Alteration of Stool Microbiota in Preterm Infants Less Than 32 Weeks With Anemia, and Following Blood Transfusion
Conditions: Anemia, Neonatal, Necrotizing Enterocolitis, Dysbiosis
Sponsor: University of Texas Southwestern Medical Center

Study Title: Alteration of Stool Microbiota in Preterm Infants Less than 32 Weeks with 
Anemia, and Following Blood Transfusion 
 
Date Approved by IRB: November 22, 2017 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

The University of Texas Southwestern Medical Center at Dallas 
Institutional Review Board 
 
 
Study Title: 
Alteration of Stool Microbiota in Preterm Infants Less than 32 Weeks with Anemia, and 
Following Blood Transfusion 
 
Investigators: 
Kshiti Shah, MD 
Julie Mirpuri, MD (PI) 
 
Type of Funding: 
Institutional 
 
Purpose: 
 To determine if the stool microbiota in preterm infants is altered with anemia. 
 To determine if the stool microbiota in preterm infants is altered after blood 
transfusion. 
 
Background: 
Neonatal gut microbiota begins developing at approximately 2 weeks of age and is less 
diverse than the microbiota of a term infant. Four bacterial classes make up more than 
90% of the microbes present in the neonate: Bacilli, Gammaproteobacteria, Clostridia, 
and Negativicutes. There are multiple factors that influence the neonatal gut microbiota, 
including mode of infant delivery, type of feeds (i.e. formula or breast milk), medications 
such as antibiotics, and exposure to pathogens in a hospital environment. 
 
Dysbiosis occurs when there is an imbalance in the proportion of bacterial pathogens 
and commensal bacteria. This can cause a shift away from homeostasis and lead to an 
inflammatory state and disease. Studies utilizing molecular methods suggest that 
dysbiosis occurs in neonates with necrotizing enterocolitis (NEC). While results vary, 
large studies and meta-analyses suggest a predominance of Gammaproteobacteria 
with NEC and a decrease in Firmicutes with NEC. There may also be a decrease in 
bacterial diversity prior to NEC onset. 
 
The relationship between transfusion and necrotizing enterocolitis has been 
controversial and frequently studied. Despite this, there is a lack of good quality studies 
on this topic with conflicted findings and the literature does not currently support a 
relationship between transfusions and NEC. There have been limited studies on the 
relationship between anemia and NEC. Some suggest that severe anemia and NEC are 
associated, however this has not been shown in other studies.  
 
While the evidence seems to support a relationship between a change in microbiota and 
NEC, there is no clear relationship between anemia or transfusion and NEC. There 
have been no studies evaluating the relationship between anemia or transfusion and a 
change in microbiota. That is the primary focus of this study. 

 
 
 
Hypothesis: 
Anemia will result in alteration of the stool microbiota. Transfusion will return microbiota 
to baseline. The specific alteration in microbiota with anemia may include: increase in 
Proteobacteria, decrease in Firmicutes, and/or decrease in microbiota diversity. 
 
Primary Outcome: 
 Change in microbiota with anemia 
 
Secondary Outcome: 
 Change in microbiota after transfusion 
 
Inclusion Criteria: 
 Preterm infants less than 32 weeks gestation at birth 
  7 days and <30 days on enrollment 
 On minimum 100mL/kg enteral feeds 
 
Exclusion Criteria: 
 NEC prior to enrollment 
 Prior surgery 
 Major congenital anomalies 
 Oxygen requirement with FiO2 >50% 
 
Methods: 
 
Definitions: 
 Severe anemia will be defined as a hematocrit < 25%. 
 Anemia will be defined as a hematocrit  25 and <30%. 
 No anemia will be defined as a hematocrit  30%.  
 
Hematocrit will be tested per patient’s care team. In this unit, hematocrit is usually 
obtained every Monday, and sometimes more frequently.  
 
Sample Collection: 
 Collect stool samples weekly (Sunday, Monday, or Tuesday) 
 Collect stool samples 1, 4, and 7 days post-transfusion 
 Stool will continue to be collected until infants are 38 weeks corrected gestational 
age (CGA) or discharge (whichever is earlier). 
 
 
 
 
 
 

Data Collection: 
Hematocrit 
Gestational Age 
Birthweight 
Apgars 
Sex 
Race 
Singleton/Multiple 
Age at stool collection and at 
hematocrit blood sampling 
Iron supplementation 
Type of feeds (breastmilk/formula 
feed) 
Calorie fortification of feeds 
Total days on antibiotics 
Number of transfusions 
Positive cultures 
NEC 
Indomethacin exposure 
 
Groups:  
Paired samples will be obtained from the same infants, one before and one after onset 
of anemia. Therefore, the patients will serve as their own controls. 
 
Sample Analysis: 
 Stool samples obtained while patient is on antibiotics will not be included in 
analysis since the microbiota is expected to be significantly changed with large 
populations wiped out. Stool samples obtained 5 days after completion of 
antibiotics will be included in analysis again since infants are expected to be 
recolonized by that time.  
 qRT-PCR of samples will be performed to quantify the major bacterial groups: 
Proteobacteria, Firmicutes, and Bacteroides.  
 Next generation sequencing will be done for qualitative analysis and 
metagenomics profiling of bacteria 
 
Informed Consent: 
New admissions to the Parkland NICU will be screened for recruitment if their birth 
gestational age (OB assigned) is less than 32 weeks 0 days and if they are at least 7 
days old. 
  
• If they meet these two criteria, they will be screened to ensure they do not meet 
exclusion criteria. 
 
• If they do not meet the exclusion criteria, then their feeds will be evaluated. If they are 
on at least 100mL/kg/day of enteral feeds, then they will be approached for recruitment. 
Infants that are not yet on 100mL/kg/day of enteral feeds will have their charts assessed 
daily until 29 days of life. If and when they achieve 100mL/kg/day of enteral feeds, they 
will be approached for recruitment. If an infant does not achieve 100mL/kg/day of 
enteral feeds before 30 days of life, they will not be approached for enrollment in this 
study. 
• If an infant meets the above criteria, and once their parent/guardian/LAR provides 
consent, then stool collection will begin for that infant from the next available defecated 
diaper. In cases where the patients are not the PI’s patients, the study team will inform 
all the physicians caring for the patients within the NICU about this research study and 

once they approve for their patients’ (infant’s) parents/guardians/LARs to be 
approached for consenting purpose to enroll the infant into this study, the bedside nurse 
will check with the parents/guardians/LARs if they would like to be approached. Once 
they let the beside nurse know if they agree to be approached, the nurse would inform 
this to Dr. Mirpuri (the PI) and Dr. Shah (study team) who will be responsible for 
recruitment of subjects and they would approach the patient’s parent/guardian/LAR for 
consenting purposes. 
 
 
Sample Size Calculation: 
Group sample sizes of 194 achieve 80% power to detect an odds ratio of 1.500 in a 
design with 2 repeated measurements having a simple covariance structure when the 
proportion for group 2 is 0.4 and group 1 is 0.5. The alpha level is 0.05. 
 
Statistical Analysis: 
Descriptive analysis will use percentages, means, medians, and measures of variability 
to describe demographic data. Categorical factors will be evaluated using Chi-square or 
Fisher exact test between the groups. Continuous factors will be evaluated using 
Student’s t-test or Mann-Whitney U test if the data is non-normal distributed between 
the groups. Repeated measurements will use analysis of variance (ANOVA) to compare 
groups. The statistical significance level will be 0.05 for all statistical tests.