The Effect of Vitamin D Administration on Sepsis Score and C-Reactive Protein Levels in Preterm Infants With Neonatal Sepsis
Phase: PHASE4
Conditions: Neonatal Sepsis, Preterm Infants
Sponsor: Universitas Padjadjaran

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Study Protocol and Statistical Analysis Plan 
 
 
Effect of Vitamin D as Adjuvant Therapy in 
Preterm Infants With Neonatal Sepsis 
Study Protocol and Statistical Analysis Plan 
Version 1 
1st June 2025 
 
NCT Number: NCT07245277 
Unique Protocol Id: DP. 04.03/D.XIV.6.5/337/2025 
 
Authors: 
Michelle Angelica Wijaya1 
Reni Ghrahani1 
Fiva Aprilia Kadi1 
 
Investigators: 
Principal Investigator: Michelle Angelica Wijaya1 
Other Investigators: 
Reni Ghrahani1 
Fiva Aprilia Kadi1 
 
 
1Department of Child Health, Universitas Padjadjaran Medical School/Dr. Hasan Sadikin Hospital, Bandung, West 
Java

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Study Protocol and Statistical Analysis Plan 
 
 
22 August 2022 
Signature Page 
 
This statistical analysis plan is approved by: 
 
 
 
 
 
 
dr. Michelle Angelica 
Wijaya 
Principle Investigator 
Author of Statistical Analysis Plan v1 
1 June 2025 

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Study Protocol and Statistical Analysis Plan 
 
 
1. ADMINISTRATIVE INFORMATION 
Protocol: Version 1 dated 1 June 2025 
ClinicalTrials.gov register Identifier: NCT07245277 
 
1.1 Document Version History 
 
Version Date 
Version 
Author 
Signature 
Change Description 
Reason/Comment 
1.0 
Initial release. 
Not applicable. 
 
2. STUDY SYNOPSIS 
This will be a randomized, multi-centered clinical trial. Participants will include preterm neonates 
diagnosed with neonatal sepsis who are treated with antibiotics. All eligible patients with parental 
consent, who cannot tolerate enteral feed will be assigned as Group 1 (antibiotic only). All eligible 
patients with parental consent, who can tolerate enteral feed enrolled into the trial will be 
randomized into two treatment groups. Group 2 will receive vitamin D as adjuvant therapy (400 IU) 
for seven days. Group 3 will receive vitamin D as adjuvant therapy (800 IU) for seven days. Each 
patient will undergo clinical assessments and blood examination at the beginning and end of each 
treatment period. 
 
Study assessments will include the sepsis score (Tollner score and sepsis prediction score) and C-
reactive protein examination. 
2.1. 
Primary Objective 
 
1. To determine the effect on the modified Tollner sepsis score among groups of preterm neonates 
with sepsis receiving adjuvant Vitamin D therapy at doses of 400 IU/day and 800 IU/day, 
compared to those receiving antibiotics alone. 
 
2. To determine the effect on the Sepsis Prediction Score among groups of preterm neonates with 
sepsis receiving adjuvant Vitamin D therapy at doses of 400 IU/day and 800 IU/day, compared to 
those receiving antibiotics alone. 
 
2.2. 
Secondary Objectives 
 
To determine the effect on C-reactive protein (CRP) levels among groups of preterm 
neonates with sepsis receiving adjuvant Vitamin D therapy at doses of 400 IU/day and 800 
IU/day, compared to those receiving antibiotics alone. 

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Study Protocol and Statistical Analysis Plan 
 
 
2.3. 
Study Population 
2.3.1 Number of Participants 
We aim to recruit minimum of fifty-seven neonates (19 neonates of each group) aged between 0 
and 28 days old or maximum post menstrual age 42 weeks with sepsis. 
2.3.2 Eligibility Criteria 
Participants will be assigned to a randomised study treatment only if they meet all inclusion criteria 
and none of the exclusion criteria. 
2.3.3 Inclusion criteria 
 
The inclusion criteria for the case group in this study comprise neonates (aged 0–28 days or a 
maximum post-menstrual age of 42 weeks) with a gestational age of 28–36 weeks who are admitted 
to the neonatal high care unit (NHCU) and neonatal intensive care unit (NICU) of Dr. Hasan Sadikin 
General Hospital, Bandung, and Bandung Kiwari Regional Hospital, and meet the following criteria: 
▪ 
Patients diagnosed with neonatal sepsis confirmed by blood or cerebrospinal fluid (CSF) 
culture, OR patients meeting the criteria for suspected sepsis based on a modified Tollner 
sepsis score of ≥ 5, OR Sepsis Prediction Score ≥ 3. 
▪ 
Parents or legal guardians are willing to participate in the study and have signed the 
informed consent form. 
 
2.3.4 Exclusion criteria 
Participants meeting any of the following criteria will be excluded from the study: 
 
• 
Patients with major congenital malformations, including anencephaly, encephalocele, 
holoprosencephaly, hydrocephalus, meningomyelocele, spina bifida, omphalocele, gastroschisis, or 
congenital heart disease. 
• 
Patients who were initially able to tolerate enteral feeding but developed a medical condition 
requiring fasting (NPO/Nil Per Os) during the 7-day monitoring period. 
• 
Patients who initially required fasting (due to a medical condition) but became able to tolerate 
enteral feeding during the 7-day monitoring period. 
 
2.4. 
Design and Intervention 
 
This study is an analytical experimental study with a randomized controlled trial (RCT) design. All samples 
that meet the inclusion and exclusion criteria will be enrolled as study subjects. Subject characteristics data 
will be obtained from the patient's medical records. This was an open-label trial due to the nature of the 
nutritional intervention (NPO vs. enteral). 
 
Subjects were subsequently allocated into three groups: 
 
• 
Group 1: Subjects requiring medical fasting (NPO) due to their clinical condition, who received 
antibiotics alone. 
 
• 
Group 2: Subjects able to tolerate enteral nutrition, who received antibiotics plus Vitamin D3 
supplementation of 400 IU once daily for 7 days. 
 
• 
Group 3: Subjects able to tolerate enteral nutrition, who received antibiotics plus Vitamin D3 
supplementation of 800 IU once daily for 7 days. 
 
 

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Study Protocol and Statistical Analysis Plan 
 
 
 
2.5. 
Randomisation and Blinding 
2.5.1 Randomisation Procedures 
Randomization was performed using a computer-generated random number sequence and allocated by the 
author.  
 
2.6. 
Sample Size 
 
Samples will be drawn from study subjects who meet the inclusion criteria. The determination of the 
sample size is tailored to the study objectives and the type of data involved. This study utilizes a specific 
design, namely unpaired (non-paired) categorical-numeric analytical design. Therefore, the 
determination of the sample size is based on statistical calculation, setting a 95% confidence level and an 
80% power of test (power). Thus, the minimum sample size for each group is 19 subjects. Therefore, the 
total minimum sample size for the three groups is 57 patients. 
 
2.7. 
Study Procedures 
 
Research Design and Data Source 
The research will be conducted using an analytical experimental quantitative design. The data source used in 
this research is primary data, collected while neonates are hospitalized at Dr. Hasan Sadikin General Hospital 
in Bandung, Indonesia and Bandung Kiwari Regional Hospital, Bandung, Indonesia. 
 
Study Protocol: 
1. Sampling Stage: Sampling will be conducted in the NHCU and NICU wards of Dr. Hasan Sadikin 
General Hospital Bandung and Bandung Kiwari Regional Hospital Bandung. 
2. Informed Consent: A verbal and written explanation of the entire study (informed consent) will be 
provided to the parents/guardians of the study subjects. 
3. Enrolment: The parents/guardians of the study subjects who agree to the study procedures will then 
sign the consent form. 
4. Screening: The researcher will conduct interviews to obtain general data, maternal history, and birth 
history to identify subjects who meet the inclusion and exclusion criteria. 
5. Blood Sample Collection: Peripheral venous blood samples will be collected from the case and 
control subjects twice (on Day 0 and Day 7). Each collection will be approximately for the examination 
of Vitamin D levels (i.e., 25(OH)D), hematology, peripheral blood smear morphology, and CRP. 
6. Intervention: The subject groups who can tolerate enteral feeding will receive Vitamin D3 
supplementation of either 400 IU or 800 IU once daily for 7 days. 
7. Sample Processing: Blood samples will be sent to the Clinical Pathology Laboratory at Dr. Hasan 
Sadikin General Hospital Bandung for sample preparation. The 25(OH)D level examination will utilize 
a stored biological material (SBM), which will be stored at a temperature of -80°C. 
 
 
 
 

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Study Protocol and Statistical Analysis Plan 
 
 
3 GENERAL STATISTICAL METHODOLOGY 
3.1. 
Objectives of Analysis Plan 
This analysis plan covers the analysis of all objectives. 
3.2. 
Analysis Software 
Analysis will be conducted using the Statistical Product and Service Solution (SPSS) for Windows, version 
27.0 (IBM Corp., Armonk, NY, USA). 
 
 
3.3. 
Definition of Baseline 
The baseline is taken as the measures and assessments recorded at day-0. 
3.4. 
Definition of analysis populations 
Data from this study will be analysed as a per-protocol analysis. Data from all participants who are 
randomised will be included in the per-protocol analysis. 
 
3.5. 
Definitions related to Adverse Events (AEs) 
Other than standard adverse effects, the following specific adverse events will be reported: 
• 
Gastrointestinal symptoms: diarrhoea, vomiting, bloating, increased abdominal circumference 
>2 cm 
• 
Hypercalcemia 
 
3.6. 
Adjustment for Multiplicity 
There are no plans to adjust for multiplicity. 
 
3.7. 
Interim Analyses 
Interim analysis was not performed. 
3.8. 
Handling of Missing Data 
It is inevitable that some data will be missing. If less than ten percent of the total data is missing then 
no change to the analysis will be made. 

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Study Protocol and Statistical Analysis Plan 
 
 
4 
DESCRIPTIVE STATISTICS 
4.1. 
Recruitment and Follow‐up 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Enrollment 
Allocation 
Analysis 
Recruitment (n=…) 
NPO/nil per os, antibiotic only 
(n=…) 
Enteral fed (n=…) 
Excluded (n=…) 
- Death (n=…) 
- Subsequently achieved 
tolerance to enteral 
nutrition during the 
monitoring period (n=…) 
Analyzed (n=…) 
Randomization 
Vitamin D 400 IU 
7 days 
Vitamin D 800 IU 
7 days 
Excluded (n=…) 
- Death (n=…) 
- Subsequently required 
medical fasting (NPO 
status) during the 
monitoring period (n=…)
Excluded (n=…) 
- Death (n=…) 
- Subsequently required 
medical fasting (NPO 
status) during the 
monitoring period (n=…) 
Analyzed (n=…) 
Analyzed (n=…) 

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Study Protocol and Statistical Analysis Plan 
 
 
4.2. 
Baseline Characteristics 
Baseline population will be summarised using mean and standard deviations or median and 
interquartile range. 
 
Variable 
Control 
(n=…) 
Vitamin D 400 IU 
(n=…) 
Vitamin D 800 IU 
(n=…) 
p value 
Gender, n (%) 
 
 
 
 
Male 
 
 
 
 
Female 
 
 
 
 
Age (days), 
Mean/Median 
(SD/IQR) 
 
 
 
 
Gestational age 
(weeks), 
Mean/Median 
(SD/IQR) 
 
 
 
 
Gestational age, n 
(%) 
 
 
 
 
32–<37 weeks 
 
 
 
 
28–<32 weeks 
 
 
 
 
Birth weight (gram), 
Mean/Median 
(SD/IQR) 
 
 
 
 
Birth weight, n (%) 
 
 
 
 
<1000 gram 
 
 
 
 
1000–<1500 gram 
 
 
 
 
1500–<2500 gram 
 
 
 
 
≥2500 gram 
 
 
 
 
Respiratory support, 
n (%) 
 
 
 
 
Low Flow 
 
 
 
 
CPAP/NIV 
 
 
 
 
Ventilator 
 
 
 
 
Vitamin D (ng/mL), 
Mean/Median 
(SD/IQR) 
 
 
 
 
Classification of vitamin D status 
 
   Sufficient 
 
 
 
 
   Insufficient 
 
 
 
 
   Deficient 
 
 
 
 
 
 
 
 
 

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Study Protocol and Statistical Analysis Plan 
 
 
 
5 REFERENCES 
 
1. Fort P, Salas AA, Nicola T, Craig CM, Carlo WA, Ambalavanan N. A Comparison of 3 Vitamin D Dosing 
Regimens in Extremely Preterm Infants: A Randomized Controlled Trial. J Pediatr. 2016 Jul;174:132-
138.e1. doi: 10.1016/j.jpeds.2016.03.028. Epub 2016 Apr 11. 
2. Kamsiah K, Hasibuan BS, Arto KS. The relationship between vitamin D levels and clinical outcomes of 
neonatal sepsis in Haji Adam Malik Hospital Medan, Indonesia. Open Access Maced J Med Sci. 
2021;9(B):698-703. 
3. Cetinkaya M, Cekmez F, Buyukkale G, Erener-Ercan T, Demir F, Tunc T, Aydin FN, Aydemir G. Lower 
vitamin D levels are associated with increased risk of early-onset neonatal sepsis in term infants. J 
Perinatol. 2015 Jan;35(1):39-45. doi: 10.1038/jp.2014.146. Epub 2014 Aug 7. 
4. Hagag AA, El Frargy MS, Houdeeb HA. Therapeutic Value of Vitamin D as an Adjuvant Therapy in 
Neonates with Sepsis. Infect Disord Drug Targets. 2020;20(4):440-447. doi: 
10.2174/1871526519666190626141859.