when fever and fast breathing were absent for 2 consecutive days. 
Outcomes assessment  
Outcomes were assessed 7 days after vitamin D injection, the first day whereas vitamin D reaches its 
maximum level in the blood [19] to guarantee the assessment of all participants before their 

6 
 
discharge.  The primary outcome variables were changed in serum level of 25(OH)D, PaO2/FIO2, 
serum creatinine, C reactive protein, and serum bilirubin levels and platelets count. Also, the SOFA 
score for severe cases. The secondary outcomes included the fate of the cases (improvement or death) 
and the duration between enrolment and hospital discharge for improved cases.   
Statistical analysis: 
Patients' percent change of Vitamin D (25 (OH)2D)   was calculated to quantify the change levels 
before and after vitamin D3 (Cholecalciferol) administration through the equation: 
Pediatric, Blood Vitamin D level (BVD) %change = 
[(BVD after – BVD before) ÷ BVD before] × 100 
Collected data were entered and analyzed using the Statistical Package for Social Science Software 
(SPSS) program, version 21.0 IBM. Tests of normality of data (like Shapero-Wilk test) revealed that 
data isn’t normally distributed. That’s why non-parametric tests like MannWhitney and Kruskal-
Wallis tests were used in univariable comparisons to quantify the associations of continuous 
variables. Data were summarized using the median and interquartile range for quantitative variables. 
Spearman correlation test was used to detect the relationship between continuous variables. P values 
below 0.05 were considered statistically significant. A multivariate Cox regression analysis model 
was conducted to explore the independent effect of vitamin D3 supplementation on the overall 
survival of pneumonic children.  
Given the short period of the study follow-up and single intervention, there was no stopping rule or 
interim analyses.  
All children randomized were included in the analysis on an intention-to-treat basis unless the 
outcome measures were missing (n = 0) or reported to have recovered or lost within 24 h (n = 0). 
Meantime to recovery for the episode of pneumonia at recruitment was compared for the vitamin D 
group and control group. Kaplan–Meier plots and log-rank tests were used to compare the time to 
recover from the index episode of pneumonia between the vitamin D and placebo groups. 
Ethical consideration  
The study protocol and the consent form have been reviewed and approved on July, 2019 before 
study implementation by the department of public health and community medicine and pediatrics at 
Kasr Alaini faculty of medicine. The study protocol was also approved by Cairo University Ethical 
committee. Written consent was obtained from one of the child’s parents if the child met the study 
criteria after explaining the study objectives, the nature of the intervention, risk and benefits from 
participating in the study and alternatives in case of participation refusal. 
References: 
1- Kliegman Robert M., Geme Joseph St. Nelson Textbook of Pediatrics 21th Edition. Elsevier;2019: 
2088 -94. 

7 
 
2- K. Tiewsoh, R. Lodha, R.M. Pandey, S. Broor, M. Kalaivani. Factors determining the outcome of 
children hospitalized with severe pneumonia. BMC Pediatr. 2009; 9:15. doi: 10.1186/1471-2431-9-
15. 
3- UNICEF. Levels & Trends in Child Mortality. Estimates developed by the UN Inter-agency Group 
for Child Mortality Estimation. Report 2019. https://childmortality.org/wp-
content/uploads/2019/10/UN-IGME-Child-Mortality-Report-2019.pdf  
4- Norman AW. From vitamin D to hormone D: fundamentals of the vitamin D endocrine system 
essential for good health. Am J Clin Nutr. 2008; 88(2):491S-499S. 
5- Underwood MA, Bevins CL. Defensin-barbed innate immunity: clinical associations in the 
pediatric population. Pediatrics. 2010; 125(6):1237-47. doi: 10.1542/peds.2009-3289. 
6- Nirvana Tavahen, Zahra Pourmoghaddas, BehnooshEsteki, NahidAslani, Hamid Rahimi. Active 
form and Reservoir form of Vitamin D in Children with Acute Lower Respiratory Infections and Its 
Association with Severity of the Infection. Arch Pediatr Infect Dis. 2019; 7 (4); e83431. doi: 
10.5812/pedinfect.83431 
7- Larkin A, Lassetter J. Vitamin D deficiency and acute lower respiratory infections in children 
younger than 5 years: Identification and treatment. J Pediatr Health Care. 2014;28(6):572–82. quiz 
583-4. doi: 10.1016/j.pedhc.2014.08.013 
8-  Bergman P, Lindh AU, Bjorkhem-Bergman L, Lindh JD. Vitamin D and respiratory tract 
infections: A systematic review and meta-analysis of randomized controlled trials. PLoS One. 
2013;8(6). e65835. doi:10.1371/journal.pone.0065835.  
9- Kana Ram Jat. Vitamin D deficiency and lower respiratory tract infections in children: a 
systematic review and meta-analysis of observational studies. Trop Doct. 2017;47(1):77-84. 
10- Banajeh SM. Nutritional rickets and vitamin D deficiency--association with the outcomes of 
childhood very severe pneumonia: a prospective cohort study. Pediatr Pulmonol. 2009;44(12):1207-
15. doi: 10.1002/ppul.21121. 
 11- Albanna EAM, Ali YF, Elkashnia RAM. Vitamin D and LL-37 in children with pneumonia 
Egypt J Pediatr Allergy Immunol 2010;8(2):81-86. 
12- Hashemian H, Heidarzadeh A. Role of Vitamin D [25(OH) D] Deficiency in Development of 
Pneumonia in Children, Arch Pediatr Infect Dis. 2017; 5(3):e57276. doi: 10.5812/pedinfect.57276. 
13- 
MedCalc 
Statistical 
Software 
version 
18.2.1.MedCalc 
Software 
bvba, 
Ostend, 
Belgium;   http://www.medcalc.org; 2018.   
14- Basu S, Gupta R, Mitra M, Ghosh A. Prevalence of Vitamin D Deficiency in a Pediatric Hospital 
of Eastern India. Indian J Clin Biochem. 2015; 30(2):167-73. doi: 10.1007/s12291-014-0428-2.  

8 
 
15- Vijay Ganji, Xu Zhang, Vin Tangpricha, Serum 25-Hydroxyvitamin D Concentrations and 
Prevalence Estimates of Hypovitaminosis D in the U.S. Population Based on Assay-Adjusted Data. J 
Nutr. 2012;142(3):498-507. doi: 10.3945/jn.111.151977.   
16- World Health Organization. Technical Bases for the WHO Recommendations on the 
Management of Pneumonia in Children at First-Level Health Facilities. (1995)Report No. WHO⁄ARI 
⁄91.20. WHO, Geneva. https://www.who.int/maternal_child_adolescent/documents/ari_91_20/en/ 
17- Bradley JS, Byington CL, Shah SS, Alverson B, Carter ER, Harrison C, et al.The Management of 
Community-Acquired Pneumonia in Infants and Children Older Than 3 Months of Age: Clinical 
Practice Guidelines by the Pediatric Infectious Diseases Society and the Infectious Disease Society of 
America. Clinical Infectious Disease. 2011 July. e1-e52. 
18- Revised WHO classification and treatment of childhood pneumonia at health facilities • 
EVIDENCE SUMMARIES, 2012 
19- Jenny E Han , Jennifer L Jones , Vin Tangpricha , Mona A Brown , Lou Ann S Brown , Li Hao  et 
al. High Dose Vitamin D Administration in Ventilated Intensive Care Unit Patients: A Pilot Double 
Blind Randomized Control Trial. J Clin Transl Endocrinol .2016, 4, 59-65.