Effect of Probiotic Lacobacillus Reuterii on Serum Bilirubin Levels in Preterm Infants
Phase: NA
Conditions: Hyperbilirubinemia, Neonatal
Sponsor: Hasanuddin University

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
EFFECT OF PROBIOTIC LACTOBACILLUS REUTERII ON SERUM BILIRUBIN LEVELS IN 
PRETERM INFANTS 
January 1st 2024 
 
 
 
 
 
 
 
 
 
 
 
 
 

EFFECT OF PROBIOTIC LACTOBACILLUS REUTERII ON SERUM BILIRUBIN LEVELS IN 
PRETERM INFANTS 
 
Protocol 
 
Responsible Personnel 
Principal Investigator Contact Information 
Name: Nurwahyuni Rachim 
Title: MD, 
Institution: Department of Pediatrics, Faculty of Medicine, Hasanuddin University 
Address: Jln. Perintis Kemerdekaan KM 11 Makassar, South Sulawesi, Indonesia, 90245 
Telephone: +62(411)584461 
Email: rachim.nurwahyuni@yahoo.co.id 
 
 
Summary:  
 
This study aims to assess the effect of probiotic Lactobacillus reuteri administration on 
serum bilirubin levels in preterm infants. It was a randomized controlled trial conducted on 40 
preterm infants (32 to <37 weeks gestation, 1000-2499g birth weight) admitted to the NICU at Dr. 
Wahidin Sudirohusodo Hospital in Makassar, Indonesia. The infants were randomly divided into 
two groups of 20 each - one receiving L. reuteri probiotic and a control group. Serum bilirubin 
levels were measured at baseline and after 7 days of intervention, the need for phototherapy 
following hyperbilirubinemia will also be assessed. 
 
Introduction:  
 
The global neonatal mortality rate stands at 18 deaths per 1,000 live births, with Indonesia 
ranking fifth highest among Southeast Asian countries. Prematurity is a leading cause of neonatal 
death in Indonesia, which ranks fifth worldwide for the number of premature births. One significant 
complication of premature birth is hyperbilirubinemia, occurring in 80% of premature babies in 
developing countries like Indonesia. This condition can lead to severe neurological complications 
if left untreated. 
Probiotics have emerged as a potential management strategy for hyperbilirubinemia in 
preterm infants. These live microorganisms can improve intestinal microbial colonization and 
regulate gastrointestinal motility. Several studies have shown promising results in reducing serum 
bilirubin levels and the need for phototherapy in newborns through probiotic administration. 
However, research findings have been mixed, with some studies showing significant reductions 
in bilirubin levels while others found no difference. 
Given the potential benefits of probiotics and the lack of comprehensive research on their 
effect on bilirubin levels in premature infants, particularly in Indonesia, further investigation is 
warranted. This study aims to explore the impact of probiotic Lactobacillus reuteri on serum 
bilirubin levels in premature infants, with the goal of providing an alternative approach to prevent 
and manage hyperbilirubinemia in this vulnerable population. As the first study of its kind in South 
Sulawesi and Indonesia, it is expected to contribute valuable knowledge for future clinical 
applications. 
 
 

Objectives of Trial: 
1. Primary Objective 
a. To assess the effect of probiotic lactobacillus reuteri on serum bilirubin levels in 
preterm infants. 
2. Secondary Objective 
a. To assess serum bilirubin levels of preterm infants before and after receiving 
probiotic lactobacillus reuteri in the probiotic group and the non-probiotic group. 
b. To compare serum bilirubin levels in preterm infants before and after receiving 
probiotic lactobacillus reuteri in the probiotic group with the non-probiotic group  
c. To compare changes in serum bilirubin levels of preterm infants the probiotic group 
with the non-probiotic group. 
d. To assess the percentage of hyperbilirubinemia requiring phototherapy in the 
probiotic group  
e. To assess the percentage of hyperbilirubinemic events requiring phototherapy in 
the non-probiotic group 
f. To compare changes in serum bilirubin levels of preterm infants requiring 
phototherapy in the probiotic group with the non-probiotic group.  
 
Hypothesis: 
H0: Bilirubin levels in preterm infants in the probiotic group are lower compared to those 
in the non-probiotic group 
H1: The incidence of hyperbilirubinemia requiring phototherapy in the probiotic group is 
less than in the non-probiotic group 
 
Study Design: 
 
Interventional study 
 
Study Population: 
 
Number of samples: 
 
40 subjects 
 
Inclusion criteria: 
• 
Premature Infants (32 - < 37 weeks gestation) 
• 
Baby Weight 1000 - 2499 grams 
• 
Sick babies who are treated in the NICU 
• 
Preterm infants who received enteral feeding > 30 cc/kgBB less than 72 hours after 
birth, without jaundice or jaundice kremer 1,2, and 3  
• 
Parents have signed their child's consent to participate in the study 
Exclusion criteria: 
• 
Infants with multiple congenital abnormalities 

• 
Infants with jaundice before 24 hours or having jaundice cremer > 4 less than 72 
hours from birth 
• 
Infants with Sepsis, Metabolic acidosis 
 
Product Description: 
Lactobacillus reuterii in the form of Interlac, contains: Lactobacillus reuteri DSM 17938 
100.000.000 CFU 
 
Outcomes:  
 
The main outcome being measured is serum bilirubin levels in preterm infants. 
Specifically, serum bilirubin screening will be conducted by taking blood samples from the radial 
or femoral artery of the infants, collecting approximately 1 cc of blood to measure total and direct 
bilirubin levels. Samples will be taken at two time points: after infants receive enteral feeding >30 
cc/kg body weight and after 7 days of intervention or when Kramer scale reaches IV/V.  
The study will compare bilirubin levels between the probiotic group and control group, 
looking at initial and final levels within each group as well as the change (delta) in levels from 
initial to final measurement between groups. Additionally, the percentage of infants requiring 
phototherapy will be compared between the probiotic and control groups. Statistical analysis will 
utilize paired and unpaired t-tests to analyze differences in bilirubin levels between and within 
groups, while a chi-square test will compare phototherapy requirements. The primary outcome is 
the difference in serum bilirubin levels between the probiotic and control groups after 7 days of 
intervention, with secondary outcomes including the need for phototherapy and the rate of bilirubin 
decline between groups. Overall, this methodology aims to comprehensively evaluate the effect 
of probiotic supplementation on bilirubin levels and management in preterm infants. 
 
Conduct of Trial: 
This study will be conducted as a randomized controlled trial (RCT) to evaluate the effect 
of probiotic Lactobacillus reuteri administration on serum bilirubin levels in preterm infants. The 
trial will take place in the Neonatal Intensive Care Units of Dr. Wahidin Sudirohusodo Hospital 
and Cahaya Medika Hospital in Makassar, South Sulawesi. Eligible participants will be preterm 
infants born at 32 to <37 weeks gestation with birth weights between 1000-2499 grams. Written 
informed consent will be obtained from the parents of eligible infants after providing a detailed 
explanation of the study purpose and procedures. Using consecutive sampling, 40 infants meeting 
the inclusion criteria will be randomly allocated into two groups of 20 each - an intervention group 
receiving probiotic L. reuteri and a control group. The intervention group will receive 5 drops of 
Interlac (containing 1x108 CFU L. reuteri) daily for 7 days, while the control group will receive 
standard care without probiotics. Serum bilirubin levels will be measured at baseline (after infants 
receive >30 cc/kg enteral feeding) and again after 7 days of intervention or when Kramer scale 
reaches IV/V. Blood samples of approximately 1 cc will be collected from the radial or femoral 
artery to measure total and direct bilirubin levels. The primary outcome will be the difference in 
serum bilirubin levels between groups after 7 days, with secondary outcomes including the need 
for phototherapy and rate of bilirubin decline. Statistical analysis will be performed to compare 
outcomes between the two groups. 
 
 

 
Figure 1. Study Procedure 
 
Statistical Aspects and Considerations: 
This study employs a randomized controlled trial (RCT) design to evaluate the effect of 
probiotic Lactobacillus reuteri on serum bilirubin levels in preterm infants. The sample size was 
calculated using a formula for a two-sided test, with a 99% confidence level (α=0.01) and 90% 
power (β=0.10). The standard deviation of bilirubin levels was estimated at 3.20 based on a 
previous study. Subjects meeting inclusion criteria will be randomly allocated to intervention and 
Infants born or admitted to the 
NICU at 32-<37 weeks gestation 
and 1000-2499 grams birth 
Meet inclusion criteria and do 
not meet exclusion criteria 
Measurement of serum Bilirubin levels after enteral feeding > 30 cc/kgBB  
(within 72 hours of age)   
Probiotic Administration 
Not given probiotics 
Bilirubin level measurement after intervention on day 7 or Kremer IV/V 
Data Collection 
Data analysis 
Results 
Reporting 

control groups using consecutive sampling. Statistical analysis will include both univariate and 
bivariate methods. Univariate analysis will describe basic data characteristics using frequencies, 
means, standard deviations, and ranges. Bivariate analysis will employ paired and unpaired t-
tests for normally distributed data, and Wilcoxon and Mann-Whitney tests for non-normally 
distributed data. Chi-square tests will be used for categorical variables. The significance level is 
set at p≤0.05 for all analyses. This comprehensive statistical approach aims to rigorously assess 
the efficacy of probiotic intervention on bilirubin levels in preterm infants while controlling for 
potential confounding factors. 
 
 
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