Clinical trial NCT01756040
Conditions: Prematurity Intestinal Permeability
Keywords: intestinal permeability preterm infants dual sugar test intestinal microbiota
Sponsor: University of Maryland, Baltimore
Start date: 2013-02-01; Completion date: 2021-08-31
Enrollment: 211 participants; Allocation: NA; Intervention model: SINGLE_GROUP

Brief summary:
Necrotizing enterocolitis (NEC) is a life-threatening, gastrointestinal emergency characterized by increased intestinal permeability, affects approximately 7 to 10% of infants \<1500 g birthweight, and typically occurs within 7 to 14 days of birth. Mortality is as high as 30-50%. Prematurity is the greatest risk factor for the development of NEC due to the physiological immaturity of the gastrointestinal tract and altered or abnormal gut microbiota. Several studies have demonstrated that the initiation of an intense systemic and local inflammatory cascade leads to intestinal necrosis. The human intestine is lined by a single layer of cells exquisitely responsive to multiple stimuli and is populated by a complex climax community of microbial partners. Under normal circumstances, these intestinal cells form a tight but selective barrier to "friends and foes": microbes and most environmental substances are held at bay, but nutrients are absorbed efficiently. Epithelial barrier integrity is itself dynamic and matures over time starting soon after birth, though the mechanisms regulating dynamic permeability are poorly understood. Low birth weight, prematurity, and early postnatal age are associated with a leaky gut. Although intestinal permeability is higher at birth in preterm than term infants, there is usually rapid maturation of the intestinal barrier over the first few days of life in both populations. The investigators hypothesize that increased levels of measures of intestinal permeability (urine lactulose/rhamnose (LA/Rh), and fecal alpha1- antitrypsin will identify infants at high risk for NEC and that intestinal probiotic strains will be associated with intestinal barrier maturation. The purpose of the study is to determine whether clinical factors in combination with non-invasive stool test such as antitrypsin (A1AT) and microbiota composition profile are associated with intestinal permeability determined by excretion of non-metabolized sugar probes in urine (LA/Rh ratio). These studies may lead to a non-invasive screening test to identify preterm infants at risk for NEC.

Detailed description:
The proposed study will evaluate the intestinal permeability measured by the urinary La/Rh ratio at one timepoint between d7-10 of life in 200 preterm infants 24-32 weeks gestation in preparation for a future study of probiotics to improve intestinal permeability in this population.

Primary Objective: To estimate mean and variance in IP measured by urinary Lactulose/Rhamnose ratio at 7-10d of life in neonates born between 24 and 32 weeks of gestational age.

Secondary Objectives

1\) To assess stool microbiome characteristics in association with intestinal permeability in preterm infants measured by the urinary lactulose/rhamnose ratio.

Arms: arm 1: Preterm Infants age 24-32 weeks gestation
Interventions: intervention 1: Lactulose -rhamnose solution
Intervention descriptions: intervention 1: Measurement of intestinal permeability by use of mon- digestible sugars known not to cross the intestinal barrier in normal healthy intestinal tissue

Protocol documents:
Prot_SAP_000.pdf: 
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FULL PROTOCOL TITLE   
Measures of Intestinal Permeability in Preterm Neonates 
Other Identifying Numbers: 
UMB IRB: HP-00049647 
IND 116718 
IND Sponsor-Investigator: Rose Marie Viscardi, MD 
 
Version Number: 2.1 
6 August 2019 
 


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Statement of Compliance 
This study will be carried out in accordance with the US Code of Federal Regulations (CFR), 
local regulations, and Good Clinical Practice (GCP) as required by the following: 
• U.S. Code of Federal Regulations applicable to clinical studies (45 CFR 46; and 21 CFR 
including part 50 and 56 concerning informed consent and IRB regulations, and 21 CFR 11 
concerning electronic records. 
• International Conference on Harmonisation (ICH E6); 62 Federal Register 25691 (1997)  
• NCCAM Clinical Terms of Award  
All individuals responsible for the design and conduct of this study have completed Human 
Subjects Protection Training and are qualified to be conducting this research prior to the 
enrollment of any subjects. CVs for all investigators and sub-investigators participating in this 
trial are on file in a central facility (21 CFR 312.23 [a] [6] [iii] [b] edition).  
 


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• Signature Page 1 
The signature below constitutes approval of this protocol and the attachments, and provides the 
required assurances that this trial will be conducted according to all stipulations of the protocol, 
including all statements regarding confidentiality, and according to local legal and regulatory 
requirements, applicable US federal regulations and (ICH E6) guidelines.  
 
Principal Investigator: __Rose Marie Viscardi, M.D.___________________________________ 
 
 
Print/Type 
 
Signed: 
 
 
Date: 
 
Name/Title 
 
 
The Lead Principal Investigator (Protocol Chair) should sign Signature Page 1.  A copy of this 
Signature Page 1 should be filed with the holder of Regulatory documents and a copy should be 
maintained at the site. 


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Signature Page 2 
The signature below constitutes approval of this protocol and the attachments, and provides the 
required assurances that this trial will be conducted according to all stipulations of the protocol, 
including all statements regarding confidentiality, and according to local legal and regulatory 
requirements, applicable US federal regulations and (ICH E6) guidelines.  
 
Investigator(s) of Record: ___Sripriya Sundararjaran, M.D.____________________________  
 
 
Print/Type 
 
Signed: 
 
 
Date: 
 
Name/Title 
 
 
Additional Investigators: _____________________________  
 
 
Print/Type 
 
Signed: 
 
 
Date: 
 
Name/Title 
 
 
 
The Principal Investigator or Investigator(s) of Record from each participating clinical site 
should sign the Signature Page 2 as appropriate. This Signature Page 2 should be maintained at 
each clinical site. 


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Tool Revision History 
Version Number 1.1 
Version Date 10/30/13 
Summary of Revisions Made:  
1) Clarified determination of adverse event severity with reference to neonatal toxicity 
tables (Appendix D) and additional guideline for assessing laboratory values (Appendix 
E) (see section 7.4.1). 
 
Version Number 2.0 
Version Date 6/30/2018 
Summary of Revisions Made: 
1) Changes in IND personnel. Dr. Viscardi will assume responsibilities as the sponsor-
investigator (see letters from Drs. Fasano and Viscardi). New study personnel have 
been added. 
2) Changed the number and timing of visits. Since the differentiation between preterm 
infants with normal intestinal barrier maturation and those with delayed maturation 
was significant at the second timepoint (Study d8) or approximately 7-10 days of 
age, we will limit the dual sugar solution administration to one administration per 
subject between d7 and 10 of postnatal age. 
3) Eliminate blood draws for zonulin measurement. There was no association of serum 
zonulin and the gold standard urinary Lactulose/Rhamnose ratio in the initial cohort 
of 43 subjects, so no further blood draws will be done.  
4) Safety Monitoring: The U. Maryland IRB has approved the change from a DSMB to a 
single safety monitor. 
5) Stool microbiome: Addition of stool microbiome analysis in relation to intestinal 
permeability measured by La/Rh ratio. 
 
Version Number 2.1 
Version Date 8/6/2019 
Summary of Revisions Made: 
1) Increase the sample size: Sample size will be increased to total 200 based on 
sample size calculation. A total of 200 sample size was calculated to obtain high 
precision in correlations between features of the stool microbiota and intestinal 
permeability (IP). Since 83% subjects in the first cohort had high IP (La/Rh ≥ 0.05) at 
study day 8, a sample size of 166-236 provides marginal error of 0.05 (maximum 
sampling error of 5%) and alpha of 0.05 (95% confidence level) to detect correlations 
between features of the microbiota and changes in IP and to achieve an expected 
sensitivity of 0.8-0.9 and expected specificity at 0.9-0.95. 


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2) Change in stool collection frequency: Instead of collecting stools twice per day from 
enrollment until postnatal d21, stools will be collected twice per day from enrollment 
until postnatal d14 and once on postnatal d21. The rationale for this change is to 
concentrate stool collections around day of IP measurement by urinary La/Rh ratio to 
analyze the correlations of stool microbiota composition and IP. 
3) Delete site Mercy Medical Center: Due to limited study personnel and adequate 
enrollment at main site University of Maryland Medical Center, Mercy Medical Center 
will be dropped as an active site. Enrollment will continue at University of Maryland 
Medical Center. 
 
 


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Table of Contents 
1 
Key Roles ..............................................................................................................................13 
2 
Background Information and Scientific Rationale ...............................................................15 
2.1 
Background Information ............................................................................................ 15 
2.2 
Scientific Rationale ................................................................................................... 16 
2.3 
Potential Risks and Benefits ....................................................................................... 18 
2.3.1 
Potential Risks .............................................................................................. 18 
2.3.2 
Known Potential Benefits .............................................................................. 19 
2.3.3 
Risk/Benefit Ratio ......................................................................................... 19 
2.4 
Study Objectives ....................................................................................................... 19 
3 
Study Design .........................................................................................................................21 
3.1 
ENDPOINTS ........................................................................................................... 21 
3.2 
Study Outcome Measures .......................................................................................... 21 
3.3 
Description of Sub-studies: Not applicable .................................................................. 21 
4 
Study POPULATION ...........................................................................................................22 
4.1 
Inclusion Criteria ...................................................................................................... 22 
4.2 
Exclusion Criteria ..................................................................................................... 22 
4.3 
Randomization ......................................................................................................... 22 
4.4 
Blinding ................................................................................................................... 22 
4.5 
Withdrawal .............................................................................................................. 23 
4.5.1 
Reasons for Withdrawal ................................................................................ 23 
4.5.2 
Handling of Withdrawal ................................................................................ 23 
4.6 
Termination of the Study ........................................................................................... 23 
5 
Investigational product(s) .....................................................................................................24 
5.1 
Study product description .......................................................................................... 24 
6 
Study Procedures / Evaluations / Schedule ...........................................................................25 
6.1 
Clinical/Laboratory Evaluations and Study Schedule ................................................... 25 
6.2 
Screening (include allowable time window) ................................................................. 25 
6.3 
Enrollment/Baseline (include allowable time window) ................................................. 25 
6.4 
Study Visits (include allowable time window for each study visit) ................................. 25 
6.5 
Follow-up Visit(s) (include allowable time window) .................................................... 26 
6.6 
Unscheduled Visit(s) ................................................................................................. 26 
6.7 
Early Termination (include allowable time window) ..................................................... 26 
6.8 
Final Study Visit (include allowable time window) ...................................................... 26 
6.9 
Product Administration (include allowable time windows) ............................................ 26 
6.10 
Rescue Therapy- NA ................................................................................................. 27 
6.11 
Define Specimen Preparation, Handling, and Shipping ................................................. 27 
6.11.1 Instructions for Specimen Preparation, Handling, and Storage ........................... 27 
6.11.2 Specimen Shipment ....................................................................................... 27 
7 
Safety reporting and Safety monitoring ................................................................................28 


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7.1 
Responsibilities ........................................................................................................ 28 
7.2 
Definitions ............................................................................................................... 28 
7.3 
Safety Reporting Requirements .................................................................................. 29 
7.3.1 
Reporting Interval ......................................................................................... 29 
7.3.2 
Notification of the Sponsor of Serious Adverse Events ..................................... 29 
7.3.3 
Regulatory Reporting for Studies Conducted Under Sponsored IND .................. 30 
7.4 
Investigator’s Assessment of Adverse Events .............................................................. 30 
7.4.1 
Assessment of Seriousness ............................................................................. 30 
7.4.2 
Assessment of Severity .................................................................................. 30 
7.4.3 
Assessment of Association ............................................................................. 30 
7.4.4 
Assessment of Reactogenicity (if applicable) ................................................... 31 
7.5 
Safety Monitoring ..................................................................................................... 31 
7.5.1 
Interruption or Discontinuation of Study Enrollment and Study Product 
Administration for all Subjects in the Study ..................................................... 31 
7.6 
Halting Criteria/Rules ............................................................................................... 31 
8 
Clinical Management of Events ............................................................................................33 
8.1 
Adverse Event Management ...................................................................................... 33 
8.1.1 
Temporary Interruption of Study Product in an Individual Subject ..................... 33 
8.1.2 
Pregnancy (if applicable) ............................................................................... 33 
9 
Clinical Monitoring/Site Monitoring Plan ............................................................................34 
10 
Statistical Considerations ......................................................................................................35 
10.1 
Sample Size Considerations ....................................................................................... 35 
10.2 
Planned Interim Analyses (if applicable) ..................................................................... 35 
10.3 
Safety Review (if applicable) ..................................................................................... 35 
10.4 
Final Analysis Plan ................................................................................................... 35 
11 
DATA HANDLING/RECORD KEEPING/Source Documents ..........................................36 
11.1 
Data Capture Methods ............................................................................................... 37 
11.2 
Types of Data ........................................................................................................... 37 
11.3 
Study Records Retention ........................................................................................... 37 
11.4 
Source Documents .................................................................................................... 38 
11.5 
Protocol Deviations ................................................................................................... 38 
12 
Quality Control and Quality Assurance ................................................................................39 
13 
Ethics/Protection of Human Subjects ...................................................................................40 
13.1 
Ethical Standard ....................................................................................................... 40 
13.2 
Institutional Review Board ........................................................................................ 40 
14 
Informed Consent Process ....................................................................................................41 
14.1 
Assent Process (Minor)/Parental Permission/Consent (If applicable) ............................. 41 
14.2 
Subject Confidentiality .............................................................................................. 41 
14.3 
PI Responsibility When Subject Withdraws or Is Discontinued ..................................... 41 
14.4 
Future Use of Stored Specimens ................................................................................. 42 


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15 
Publication Policy .................................................................................................................43 
16 
Literature References ............................................................................................................44 
Appendix A: Schedule of Events ...................................................................................................50 
Appendix B-Modified BEll staging of NEC ..................................................................................51 
Appendix c: Quality Management Plan .........................................................................................52 


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List of Abbreviations 
AE 
Adverse Event/Adverse Experience 
A1AT 
Alpha 1 antitrypsin 
CFR 
Code of Federal Regulations 
CIOMS 
Council for International Organizations of Medical Sciences 
CLIA 
Clinical Laboratory Improvement Amendments  
CONSORT 
Consolidated Standards of Reporting Trials 
CRF 
Case Report Form 
DCC 
Data Coordinating Center 
DHHS 
Department of Health and Human Services 
DSMB 
Data and Safety Monitoring Board 
FDA 
Food and Drug Administration 
FDAAA 
Food and Drug Administration Amendments Act 
FIH 
First in Human 
FWA 
Federalwide Assurance 
GCP 
Good Clinical Practice 
HIPAA 
Health Insurance Portability and Accountability Act 
IATA 
International Air Transport Association 
ICF 
Informed Consent Form 
ICH 
International Conference on Harmonisation 
ICMJE 
International Committee of Medical Journal Editors 
IND 
Investigational New Drug  
IoR 
Investigator of Record 
IRB 
Institutional Review Board 
ISM 
Independent Safety Monitor 
La/Rh 
Lactulose Rhamnose Test 
LAR 
Legally Authorized Representative 
MedDRA® 
Medical Dictionary for Regulatory Activities 
MOP 
Manual of Procedures 
N 
Number (typically refers to subjects) 
NCCAM 
National Institute of Complementary and Alternative Medicine  
NDA 
NG 
New Drug Application 
Naso-gastric 
NICU 
Neonatal Intensive Care Unit 
NIH 
National Institutes of Health 


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NLM 
National Library of Medicine 
OCRA 
OG 
Office of Clinical Research Affairs, NIH, DHHS 
Oro-gastric 
OHRP 
Office for Human Research Protections, DHHS 
OHSR 
Office for Human Subjects Research, NIH, DHHS 
ORA 
Office of Regulatory Affairs, NIH, DHHS 
PHI 
Protected Health Information 
PI 
Principal Investigator 
QA 
Quality Assurance 
QC 
Quality Control 
SAE 
Serious Adverse Event/Serious Adverse Experience 
QM 
Quality Management 
SMC 
Safety Monitoring Committee 
SOP 
Standard Operating Procedure 
US 
United States 
WHO 
World Health Organization 


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Protocol Summary  
Title: 
Measures of Intestinal Permeability in Preterm Neonates 
Abbreviated Title 
Intestinal Permeability in Preterm Neonates 
Phase: 
1 
Population: 
A sample of N= 200 inpatient neonates between 24-32 week 
gestational age. No gender or ethnic exclusions apply.  
Number of Sites: 
1 
Study Duration: 
24 months  
Subject Participation 
Duration: 
28 days 
Agent or Intervention: 
Lactulose/rhamnose ratio measurement of intestinal permeability 
in preterm neonates. 
Objectives: 
To assess intestinal permeability in preterm infants with the 
lactulose/rhamnose ratio in urine and relationship to stool 
microbiome characteristics 
Endpoints: 
 
Onetime measurement of in vivo permeability determined by the 
urine La/Rh ratio between 7-10 days of age and serial stool 
microbiome changes between postnatal d5-d14 and at PND21 in 
a cohort of preterm neonates. The goal is to determine the 
changes in the intestinal microbiome characteristics in 
association with impaired intestinal barrier function (La/Rh 
≥0.05) at 7-10 days in in preterm neonates. 
 
 
 


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Descriptive Schematic of Study Design  
  PND5 
PND7-10 
PND21 
Enroll 200 preterm infants 
Inclusion: 240-326 weeks, 
<5d age 
Exclusion: nonviable or 
planned withdrawal of care, 
severe asphyxia, 
chromosome abnormalities, 
cyanotic congenital heart 
disease, intestinal atresia or 
perforation, abdominal wall 
defects, significant GI 
dysfunction, galactosemia 
or other forms of 
galactose intolerance 
§
In vivo permeability tests: Perform urinary lactulose/rhamnose 
(La/Rh) intestinal permeability test PND7-10 by HPLC, 
§
Collect daily stool PND5-14 and PND21 for calprotection, sIgA, and 
lactoferrin by ELISA and microbiome analysis 
§
Compare La/Rh ratio in urine and stool microbiome relative 
abundance 
§
Compare La/Rh ratio in urine and stool calprotectin, sIgA, lactoferrin 


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1 
KEY ROLES 
List if applicable: 
Principal Investigator (Protocol 
Chair) (name, degree, title) 
Rose M. Viscardi, M.D.,  
Professor of Pediatrics 
University of Maryland  
110 S. Paca Street, 8th floor 
Baltimore, MD 21201 
Office phone: 410-706-1913 or 410-328-6003 
Fax: 410-706-0404 or 410-328-1076 
e-mail: rviscard@som.umaryland.edu 
Site Investigator(s) (name, 
degree, title) 
 
Sripriya Sundararajan, M.D. 
Assistant Professor 
University of Maryland  
Neonatology 
110 S. Paca Street, 8th floor 
Baltimore, MD 21201  
410 328 6003 
ssundararajan@som.umaryland.edu 
 
Safety Monitor 
Rose M. Viscardi, M.D.,  
Professor of Pediatrics 
University of Maryland  
29 S. Greene St., RM GS110 
Baltimore, MD 21201 
Office phone: 410-706-1913 or 410-328-6003 
Fax: 410-706-0404 or 410-328-1076 
e-mail: rviscard@som.umaryland.edu 
 


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Statistician (name, degree, title)  
Larry Magder, PhD, Professor 
University of Maryland 
EPH Biostatistics/Informatics 
John Eager Howard Hall 
Room 114B  
Baltimore MD 21201 
410 706 3253 
lmagder@som.umaryland.edu 
 
 


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2 
BACKGROUND INFORMATION AND SCIENTIFIC RATIONALE 
2.1 
Background Information 
Intestinal barrier: The human intestine is lined by a single layer of cells exquisitely responsive 
to multiple stimuli, and is populated by a complex climax community of microbial partners.  
Under normal circumstances, these intestinal cells form a tight, but selective barrier to “friends 
and foes”: microbes and most environmental substances are held at bay, but nutrients are 
absorbed efficiently (1, 2). Environmental antigen(s) that gain access to the intestinal submucosa 
via paracellular passage from the intestinal lumen across an aberrant mucosal barrier trigger a 
host inflammatory response. There may be genetic variation in susceptibility to recognize, and 
potentially misinterpret, these non-self antigens (1). In all cases, increased permeability precedes 
disease and causes an abnormality in antigen delivery that triggers the multi-organ process 
leading to the inflammatory response (1).   
Measurement of Intestinal Permeability: The measurement of the urinary excretion of orally 
administered isotonic solution of non-metabolized sugar probes have been used extensively for 
30 years to assess intestinal permeability in health and disease in adults adults (3-9) and in 
preterm (10-13) and term infants (13-15) as well as older children. Lactulose, a disaccharide is a 
constituent of infant formulas(10) and is safe treatment for constipation(16) at doses 8-15 times 
higher  than used in the assessment of intestinal permeability. L-Rhamnose is a naturally 
occurring monosaccharide without known biologic effects. It is found in the polysaccharides of 
gums, cardiac glycosides, and foods such as oranges, French beans, winter cabbage, and carrots 
(17) and is used in food flavoring(10). It is not absorbed by the human small intestine and 
reaches the colon where fermentation may produce propionate. The percent urinary excretion of 
orally administered lactulose and rhamnose are markers of the intestinal paracellular and 
transcellular pathways, respectively. The sugar probe tests have been used to safely estimate 
intestinal permeability in pediatric conditions including celiac disease (18), atopic eczema (19, 
20), cystic fibrosis (21, 22), major burns (23) and diarrhea (24-26). The sugar probe has also 
been safely used to assess intestinal permeability in ill newborns with birth asphyxia (10), 
necrotizing enterocolitis (NEC) (11) and congenital heart disease (15, 27). A recent review 
confirmed that the use of the sugar absorbtion tests have been found to be safe in newborns (28). 
We have summarized the literature of the use of lactulose and L-rhamnose as sugar probes in 
studies of preterm and term infants (Table 1) and older infants and children (Table 2) (see IND 
section 6.2). 
Intestinal functions and prematurity: Epithelial barrier integrity is itself dynamic and matures 
over time starting soon after birth, though the mechanisms regulating dynamic permeability are 
poorly understood. Low birth weight, prematurity, and early postnatal age are associated with a 
leaky gut (34). Although intestinal permeability (IP) is higher at birth in preterms than term 
infants, there is usually rapid maturation of the intestinal barrier over the first few days of life in 
both populations (34). Diet also affects intestinal permeability with breast milk feeding lowering 


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IP more rapidly in the first month of life compared with cow milk-based formula in term (35) 
and in preterm neonates (36). 
NEC and its pathogenesis: NEC, a life-threatening, GI emergency characterized by increased IP, 
affects approximately 7 to 10% of preterm neonates, and typically occurs within 7 to 14 days of 
birth (37, 38) with mortality as high as 30-50% (39). NEC symptoms mainly involve GI 
dysfunction, such as abdominal distension and feeding intolerance, but the presentation can be 
non-specific with few warning signs. Current therapies may be invasive, including surgical 
interventions that are often ineffective due to the rapid progression of the disease. Prematurity is 
the greatest risk factor for development of NEC (40, 41), due to physiological immaturity of the 
GI tract and altered levels of the normal GI microbiota.  Several studies suggest that the initiation 
of an intense systemic and local inflammatory cascade leads to intestinal necrosis (42-47). 
Antenatal exposure to infection/inflammation may predispose the developing intestinal mucosa 
to subsequent injury or dysregulated inflammatory responses. Previous studies have linked 
presence of amniotic fluid infection/elevated cytokines, (48) cord blood cytokines, (49, 50) and 
umbilical cord inflammation (51) with risk for NEC in preterm neonates. In a rat model of NEC, 
maternal prenatal exposure to microbial LPS led to increased frequency and severity of intestinal 
injury (52). Taken together, these observations suggest that intestinal injury may be initiated in 
utero and contributes to increased IP at birth in the preterm neonate. Many of the defense 
mechanisms present in the mature intestine, such as peristalsis and tight junctions between 
intestinal epithelial cells (37) are decreased in an immature intestine, and thus bacteria normally 
confined to the intestinal lumen are able to reach systemic organs and tissues. Bacterial 
translocation triggers the activation of an exaggerated inflammatory response, which leads to 
further epithelial damage.  
2.2 
Scientific Rationale 
Despite improvements in neonatal intensive care, the birth weight-specific incidence of NEC has 
not changed over the past 2 decades. However, the total burden of disease is increasing due to 
increased survival of very immature infants. Mortality remains high and survivors experience 
significant morbidity including post-surgical short bowel syndrome and its consequences 
(prolonged dependence on total parenteral nutrition, recurrent infections, poor growth, and liver 
failure), prolonged hospitalization, and long-term neurodevelopmental impairments. Intestinal 
barrier immaturity is the proximate cause of susceptibility to NEC in preterm neonates. Although 
intestinal permeability is higher at birth in preterm compared to term infants (34), in utero 
infection/inflammation and post-natal acquisition of pathogenic bacteria may further exacerbate 
intestinal injury (48-53). Despite intensive efforts, no clinical factor or routine laboratory test 
alone or in combination has been described that predicts the 7-10% preterm neonates at-risk for 
this potentially life-threatening complication (54). Although increased intestinal permeability is a 
major risk factor for NEC and in vivo intestinal permeability has been assessed in infants with 
the urine LA/MA ratio or urine La/Rh ratio, few preterm infants <30 wk gestation have been 
included in the prior studies of intestinal permeability.  


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Since current therapies for NEC may be invasive, including surgical interventions that are often 
ineffective due to the rapid progression of the disease, there has been considerable focus on 
developing preventive therapies. Probiotic therapy is a promising, low-cost, and likely safe 
intervention to reduce intestinal permeability in at-risk infants. There have been at least 11 RCTs 
and a recent meta-analysis of probiotic supplementation to prevent NEC in preterm neonates
(55). Although there was a 30% reduction in NEC incidence in these trials, various formulations, 
doses, and duration of therapy were used, infants <1000 g BW with the highest NEC incidence 
were under-represented, and no FDA-approved products are available to assure quality and 
safety under good manufacturing practices. In preparation for future trials of the efficacy of 
probiotics to prevent NEC in preterm infants, the proposed observational study will utilize the 
La/Rh ratio to determine in vivo intestinal permeability in infants 24-32 weeks gestation during 
the 2 weeks of life.  
2.3 Preliminary Data 
Our analysis of the initial cohort of 43 preterm 
infants recruited for the IND protocol, and 
others’ previous studies have shown that IP is 
high at birth in preterms (<33wk gestation) with 
a rapid maturation of the intestinal barrier over 
the first 2 weeks. However, in some infants, high 
IP persisted and/or recurred in association with 
altered levels of the normal microbiota (bacteria 
community 
composition). 
Specifically, 
we 
observed that (1) rapid maturation of intestinal 
barrier function, characterized by decreased IP, 
correlates with increased microbial community 
diversity (Figure 1), and most outstandingly, the 
increased abundance of beneficial bacteria 
Clostridiales (Figure 2); (2) Clostridiales is 
highly transcriptionally active and co-active with 
the probiotic bacterium Bifidobacterium; (3) 
neonatal factors, including early introduction of 
breast 
milk, 
shorter 
period 
of 
antibiotic 
exposure, and later gestational age, favor the 
early colonization of the gut microbiota by 
members of Clostridiales and Bifidobacterium, 
which altogether are associated with improved 
intestinal barrier in preterm infants; (4) low 
Clostridiales spp. abundance (<5%) and early 
gestational age (<31.7wk) were identified to be the most discriminatory features for elevated IP 
by supervised learning scheme, reaching an accuracy of 86.1%. (5) Clostridiales and 
Bifidobacteriales are the most abundant bacteria groups in later stages (phase II/III at 6-18 
Figure 1. Microbial community diversity and intestinal 
permeability at study time points day 1, 8, and 15 for a 
cohort of 43 preterm infants (<33 weeks gestational age). 
Microbial community diversity is calculated as OTU 
richness based on 16S rRNA gene amplicon high-
throughput sequencing. Intestinal permeability was 
calculated as the ratio of non-metabolized sugar probes 
l
t l
d h
Day 1
Day 8
Day 15
2
4
6
8
10
12
Microbial Community Diversity
0.0
0.1
0.2
0.3
0.4
0.5
Intestinal Permeability 
Microbial community diversity (OTU richness)
Intestinal Permeability (Lactulose/Rhamnose ratio)


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months of age) as shown in Figure 2, suggesting a process of gaining prosperity of these two 
bacterial groups during intestine development after birth. Altogether our preliminary results 
suggest the early colonization of the natural occurring probiotics strains Clostridiales and 
Bifidobacterium strongly associate with rapid maturation of intestinal barrier function, and their 
measurement are highly promising for early detection and as potential nutritional supplement to 
prevent NEC in the high-risk preterm population. 
We propose in this study to recruit additional 157 preterm subjects and to combine with our 
existing 43 preterm subjects to form a statistically powered cohort at 200 (justified in study size 
analysis in research design), to address our hypothesis that the two naturally occurring beneficial 
bacteria Clostridiales and Bifidobacterium are rapidly gaining prosperity during normal intestine 
development in association with improved barrier function measured by La/Rh ratio. Overall, 
this continuation of the initial study builds on the previous findings that identified commensal 
bacteria Clostridiales and Bifidobacterium species as a strong indicator to the lowered IP and 
rapid maturation of intestinal barrier, to substantiate measurement of these probiotic strains 
combined with associated neonatal factors to form an accurate, rapid detection of intestinal 
permeability abnormality.  
2.4
Potential Risks and Benefits 
2.4.1
Potential Risks 
The research involves minimal risks. 
Phase II/III
High IP (La/Rh >= 0.05)
Low IP (La/Rh ratio < 0.05)
Low IP
0
5
10
15
Actinobacteria
Alphaproteobacteria
Bacilli
Bacteroidia
Betaproteobacteria
Clostridia
Coriobacteriia
Erysipelotrichi
Fusobacteriia
Gammaproteobacteria
Mollicutes
TM7−3
Verrucomicrobiae
Actinobacteria
Alphaproteobacteria
Bacilli
Bacteroidia
Betaproteobacteria
Clostridia
Coriobacteriia
Erysipelotrichi
Fusobacteriia
Gammaproteobacteria
Mollicutes
TM7−3
Verrucomicrobiae
Actinobacteria
Alphaproteobacteria
Bacilli
Bacteroidia
Betaproteobacteria
Clostridia
Coriobacteriia
Erysipelotrichi
Fusobacteriia
Gammaproteobacteria
Mollicutes
TM7−3
Verrucomicrobiae
Abundance
0.0
2.5
5.0
7.5
0.0
2.5
5.0
7.5
0.0
2.5
5.0
7.5
Day 1
Actinobacteria
Alphaproteobacteria
Bacilli
Bacteroidia
Betaproteobacter ia
Clostridia
Coriobacteriia
Erysipelotrichi
Fusobacteriia
Gammaproteobacteria
Mollicutes
TM7−3
Actinobacteria
Alphaproteobacteria
Bacilli
Bacteroidia
Betaproteobacter ia
Clostridia
Coriobacteriia
Erysipelotrichi
Fusobacteriia
Gammaproteobacteria
Mollicutes
TM7−3
Abundance
High IP
Phylum
Actinobacteria
Bacteroidetes
Firmicutes
Fusobacteria
Proteobacteria
Tenericutes
TM7
Day 8
Day 15
Phase I
Low IP
High IP
Phase I
Figure 2: Bar graph of the accumulative relative abundance of bacterial groups. (A) accumulative abundance between phase II/III 
subjects (6-18 months of age) and phase I infants (within first two weeks of life), (B) stratified abundance profiles by different study 
day at day 1, 8, and 15 for phase I subjects. The most outstanding difference between high and low IP in preterm infants is in the 
Clostridiales (arrows), the only bacteria group of Clostridia in this study, which is the most abundant bacterial group in phase II/III 
eobacter ia
a
teriia
trtrt irirchi
ici
dia
eobacteria
a
cteriia
rir
otririr chi
dia
eobacteria
a
teriia
rir
tririr chi
dia
eobacteria
teriia
rir
tririr chi
eobacter ia
a
teriia


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Loss of confidentiality presents minimal risk. Specimens will be identified by subject ID only 
linking data for subject ID will be kept in locked cabinet accessible only to the clinical research 
staff.  
Fecal sample will be collected from the diaper and presents no risk to the participant.  
Sugar solution- No risks have been reported in the literature from ingesting an isotonic solution 
of sugar (3-15). A similar simple sugar, sucrose (56), is given in higher doses for pain relief with 
fewer adverse effects than water placebo.  
2.4.2 Known Potential Benefits 
There are no known potential benefits to participants. The ability to measure increased intestinal 
permeability would indicate an infant has a leaky gut, and may be at increased risk for NEC. The 
potential risks from specimen collection are equivalent to those of normal clinical care.  
2.4.3 Risk/Benefit Ratio 
Despite improvements in neonatal intensive care, the birth weight-specific incidence of NEC has 
not changed over the past 2 decades. However, the total burden of disease is increasing due to 
increased survival of very immature infants. Mortality remains high and survivors experience 
significant morbidity including post-surgical short bowel syndrome and its consequences 
(prolonged dependence on total parenteral nutrition, recurrent infections, poor growth, and liver 
failure), 
prolonged 
hospitalization, 
and 
long-term 
neurodevelopmental 
impairments. 
Development of an in vivo measurement to identify infants with increased intestinal 
permeability, who are at high risk for NEC, will facilitate clinical trials of potential preventive 
therapies. There may be risks from the research that are not known. 
2.5 
Study Objectives 
The proposed study will evaluate the intestinal permeability measured by the urinary La/Rh ratio 
at one timepoint between d7-10 of life in 200 preterm infants 24-32 weeks gestation in 
preparation for a future study of probiotics to improve intestinal permeability in this population.  
Primary Objective: To estimate mean, variance, and quantiles in IP measured by urinary 
Lactulose/Rhamnose ratio at 7-10d of life in neonates born between 24 and 32 weeks of 
gestational age. 
Secondary Objectives:  
1) To assess stool microbiome characteristics in association with intestinal permeability in 
preterm infants measured by the urinary lactulose/rhamnose ratio  


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2) To analyze the correlation of stool calprotectin, sIgA, and lactoferrin to urinary 
lactulose/rhamnose ratio and stool microbiome characteristics in neonates born between 
24 and 32 weeks of gestational age. 
 


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3 STUDY DESIGN 
The study is a specimen collection study. Participants will include neonates admitted to the 
Neonatal Intensive Care Unit at the University of Maryland Medical Center, Baltimore, MD. The 
study will not test any type of device or treatment but is designed to accrue information. 
3.1 ENDPOINTS 
3.2 Study Outcome Measures  
The study outcome measures will be intestinal permeability (quantitative) and leaky gut (binary),  
1) increased levels of urine Lactulose/Rhamnose [La/Rh] ratio >0.05 will identify infants with 
increased intestinal permeability (IP). 
3.3 Description of Sub-studies: Not applicable 


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4 STUDY POPULATION 
All admissions to the University of Maryland Medical Center (UMMC) NICU <5 d age and 
gestational age 240-326 weeks will be screened for study eligibility and parental consent of 
eligible subjects will be obtained. Two hundred total subjects will be recruited. The combined 
NICU population is approximately 59% male with an ethnic distribution of 65% African-
American, 33% Caucasian and 2% other. Subjects will not be excluded based on gender, race or 
ethnicity. 
4.1 Inclusion Criteria 
Inclusion criteria will include 
1) < 5days 
2) Gestational age 240-326 weeks 
4.2 Exclusion Criteria 
Exclusion criteria will include:  
1) Non-viability or planned withdrawal of life support; 
2) Triplet or higher order multiple; 
3) Severe asphyxia (Apgar <3 at 5 min and cord pH <7.0); 
4) Lethal chromosome abnormalities;  
5) Cyanotic congenital heart disease;  
6) Intestinal atresia or perforation;  
7) Abdominal wall defects; 
8) Significant GI dysfunction (e.g. heme-positive stools, abdominal distension (girth >2 cm 
baseline), or bilious emesis/aspirates. 
9) Infants with galactosemia or other forms of galactose intolerance 
4.3 Randomization 
Not randomized 
4.4 Blinding 
Not blinded 


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4.5 Withdrawal 
4.5.1 Reasons for Withdrawal 
Subjects may be withdrawn from the study for the following reasons:  
1) At the request of the subject’s parent(s) or guardian(s), or at the request of other legally 
authorized representative; 
 2) If, in the investigator’s opinion, continuation in the study would be detrimental to the 
subject’s well-being; 
 3) At the request of the IRB or Safety Monitor. 
4.5.2 Handling of Withdrawal 
Subjects who discontinue due to being withdrawn or lost to death will be replaced. Data and 
specimens will be de-identified and remain as part of the study.   
4.6 
Termination of the Study 
The study may be terminated:  
1) At the request of the IRB or Safety Monitor 
2) If the principal investigator is unable to continue with the study and a suitable replacement is 
not identified. 


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5
INVESTIGATIONAL PRODUCT(S) 
5.1 Study product description 
A 1mL/kg La/Rh solution (8.6 g of lactulose+140 mg of rhamnose/100mL) will be used to 
assess intestinal permeability.  Lactulose is a synthetic disaccharide formed from fructose and 
galactose.  Rhamnose is a naturally occurring sugar. 
Lactulose will be obtained as Kristalose from Cumberland Pharmaceuticals 2525 West End 
Avenue, Suite 950, Nashville, TN 37203 (www.cumberlandpharma.com) and is listed in the U.S. 
Pharmacopoeia. 
Rhamnose will be obtained from Saccharides, Inc. 205, 259 Mid Park Way S.E., Calgary Alberta 
T2X 1M2 Canada.
Detailed instructions for preparation, storage and handling of the sugar solution can be found in 
the Manual of Procedures. 
Administration details can be found in Section 6. 


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6   STUDY PROCEDURES / EVALUATIONS / SCHEDULE 
6.1 Clinical/Laboratory Evaluations and Study Schedule  
Informed consent will be obtained before any clinical evaluations are performed. Specific details 
of assay performance and calibration are listed in the MOP. 
6.2 Screening (include allowable time window) 
After receiving a HIPAA waiver from the IRB, clinical PI and staff will screen all new 
admittances to the NICU for eligibility criteria. Study interventions must be able to begin within 
24 hours of enrollment. Informed consent must be signed before any study procedures are begun.  
6.3 Enrollment/Baseline (include allowable time window) 
After consent and HIPAA forms are signed, chart will be reviewed and history will be recorded. 
Medical history will be obtained from the chart and eligibility criteria will be confirmed.  
Prior to Lactulose/rhamnose sugar probe dosing, the subject will have a complete physical exam 
including vital signs, weight, height, and head circumference.  
6.4 Study Visits (include allowable time window for each study visit) 
Postnatal day 5 (or within 24 hours of enrollment) 
• Begin collecting available stool samples for microbiome, calprotectin, sIgA, and 
lactoferrin analysis  
• Medication history will be collected from the chart. 
• Complete physical exam including vital signs, weight, height, and head 
circumference. 
Postnatal d7-10  
• In vivo IP will be determined by means of the urine La/Rh test.  Briefly, preterm 
neonates will be administered a 1mL/kg La/Rh solution (8.6g of lactulose+140 mg 
of rhamnose/100mL) via a clinically indicated OG or NG tube. The La/Rh solution 
will be prepared by the Investigational Drug Pharmacy (IDS) and administered by 
gavage.  Starting at the time of the dose of the La/Rh solution, urine will be collected 
for a 4-hour period with cotton balls. If a participant does not require an OG/NG tube 
the solution will be administered orally by nippling. 
• Continue collecting available stool samples daily until PND14 and single sample on 
PND21 for microbiome, calprotectin, sIgA, and lactoferrin analysis. Record daily 
weight, and weekly height, and head circumference. 


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• Demographic and clinical data will be collected from the chart. 
• Collect adverse events from medical chart. 
6.5
Follow-up Visit(s) (include allowable time window) 
Not applicable. 
6.6
Unscheduled Visit(s) 
Not applicable
6.7
Early Termination (include allowable time window) 
If early termination occurs the assessments from the final visit should be completed if the 
parent/LAR is willing. The physical exam and data collection regarding adverse events 
medication history should be collected at a minimum. If possible, the La/Rh should be 
administered and urine and serum should be collected.  
6.8
Final Study Visit (include allowable time window) 
PND21 (± 2d) or discharge/transfer whichever comes first 
• Final collection of stool samples for analysis 
• Medication history including the number of packed red blood cell transfusions and 
total volume received will be collected from the chart 
• Record daily weight, and weekly height, and head circumference. 
• Collection of adverse events from medical chart. 
6.9
Product Administration (include allowable time windows) 
Each enrolled preterm neonates will be administered a 1mL/kg La/Rh solution (8.6 g of lactulose
+140 mg of rhamnose/100mL) via a clinically indicated OG or NG tube once between 7-10 d of 
age. The La/Rh solution will be administered by gavage with an enteral feed. Total volume of 
La/Rh ingested will be recorded. Urine will be collected for a 4-hour period with cotton balls. 
Total volume of urine during the 4 hour collection will be recorded.  The urine will be aliquoted 
and 4 ml of urine saved for analysis. Nursing staff in the NICU will be trained in the procedures 
prior to study initiation. Only trained NICU staff will be allowed to be responsible for 
administering the La/Rh solution and collecting the urine. A minimum of 2 ml of urine will be 
collected. If urine leaks around the cotton balls, then the diaper will be weighed and the diaper 
weight will be used to estimate the urine volume for the 4 hour collection period. If less than 2 
ml urine is collected, then the test will be repeated on another day within the 7-10d window by 


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administering 1 ml/kg La/Rh solution (8.6g of lactulose+140 mg of rhamnose/100mL) via an 
OG/NG tube followed by a 4 hour urine collection. The urine will be stored at -80°C until 
processed. The sugars will then be measure in urine by HPLC. 
Urine La and Rh will be measured in the lab of our collaborator Dr. Jon Meddings from the 
University of Calgary, Canada. 
Stool calprotectin, sIgA,and  lactoferrin will be mesured by commercial ELISA kits.  
Rescue Therapy- NA
6.10 Define Specimen Preparation, Handling, and Shipping 
6.10.1 Instructions for Specimen Preparation, Handling, and Storage 
Specimen management will be detailed in the MOP. 
6.10.2 Specimen Shipment  
Research staff will transport specimens collected at the University of Maryland Medical Center 
to the appropriate laboratory on campus. Specimens collected at Mercy Medical Center will be 
frozen and transported monthly to the laboratory of Dr. Viscardi. Aliquots of urine for La and Rh 
measurements by HPLC will be shipped to our collaborator Dr. Jon Meddings at the University 
of Calgary, Canada, for HPLC. All specimens will be accompanied by a shipping invoice 
indicating the sample ID and the chain of custody. Specific transport procedures are detailed in 
the MOP.  


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7 SAFETY REPORTING AND SAFETY MONITORING 
Regulatory requirements including the FDA regulations, ICH Guidelines for Good Clinical 
Practice, and EU Clinical Trials Directive set forth safety monitoring and reporting 
responsibilities of sponsors and investigators to ensure the safety and protection of human 
subjects participating in clinical trials. 
7.1 Responsibilities 
Investigators participating in this clinical trial are responsible for and will: 
• evaluate subject safety including assessment of adverse events (AEs) for seriousness, 
severity, and causality, 
• notify the sponsor of SAEs immediately, 
• provide detailed written reports, including necessary documentation requested by the 
sponsor or IRB, promptly following immediate initial reports, and 
• inform the IRB of AEs as required by applicable regulatory requirements. 
7.2 Definitions 
Adverse Event (AE)  
Any untoward medical occurrence clinical investigation subject who has received a study 
intervention and that does not necessarily have to have a causal relationship with this treatment. 
An AE can, therefore, be any unfavorable and unintended sign (including an abnormal laboratory 
finding, for example), symptom, or disease temporally associated with the use of a medicinal 
product, whether or not considered related to the medicinal product. 
Serious Adverse Event (SAE)  
An SAE is any adverse event that results in any of the following outcomes: 
• Death; 
• Life-threatening (immediate risk of death); 
• Inpatient hospitalization or prolongation of existing hospitalization; 
• Persistent or significant disability or incapacity; 
• Congenital anomaly/birth defect; 


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• Important medical events that may not result in death, be life threatening, or require 
hospitalization may be considered a serious adverse event when, based upon 
appropriate medical judgment, they may jeopardize the subject and may require 
medical or surgical intervention to prevent one of the outcomes listed in this 
definition. Examples of such medical events include allergic bronchospasm requiring 
intensive treatment in an emergency room or at home, blood dyscrasias or 
convulsions that do not result in inpatient hospitalization, or the development of drug 
dependency or drug abuse. 
Unexpected 
An adverse reaction, the nature or severity of which is not consistent with the applicable product 
information (e.g., investigator’s brochure for an unapproved investigational medicinal product). 
Expedited Safety Report  
Documentation in appropriate form and format summarizing an SAE that meets expedited safety 
reporting criteria, submitted within the required reporting time frame of applicable regulatory 
authorities and/or IRBs/IECs of participating countries. 
7.3 
Safety Reporting Requirements 
7.3.1 Reporting Interval 
Document all adverse events (AEs) and SAEs from enrollment through PND21.  
All SAEs will be followed until resolution even if this extends beyond the study-reporting 
period. Resolution of an adverse event is defined as the return to pre-treatment status or 
stabilization of the condition with the expectation that it will remain chronic.  
The investigator will follow all SAEs until resolution (return to pretreatment status or 
stabilization of the condition with the expectation that it will remain chronic) even if this extends 
beyond the study-reporting period.  
7.3.2 Notification of the Sponsor of Serious Adverse Events 
Any AE that meets a protocol-defined serious criterion that are associated with study product(s) 
must be submitted within 24 hours of site awareness to the sponsor, and IRB.  
Other supporting documentation of the event may be requested by the sponsor and should be 
provided as soon as possible.  


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7.3.3 Regulatory Reporting for Studies Conducted Under Sponsored IND 
Following notification from the investigator, the sponsor will report events that are both serious 
and unexpected and that are associated with study product(s) to the Food and Drug 
Administration (FDA) within the required timelines as specified in 21 CFR 312.32: fatal and 
life-threatening events within 7 calendar days (by phone or fax) and all other SAEs in writing 
within 15 calendar days. All serious events designed as “not associated” to study product(s) will 
be reported to the FDA at least annually in a summary format. 
7.4 
Investigator’s Assessment of Adverse Events  
The determination of seriousness, severity, and causality will be made by an on-site investigator 
who is qualified (licensed) to diagnose adverse event information, provide a medical evaluation 
of adverse events, and classify adverse events based upon medical judgment. This includes but is 
not limited to physicians, physician assistants, and nurse practitioners.   
7.4.1 Assessment of Seriousness 
Event seriousness will be determined according to the protocol definition of an SAE 
(Section 7.2). Severity will be determined according to neonatal toxicity table (Appendix D) and 
expanded table of severity for laboratory values (Appendix E). 
7.4.2 Assessment of Severity 
Event severity will be assigned according to the definitions below: 
• Mild: events require minimal or no treatment and do not interfere with the study 
subject’s daily activities. 
• Moderate: events result in a low level of inconvenience or concern with the 
therapeutic measures. Moderate events may cause some interference with 
functioning. 
• Severe: events interrupt a study subject’s usual daily activity and may require 
systemic drug therapy or other treatment. Severe events are usually incapacitating. 
• Life-threatening 
7.4.3 Assessment of Association 
The association assessment categories that will be used for this study are: 
• Associated – The event is temporally related to the administration of the study 
product and no other etiology explains the event. 


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• Not Associated – The event is temporally independent of the study product and/or the 
event appears to be explained by another etiology.  
The investigator must provide an assessment of association or relationship of AEs to the 
study intervention based on: 
• Temporal relationship of the event to the study intervention 
• Whether an alternative etiology has been identified; 
• Biological plausibility; 
• Existing therapy and/or concomitant medications. 
7.4.4 Assessment of Reactogenicity (if applicable) 
Not applicable. 
7.5 
Safety Monitoring  
Safety Monitoring:  For the continuation of this observational study, the sponsor-investigator 
will be the Data Safety Monitor to 1) periodically review and evaluate the accumulated study 
data for subject safety, study conduct and progress, and, when appropriate, efficacy, validity, and 
integrity and 2) make decisions concerning the continuation, modification, or termination of the 
study.   
7.5.1 Interruption or Discontinuation of Study Enrollment and Study Product 
Administration for all Subjects in the Study 
Not Applicable 
7.6 
Halting Criteria/Rules 
7.6.1  Stopping Rules for Individual Subjects 
A pre-dose checklist for evidence of feeding intolerance will be completed prior to La/Rh 
administration. The Lactulose/Rhamnose administration will be re-scheduled within the study 
window if the clinically indicated gastric residual is obtained and measures >50% of the previous 
nutritive feeding volume, and/or contains bile or gross blood, or is hemoccult-positive. If these 
GI signs are present throughout the study window, the La/Rh test will not be administered. Other 
conditions that would result in delay or discontinuation of the La/Rh test include hypotension 
requiring pharmacologic support, clinical or radiographic signs of intestinal ileus or NEC. The 
subject will continued to be followed until postnatal day 21±2 d or discharge/transfer which ever 
occurs first. 


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Plans for Study Interruption Based on Safety Monitoring: 
Since the expected in-hospital mortality rate for infants 24-32 weeks gestation is 10-15% at the 
UMMC NICU, 5-8 deaths may occur in our study population per year. If more than 10 deaths or 
7 confirmed cases of NEC >Stage 2 occur within one year or more than 4 deaths or 3 confirmed 
NEC cases >Stage 2 in consecutively recruited subjects occur within 30 d of enrollment, the 
study will be interrupted until reviewed by the safety monitor. The study may also be halted if 
the number of SAEs overall, the number of occurrences of a particular type of SAE, severe 
AEs/reactions, or increased frequency of events warrant further investigation. The safety review 
may require temporary suspension of enrollment or all study interventions.  The safety review 
may determine whether the study should continue per protocol, proceed with caution, be further 
investigated, be discontinued, or be modified and then proceed. Subsequent review of serious, 
unexpected, and related AEs by the medical monitor, IRB, the sponsor(s), or relevant local 
regulatory authorities may also result in suspension of further trial interventions at a site. The 
study sponsor(s) retain the authority to suspend additional enrollment and study interventions for 
the entire study, as applicable. 
 


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8 CLINICAL MANAGEMENT OF EVENTS 
8.1 
Adverse Event Management  
Study interventions will not influence medical care. Morbidities associated with 
prematurity are protocol-specified potential serious adverse events. These are events that 
are anticipated to occur in the preterm population even in the absence of study 
intervention. Morbidities of prematurity will be recorded on CRFs on study day 21 or 
discharge/transfer whichever comes first.  
8.1.1 Temporary Interruption of Study Product in an Individual Subject 
Not applicable. 
8..2 
Pregnancy (if applicable) 
Not applicable. 


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9 
CLINICAL MONITORING/SITE MONITORING PLAN  
In general, site monitoring is conducted to ensure that:  
• human subjects’ rights and well-being are protected; 
• data are accurate, complete, and verifiable from source documents; 
• the study complies with the protocol/amendment(s), ICH Good Clinical Practice 
(GCP) guidelines, and applicable regulatory requirements. 
The internal monitoring plan will be conducted according to the Quality Management plan in 
Appendix C.  
 


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10 STATISTICAL CONSIDERATIONS 
10.5 
Sample Size Considerations 
Sample size for primary objective: The sample size for the first aim (50 subjects) will provide 
good precision in the estimate of the distribution of levels of intestinal permeability at each time 
point. The quantiles will be accurate +/- 10 percentage points (based on a 95% confidence 
interval). Based on the sites’ combined annual admission numbers and survival rates, we expect 
120 infants to be eligible in the 6 month planned enrollment period in the first year. The 
combined NICU population is approximately 59% male with an ethnic distribution of 65% 
African-American, 33% Caucasian and 2% other. 
Sample size for microbiome analysis: The sample size of 200 was calculated to obtain high 
precision in correlations between features of the stool microbiota and intestinal permeability (IP). 
Since 83% subjects in the first cohort had high IP (La/Rh ≥ 0.05) at study day 8, a sample size of 
166-236 provides marginal error of 0.05 (maximum sampling error of 5%) and alpha of 0.05 
(95% confidence level) to detect correlations between features of the microbiota and changes in 
IP and to achieve an expected sensitivity of 0.8-0.9 and expected specificity at 0.9-0.95. 
10.2  Randomization  Not applicable 
10.3 
Blinding  Not applicable.  
10.4 
Planned Interim Analyses (if applicable) 
Not applicable 
10.5 
Safety Review (if applicable) 
10.6 
Final Analysis Plan  
Primary Objective: To estimate mean, variance, and quantiles in IP measured by urinary 
Lacutlose/Rhamnose ratio in in neonates born between 24 and 32 weeks of gestational age. 
Sample quantiles of the distribution of IP (as measured by urine LA/Rh ratio) will be 
calculated at each time point.  In addition, we will use mixed effects models to estimate the 
mean and variance of urine La/Rh.  By including other terms in the model we will be able to 
assess whether high IP (La/Rh ratio >0.05) is affected by gestational age, birthweight, sex or 
feeding type (mother’s own breast milk only, donor breastmilk, preterm formula only, or both 
breast milk and preterm formula).  
Secondary Objective:  To evaluate the performance of stool calprotectin, sIgA, and lactoferrin 
as markers for intestinal permeability in vivo as established by the gold standard urine La/Rh in 
preterm neonates. 


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Assessing the relationships between stool markers and urine La/Rh ratio:  To quantify the 
association between stool calprotectin, sIgA, and lactoferrin, and urine La/Rh ratio, we will 
calculate Pearson Correlation Coefficients.  If the distribution of either variable exhibits 
substantial skewness we will substitute Spearman Correlation Coefficients. In addition, we will 
perform sub-group analyses by gestational age or birthweight.  
Assessing the sensitivity and specificity of calprotectin, sIgA, and lactoferrin for identifying 
children with leaky gut syndrome using an ROC analysis.  Leaky gut syndrome will be 
defined as having a urinary La/Rh ratio of greater than 0.05 at 7-10 d (12).  Using this definition, 
we will assess the sensitivity, specificity, and predictive value of calprotectin, sIgA, and 
lactoferrin on each day for identifying those with leaky gut syndrome. An analogous calculation 
will be made to estimate specificity. We will perform these calculations for a large range of cut-
offs for the stool markers, and summarize the findings using an ROC curve.  The area under the 
ROC curve (AUC) will be calculated using an empirical approach to provide an overall summary 
measure of the performance of calprotectin, sIgA, and lactoferrin as measures for leaky gut 
syndrome.     
Handling missing data or dropouts.  Our analysis of the correlation between calprotectin, 
sIgA, and lactoferrin and IP will be based on an “available data” approach.  That is, if a child 
missing data due to lack of stool sample or sufficient urine collection, information from that time 
point will not be included in the analysis.  We think this is reasonable because in that situation 
both and IP will be unknown and imputating both would result in very limited additional 
information.  For the analysis of the degree to which calprotectin, sIgA, and lactoferrin can 
predict leaky gut syndrome, we will initially do an analysis based on the available data, but in a 
secondary sensitivity analysis we will see how our estimates are affected if we assume that all 
those children who dropped out due to illness or deteriorating condition actually developed leaky 
gut syndrome.  
Stool bacterial community characterization: DNA will be extracted using the protocols 
developed and validated at IGS that routinely obtain between 5 and 10 µg of high-quality whole 
genomic DNA. 16S rRNA gene amplicon sequencing will be performed on Illumina MiSeq 
platform, using standard methods developed for the NIH Human Microbiome Project and by our 
group at IGS. Bioinformatics analyses will be performed to calculate bacterial types and their 
relative abundance. A TaqMan® PanBacterial quantitative PCR assay targeting the 16S rRNA 
gene, designed and validated by our group at IGS, will be used to estimate the true bacterial 
abundance per gram of stool. Together the relative proportion and absolute copy number of each 
individual bacterium will be calculated. Metabolome profiling will be performed using LC/MS 
(Liquid chromatography/mass spectrometry) by Metabolon, Inc. from 1:2 fecal to PBS solution 
according the company’s sample preparation protocol. Fecal calprotectin biochemical test 
(CALPR) will be used as an indicator for pathological inflammation of the bowel wall.  
Discriminatory classifier computation: Three major procedures are involved to build a 
discriminatory classifier using supervised machine classifiers to identify high or low intestinal 
permeability. (1) Data collection. Upon the recruitment of preterm infants at <5d of age and 


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gestational age (GA) at 24-32 weeks, information including extensive data on demographics, 
obstetric and neonatal factors, medication exposures, and feeding practices will be obtained. 
Fecal specimen will be collected to generate bacterial community composition and structure 
using high-throughput sequencing of 16S rRNA gene amplicon and to calculate bacterial total 
copy number using quantitative PCR. Urine specimen will be used to measure the non-
metabolized sugar lactulose (La) and rhmnose (Rh) concentration using high-pressure liquid 
chromatography (HPLC), and their ratio will be used to calculate high IP (La/Rh > 0.05) and low 
IP (La/Rh <= 0.05). (2) Discriminatory classifier generation. The bacterial abundance and 
neonatal information will be used to form the data matrix and the high or low IP category will be 
used to “label” a sample as its class, which is used in supervised machine learning scheme with 
cross-validation (construct model on a sample set and evaluate on a different sample set to avoid 
over-prediction) to direct generating the classifier after data preprocessing and evaluation. The 
most discriminatory features (i.e. bacterial species and/or neonatal factors) will be characterized 
by its contribution to identify a sample in a high or low IP class. (3) We will evaluate the 
contribution of each discriminatory feature to IP classification using permutation importance 
measurement (5).  
 
10.7  DATA HANDLING/RECORD KEEPING/Source Documents 
All record keeping procedures will maintain participant confidentiality in accordance with ICH-
GCP guidelines. Paper records will be kept locked and only study staff will have access to study 
records. Accuracy will be ascertained through the study quality assurance plan. Details are listed 
in the Manual of Procedures. 
10.7.1 
Data Capture Methods 
Clinical data (including AEs, and concomitant medications) and clinical laboratory data will be 
entered on paper CRFs.  Clinical data will be entered directly from the source documents. For 
data analysis the data will be manually entered from CRFs into electronic spreadsheets and 
processed using SAS 9.2 (Copyright (c) 2002-2008 by SAS Institute Inc., Cary, NC, USA.). 
10.7.2 
Types of Data 
Data for this study will include safety, laboratory, and outcome measures (e.g., intestinal 
permeability). 
10.7.3 
Study Records Retention   
Each participating site will maintain appropriate medical and research records for this study, in 
regulatory and institutional requirements for the protection of confidentiality of subjects. Each 


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site participating in this study will permit authorized representatives of the sponsor, the 
institutional IRB, and regulatory agencies to examine (and when required by applicable law, 
copy) clinical records for the purposes of clinical site monitoring, quality assurance reviews, 
audits, and evaluation of the study safety and progress.  
10.7.4 Source Documents 
The source document is defined as the first place the data are recorded; i.e., the location where 
all study-related data are initially recorded. The source could be hard copy paper and/or 
electronic forms, lab printouts, pharmacy records, medical records, etc, onto which the data are 
first recorded. In most cases, the source documentation for this study will be the electronic 
medical record. 
The study staff will transcribe research data into the RedCap electronic database from the 
subject’s chart or other medical records. The electronic medical record will be the source 
document except for data collection forms listed below.  
 
Case Report Forms as Source Documents 
Study personnel trained in the proper completion of case report forms (CRFs) will record study data in 
RedCap database and all forms will be eletronically signed and dated by the study personnel who 
completed the form. In some instances data for which there is no prior written or electronic record will be 
recorded on electronic CRFs and these will be considered source documents. This will be limited to the 
following CRFs: 
1. IP05 (Physical Exam) 
2. IP06 (LaRh Collections) (with supplementary attachments)  
3. IP07 (Specimen Collection) 
4. IP10 (Adverse Events) (with supplementary attachments) 
5. IP11 (Serious Adverse Events) (with supplementary attachments) 
6. IP12 (Protocol Deviation Form) 
7. IP13 (Early Termination) 
10.8 
Protocol Deviations 
A protocol deviation is any noncompliance with the clinical trial protocol, Good Clinical Practice 
(GCP), or Manual of Procedures requirements. The noncompliance may be either on the part of 
the subject, the investigator, or the study site staff. As a result of deviations, corrective actions 
will be developed by the site and implemented promptly.  


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11 
QUALITY CONTROL AND QUALITY ASSURANCE 
This section summarizes the separate written quality management plan for quality control and 
quality assurance at the investigative site (site).  
Each site will have a delegation of responsibility log. The log will list the staff involved in the 
trial and their responsibilities. The PIs will ensure that all staff are adequately trained for their 
responsibilities. A log of the trainings completed by the staff will be maintained with the 
regulatory documents.  
A checklist will be created for key quality indicators such as eligibility criteria and consent. All 
records will be reviewed for these indicators. In addition, eligibility criteria will be reviewed and 
signed by the site PI. Informed consent and HIPAA forms will be reviewed by site staff for 
completeness, including date and any additional checks or signatures required. Data collected 
from protocol procedures will be reviewed monthly for compliance with procedures and 
accuracy with source documents. 
Specimen collection and storage documentation will be compared to the Manual of Procedures. 
Chain of custody documentation and laboratory storing and processing procedures will also be 
reviewed. Deviations from the Manual of Procedures may require a corrective action plan. 
A summary of review findings will be filed with regulatory documents.  


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12 
ETHICS/PROTECTION OF HUMAN SUBJECTS 
12.1 Ethical Standard 
The investigator(s) will ensure that this study is conducted in full conformity with the principles 
of the Belmont Report: Ethical Principles and Guidelines for the Protection of Human Subjects 
of Research of the National Commission for the Protection of Human Subjects of Biomedical 
and Behavioral Research (April 18, 1979) and codified in 45 CFR 46, 21 CFR 312, and/or ICH 
E6; 62 Federal Regulations 25691 (1997). The PI/Institution will hold a current FWA issued by 
OHRP for federally funded research. 
12.2 
Institutional Review Board 
Each participating institution IRB must review and approve this protocol, associated informed 
consent documents, and recruitment material. Any amendments to the protocol or consent 
materials must also be approved before they are implemented. Only institutions holding a current 
US FWA issued by OHRP are authorized to review and approve these documents 
(http://www.hhs.gov/ohrp/assurances/).  


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13 INFORMED CONSENT PROCESS  
The informed consent process will be initiated before a volunteer agrees to participate in the 
study and should continue throughout the individual’s study participation. The parent/LAR will 
sign the informed consent document before any procedures are undertaken for the study. 
Extensive explanation and discussion of risks and possible benefits of this investigation will be 
provided to the subjects in understandable language. Adequate time will be provided to ensure 
that the subject has time to consider and discuss participation in the protocol.  
Drs. Viscardi, Sundararajan, or research coordinators will review all UMMC NICU admissions 
to determine eligibility. For those babies who meet criteria, study personnel will approach the 
parents for consent. The content of the consent form and the purpose of the study will be 
explained. The risks, alternative to not participate and right to withdraw without penalty will be 
explained. The study team member will offer to answer any questions.  Consent will be 
documented by parent/LAR signature on the IRB approved consent form. The original signed 
consent form will be filed for study records and the consent process documented in the infant’s 
medical record. The parent/LAR will be given a copy of the consent form for their records. 
13.1 
Assent Process (Minor)/Parental Permission/Consent (If applicable) 
Not applicable  
13.2 
Subject Confidentiality 
Subject confidentiality is strictly held in trust by the participating investigators, their staff, and 
the sponsor(s) and their agents. This confidentiality includes documentation, investigation data, 
subject’s clinical information, and all other information generated during participation in the 
study. 
No information concerning the study or the data generated from the study will be released to any 
unauthorized third party without prior written approval of the sponsor and the subject. 
The study monitor or other authorized representatives of the sponsor or governmental regulatory 
agencies may inspect all documents and records required to be maintained by the investigator, 
including but not limited to, medical records (office, clinic, or hospital) and pharmacy records 
for the subjects in this study. The clinical study site will permit access to such records. 
13.3 
PI Responsibility When Subject Withdraws or Is Discontinued 
Subjects may be withdrawn from the study for the following reasons: 1) at the request of the 
subject’s parent(s) or guardian(s), or at the request of other legally authorized representative; 2) 


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if, in the investigator’s opinion, continuation in the study would be detrimental to the subject’s 
well being. 
13.4 
Future Use of Stored Specimens 
Specimens will be stored until the conclusion of the research. The consent form will specify if 
specimens may be stored indefinitely for future research, destroyed at the end of the research, or 
if the participants must be contacted to obtain permission for specimens to be used for other 
research. Future use may only include IRB approved protocols. Specimens will be labeled with a 
code indicating such.  


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14 PUBLICATION POLICY  
Final, peer-reviewed manuscripts upon acceptance for publication, to be made publicly available 
no later than 12 months after the official date of publication at the National Library of 
Medicine’s PubMed Central website. The clinical trial is listed at the ClinicaTrials.gov website. 


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48. 
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Giannone PJ, Nankervis CA, Richter JM, Schanbacher BL, Reber KM. Prenatal 
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Morowitz MJ, Poroyko V, Caplan M, Alverdy J, Liu DC. Redefining the role of intestinal 
microbes in the pathogenesis of necrotizing enterocolitis. Pediatrics 2010;125:777-785. 
54. 
Christensen RD, Wiedmeier SE, Baer VL, Henry E, Gerday E, Lambert DK, Burnett J, 
Besner GE. Antecedents of bell stage iii necrotizing enterocolitis. J Perinatol 2010;30:54-57. 
55. 
Deshpande G, Rao S, Patole S, Bulsara M. Updated meta-analysis of probiotics for 
preventing necrotizing enterocolitis in preterm neonates. Pediatrics 2010;125:921-930. 
56. 
Taddio A, Shah V, Hancock R, Smith RW, Stephens D, Atenafu E, Beyene J, Koren G, 
Stevens B, Katz J. Effectiveness of sucrose analgesia in newborns undergoing painful medical 
procedures. CMAJ 2008;179:37-43. 
57. 
Bland JM, Altman DG. Statistical methods for assessing agreement between two methods 
of clinical measurement. Lancet 1986;1:307-310. 
 
  
 


Interventional Protocol Template and Instructions 
12 June 2009 
 _____________________________________________________________________________________________  
 
 
 _____________________________________________________________________________________________  
49 
Supplements/Appendices 
 
 


 
 
 
 
50 
APPENDIX A: SCHEDULE OF EVENTS 
 
Study procedure 
DOL 0-5 
DOL 7-10  
DOL 21 
Visit 
1 
2 
3 
Screen for eligibility 
R 
 
 
Obtain consent 
R 
 
 
Physical exam 
 
R 
 
Review of Medical History R 
 
 
Review of Concomitant 
Medications  
R 
R 
R 
Adverse events 
 
R 
R 
Perform 
lactulose/rhamnose 
(La/Rh) intestinal 
permeability test 
Collect urine 
 
R 
 
Collect stool  
R 
R 
R 
 
 
 


 
 
 
 
51 
APPENDIX B-MODIFIED BELL STAGING OF NEC 
 
Modified Bell staging NEC 
Stage 
Classification 
of NEC 
Systemic signs 
Abdominal signs 
Radiographic signs 
IA 
Suspected 
Temperature instability, 
apnea, bradycardia, 
lethargy 
Gastric retention, 
abdominal distention, 
emesis, heme-
positive stool 
Normal or intestinal 
dilation, mild ileus 
IB 
Suspected 
Same as above 
Grossly bloody stool Same as above 
IIA 
Definite, mildly 
ill 
Same as above 
Same as above, plus 
absent bowel sounds 
with or without 
abdominal 
tenderness 
Intestinal dilation, 
ileus, pneumatosis 
intestinalis 
IIB 
Definite, 
moderately ill 
Same as above, plus mild 
metabolic acidosis and 
thrombocytopenia 
Same as above, plus 
absent bowel 
sounds, definite 
tenderness, with or 
without abdominal 
cellulitis or right 
lower quadrant mass 
Same as IIA, plus 
ascites 
IIIA 
Advanced, 
severely ill, 
intact bowel 
Same as IIB, plus 
hypotension, bradycardia, 
severe apnea, combined 
respiratory and metabolic 
acidosis, DIC, and 
neutropenia 
Same as above, plus 
signs of peritonitis, 
marked tenderness, 
and abdominal 
distention 
Same as IIA, plus 
ascites 
IIIB 
Advanced, 
severely ill, 
perforated bowel 
Same as IIIA 
Same as IIIA 
Same as above, plus 
pneumoperitoneum 
 
DIC: disseminated intravascular coagulation. Adapted from Neu J. Pediatr Clin North Am 1996; 43:411, 
1996.  


 
 
 
 
52 
APPENDIX C: QUALITY MANAGEMENT PLAN 
Quality Management Plan 
 
The combined processes of quality control (QC) and quality assurance (QA) form the 
basis of quality management and will be applied to this clinical research in accordance 
with a CCHI Quality Management Plan. This plan identifies the protocol, details the 
frequency of quality control checks, quality assurance reviews, priority of review, and the 
percentage of records selected for review.  
 
The Protocol-specific Quality Management Plan and written quality assurance 
communication (e.g., audit reports, problem resolution worksheets, regulatory checklists, 
audit summaries, QC/QA trend reports, quarterly and annual reports, etc.) will be kept in 
a separate Protocol-specific file Quality Management file.  
Quality Management Plan Audit Tools include as appropriate 
 
• Quality Assurance Audit Tool (RedCap)-  
• Consent Form Quality Assurance Tool 
• Regulatory File Review Tool   
• Quality Assurance Audit Report (RedCap) 
• Quality Assurance Annual Progress Report 
 
Quality Control (QC) and Quality Assurance (QA) 
Designated research staff will prospectively review and verify 100% of all data collected and 
entered into the participant’s study record. This review will be performed as close to the time of 
data collection as possible (“real-time”) to ensure the collection and accurate documentation of 
all key data points.  Data will undergo quality control “checks” prior to transfer from source 
document(s) to paper or electronic case report forms (CRFs) when applicable, however, where 
this is not feasible, the data will undergo quality control checks within 2-3 business days. 
Quality control checks or “QC checks” will begin upon the initiation of screening and 
recruitment activities and will continue through to study completion. These checks will entail the 
review of 100% of all study-specific consent forms, privacy authorizations, source documents 
and/or case report forms. Consent forms (screening, study-specific, stored specimen, and 
eligibility checklists will be reviewed at time of screening, on the day of enrollment and prior to 
study product administration (initial and subsequent administration). “All” other source 
documents and study-related forms will undergo quality control checks on an ongoing basis 
throughout data collection.  Twenty-five percent of all CRFs will be reviewed every 3 months for 
accuracy verified by review of source documents. 
 
The quality control procedure requires that data collected and recorded by one member of the 
research staff be “checked” for logic and edits by another member of the research staff. 
Communication of errors or problems identified during “QC checks” may be communicated 
verbally to the person who either collected and/or recorded the data and/or via written 
communication (i.e., post-it note which will be removed once the error has been corrected). 


 
 
 
 
53 
 All data corrections will be made by drawing a single line through the error, entering the correct 
information, initials, and the date on which the correction was made. This method of data 
correction is in accordance with the Good Clinical Practice Guideline (GCP) and is important for 
maintaining an audit trail. The date of corrections must reflect the date on which the correction 
was made and late entries dated the day the late “entry” is made. Corrections to data will be 
made by authorized staff only. Data and/or clerical errors noted in the source document that 
require a change to a database will be corrected by the designated data entry staff. Once the data 
has been corrected, the data entry staff will provide the coordinator written documentation of the 
change. Completion of “QC checks” will be indicated by entering “Qc’d by” in the lower right 
hand corner of the document followed by the initials of the person verifying completion of the 
“QC check” and the date.  In cases where documentation of the QC check is not permissible by 
the sponsor, another method of documentation may be implemented. 
Quality control “checks” will verify the following as applicable to the study:  
      ● HIPAA authorization obtained and documentation that study participant or authorized 
representative have been given a copy 
      ● Consent Form(s) are current, valid, IRB approved and are appropriately signed, dated and 
witnessed by the study participant or authorized representative and by the person obtaining 
consent 
      ● Consent process is documented by the person(s) obtaining consent. 
      ● Date and time of consent is documented  
      ● Eligibility checklist, medical history, physical exam forms have been signed and dated by 
the principal investigator or appropriate medical clinician as required by the sponsor  
      ● All study forms, source documents, and CRFs are complete (e.g., all data collection fields 
and header information is complete; check boxes are appropriately checked, etc.) and are signed 
by persons who obtained the data  
      ● All visits, screening through termination, are documented 
      ● Screening tests and visit procedures are completed for all visits 
      ● Missed visits and attempts to contact participant or authorized representative are 
documented 
      ● Data recorded on medical history and adverse event forms is consistent with concomitant 
medications forms 
      ● All concomitant medications as reported by the participant or authorized representative are 
recorded  
      ● Prohibited medications have been identified and documented 
      ● All forms are correctly dated and participant identifiers (PIDs) correctly recorded  
      ● Data quality is good (ALCOA - attributable, legible, contemporaneous, original, and 
accurate)  
      ● All adverse events are documented and serious adverse events (SAEs) reported to sponsor/ 
IRB 
      ● All required safety labs obtained and clinical significance assigned by the investigator to 
abnormal values 
      ● Study product temperature appropriately monitored and temperatures documented. Cold 
Chain maintained  
      ● Verify that study samples have been collected, processed, and/or stored as described in the 
protocol, MOP and/or SOP. 


 
 
 
 
54 
      ● Verify that sample storage temperatures have been monitored and recorded daily and if 
applicable, excursions have been reported as deviations to the sponsor and IRB as appropriate 
    ● Protocol deviations and/or violations, if any, (e.g., eligibility, missed visits, labs, procedures, 
out-of-window visits, etc.) are documented and appropriately reported to sponsor and IRB 
Signatures have been obtained on all documents, as required 
      ● Treatment and/or study discontinuation is adequately documented and study status form(s) 
completed.       
      ● Reason for ineligibility is adequately documented and all study-specific documents are  
completed up to the point of ineligibility determination       
      ● Study-specific logs are complete and accurate (e.g., questionnaires) are complete  
      ● Laboratory equipment is maintained according to manufacturer’s directions 
      ● Calibration data is quality checked before entry the research record 
      ● All laboratory data are checked by two people for entry in to the research record 
      ● Study product management (i.e., accountability, qualified personnel, dispensing, 
administration, disposition, secure storage, and access by authorized study personnel) follows 
protocol. 
 
Quality Assurance Process 
Quality Assurance (QA) audits of the study will be performed by a Quality Management (QM) 
Coordinator. Ten to 20% of study charts will be audited for each study, greater if deemed 
necessary by the PI or if QA findings suggest the need to increase the number of charts to be 
reviewed. The minimum number of charts to be reviewed will be no less than 10%.  
Quality assurance audits broadly assess investigator and site adherence to the IRB approved 
protocol, UMB-HRPO policies, sponsor guidelines, and other applicable regulatory authorities.  
Data and/or clerical errors or other problems identified during quality assurance audits will be 
documented on the appropriate Problem Resolution Worksheet and communicated to the PI and 
the Research Coordinator.  Data errors and/or problems may be resolved during the audit, 
however, if unresolved at the end of audit visit, will be noted on the Problem Resolution Work-
sheet, corrected by the appropriate staff and returned to the person performing the audit within 2 
weeks of the audit completion date. The investigator will be provided with a summary report for 
each protocol that is audited.   
 
“All” data and/or clerical errors identified during the quality assurance audit will be corrected in 
accordance with the Good Clinical Practice Guideline (GCP).  A single line will be drawn 
through the error, then initialed and dated. The date of data correction must reflect the date of the 
correction and late entries dated the day the late “entry” is made. This method of correction is 
essential to maintaining an audit trail. Corrections to study data will be made by authorized staff 
only.  The person performing the audit will only identify errors and/or problems and is not 
permitted to make changes to the study data, make entries into the study record (source, paper or 
electronic CRFs). 
 
Trends, significant findings, data or quality management related issues identified during quality 
assurance audits will be communicated to the research staff and discussed during routine staff 
meetings and documented in quarterly and annual reports.  Monitoring reports will be reviewed 


 
 
 
 
55 
by the PI and/or the Research Coordinator. Any findings unresolved upon completion of 
monitoring visits will be promptly addressed. Significant findings noted on monitoring reports 
will be considered for integration into quality improvement initiatives, as appropriate, re-training 
of staff, revision of existing MOPs and/or SOPs, development of new MOP and/or SOPs, and/or 
Corrective Action Preventive Action (CAPA) plans.  
 
Quality assurance audit reports, summaries, worksheets, and other communications, are not part 
of the regulatory file therefore, will be maintained in a separate protocol-specific Quality 
Management file. Quality management plans for multiple year studies will be re-evaluated on 
annual basis by the PI and the protocol-specific research team. 
  
Quality assurance audits will verify the following as applicable to the study:  
      ● The regulatory file (essential documents, IRB approvals, continuing reviews, protocol 
exceptions, etc.) 
      ● Informed consent(s) for inclusion of requirements for elements of informed consent 
      ● Documentation of the informed consent process  
      ● Eligibility (completion of screening procedures / requirements within protocol-specified 
timelines)  
      ● Adherence to protocol procedures and parameters 
      ● Documentation of adverse events (AEs) and concomitant medications 
      ● Documentation and timely reporting of internal and external Serious Adverse Events 
(SAEs)  
      ● Documentation of clinical evaluations 
      ● Documentation of study endpoints (e.g., study specimens).   
      ● Documentation of specimen collection, processing, and storage; shipping manifests, and 
freezer temperature logs.  
      ● Documentation and reporting of protocol deviations (e.g., missed visits, study labs, study 
procedures, temperature excursions) within specified timelines 
      ● Documentation and reporting of protocol violations (e.g., eligibility, deviations that impact 
participant safety, rights, well-being) within specified timelines 
      ● Documentation of treatment and study discontinuation 
      ● Signatures obtained as required 
      ● Adherence to institutional, sponsor and GCP documentation standards 
      ● Compare data consistency between source documents and electronic and/or paper CRFs 
      ● Assess data quality (ALCOA - attributable, legible, contemporaneous, original, and 
accurate) 
 
 
Data management 
When an electronic data base is used study-data will undergo quality control checks prior to 
transfer from source documents to case report forms (paper or electronic), where feasible. Every 
effort will be made to enter data into the electronic database within the timeframe specified by 
the sponsor. Data and/or clerical errors received from the data management center(s) will be 
promptly reviewed by appropriate members of the research staff and corrected.  Data will be 
corrected or amended by persons authorized to make such changes to paper or electronic CRFs. 
Persons entering data into electronic CRFs will be trained in the use of the computerized system. 


 
 
 
 
56 
 
 
 
 

ICF_001.pdf: 
Page 1 of 5
RESEARCH CONSENT FORM
Protocol Title: Measures of Intestinal Permeability in Preterm infants
Study No.: HP -00049647
Principal Investigator: Sripriya Sundararajan, M.D.
Sponsor: University of Maryland Institute for Clinical and Translational 
Research
You are being asked to allow your child to take part in a research study.  Your child does not have to take 
part in this study. You may ask questions at any time. This research is sponsored by the University of 
Maryland, Baltimore. 
PURPOSE OF STUDY
Under normal circumstances, cells from the gut form a tight but selective barrier.  Most substances are 
kept inside the intestine, but food is absorbed.  The ability to absorb food while keeping out other 
substances matures over time.  It begins soon after birth but we do not understand much about how it 
works.  Low birth weight, prematurity, and early postnatal age are associated with problems with this 
process called “leaky gut”.  The ability to allow substances to cross from the intestine is called intestinal 
permeability.  The gut is leakier at birth in preterm than term infants and allows more substances to cross 
the barrier.  We are trying to identify infants at high risk for problems associated with prematurity by 
measuring their intestinal permeability.  We are trying to find the best way to measure leaky gut in very 
low birth weight infants. 
You are being asked to allow your child to participate because his/her birth weight is very low.  Your 
child is one of approximately 230 infants that will be asked to participate in this study.  The study will 
take place at the University of Maryland Medical Center. Around one hundred and sixty participants will 
be enrolled at this local site.
PROCEDURES
If you agree to allow your child to be in this study, we will collect urine and stool samples. Everyone will 
receive the same tests.  We will measure the leakiness of your child’s gut 1 time. Your child’s part in the 
study will last 16 days.  We will do the urine test during your child’s hospitalization between 7-10 days of 
age. 
Your child will be given one dose (1/5 tsp) of a sugar solution through a feeding tube. We will collect 
urine for the next 4 hours after your child is given the solution. The urine will be tested for the amount of 
sugar that crossed from the gut. We will collect stool specimens daily from the diaper from the day of the 
study enrollment until 14 days of age and again on day of life 21. The stool will be tested for the different 
types of bacteria and whether bacteria associated with a healthy gut (probiotic bacteria) are present.   
Individual results will not be disclosed. 
HP-00049647 UM IRB Approval Date 10/20/2019
Do Not Sign this Form after this Date 10/19/2020


Page 2 of 5
The investigators will review your child’s medical records. We will use your child’s hospital record to 
collect information about your child’s race and gender.  We will also collect information about labor and 
delivery. We will record your child’s birth weight, feeding data (formula or breast milk, age of starting 
feeding, feeding difficulties, age when receiving the full amount of feeds, etc), growth during the study 
period, and illnesses related to prematurity.  We will collect some laboratory results such as blood counts 
and facts about any infections your child might develop.  
If we do not use all of your child’s samples, we plan to store them for future use.  They will be identified 
only by number.  Only the investigators will have access to the code that identifies your child. 
Please check one of the following choices and sign below:
-----My child's samples (urine and stool) may be used for this project only.  Do not use them for any other 
project and do not contact me again for permission. 
-----My child's samples (urine and stool) may be used for this project only and for other projects with my 
permission.  If my samples could be used for another project, contact me to ask my permission.
-----My child's samples (urine and stool) may be used for any scientific purposes involving this or any 
other project.  Do not contact me again for permission.  
Parent's/Guardian's signature___________________________________
You will not receive any individual test results.  The results of the study will be published in publicly 
available journals and will be made available in summary form. Individual results will not be disclosed. 
You may choose to receive a summary of the study’s results.  Please indicate below if you wish to be 
notified. If you want be told the results of the study, we will give you the contact information for the 
principal investigator, Dr. Sripriya Sundararajan. 
___ Yes I would like to be contacted by Dr. Sundararajan or one of her associates if potentially important 
results are found from my child’s participation that may be helpful in diagnosing, treating or preventing 
disease. 
___ No I do not wish to be contacted about any results obtained in the future from the study of my child’s 
genetic sample.
Parent/Guardian signature ______________________________________
WHAT ARE MY RESPONSIBILITIES IF I TAKE PART IN THIS RESEARCH?
If you take part in this research, you will be responsible to:

Ask questions about anything you do not understand

For your child to complete all research procedures unless you formally request to withdraw from 
the study

Notify study staff of all changes to your contact information including your address, phone 
numbers, and e-mail address
HP-00049647 UM IRB Approval Date 10/20/2019
Do Not Sign this Form after this Date 10/19/2020


Page 3 of 5
POTENTIAL RISKS/DISCOMFORTS:
There is a potential for the loss of confidentiality.  To prevent this risk, the results of all tests will be kept 
private.  Files will be kept in a locked room.  Only research staff can use computers or files with your 
information.  Tubes for urine and stool samples will be labeled with a number, your child’s name will not 
be used.
POTENTIAL BENEFITS
Your child will not benefit from being in this study.  Your child’s participation may help the doctors 
better understand leaky gut in very low birth weight babies.  You need to decide if your child’s 
participation in this research study is in your child’s best interest.
ALTERNATIVES TO PARTICIPATION
This is not a treatment study.  Your alternative is not to take part.  If you choose not to take part, your 
child’s healthcare will not be affected.
COSTS TO PARTICIPANTS
It will not cost you anything for your child to take part in this study.
PAYMENT TO PARTICIPANTS
You will not be paid to be part of in this research study.  If your child is hurt by being in the study, he/she 
will receive emergency medical care if needed.  Your child will receive assistance in getting other 
medical care as needed.  You or your insurance carrier will be billed for the cost of care, just as you 
would be billed for any other medical care.  If you have uninsured medical costs, you are responsible for 
them.  The study staff will give you more information about this if you have a study injury. 
CONFIDENTIALITY AND ACCESS TO RECORDS
All information collected as part of this study will be kept confidential to the fullest extent permitted by 
law.  The data from the study may be published.  However, you will not be listed by name.  We will not 
give out your personal data unless the law requires it.  Everyone using study data will work to keep your 
personal data safe.  The university or the hospital where the study takes place may assign people to 
inspect the research including the parts of your medical record related to research.  Study records may be 
reviewed by the sponsor, or the Institutional Review Board.
Efforts will be made to limit your personal information, including research study and medical records, to 
people who have a need to review this information.  We cannot promise complete secrecy.  Organizations 
that may inspect and copy your information include the IRB and other representatives of this 
organization. 
RIGHT TO WITHDRAW
Your participation in this study is voluntary.  You do not have to take part in this research. You are free to 
withdraw your consent at anytime. Refusal to take part or to stop taking part in the study will involve no 
penalty or loss of benefits to which you are otherwise entitled.  If you decide to stop taking part, if you 
have questions, concerns, or complaints, or if you need to report a medical injury related to the research, 
please contact the investigator Dr. Sundararajan at 410-328-6003.  If you decide to withdraw your child, 
you must notify Dr. Sundararajan in writing. If you withdraw from this study, already collected data may 
not be removed from the study database.  You will be asked whether the investigator can collect data 
from your routine medical care.  If you agree, this data will be handled the same as research data. 
HP-00049647 UM IRB Approval Date 10/20/2019
Do Not Sign this Form after this Date 10/19/2020


Page 4 of 5
CAN I BE REMOVED FROM THE RESEARCH?
The person in charge of the research study or the sponsor can remove you from the research study without 
your approval.  Possible reasons for removal include failure to follow instructions of the research staff if 
the person in charge decides that the research study is no longer in your child’s best interest.  The sponsor 
can also end the research study early. The study doctor will tell you about this and you will have the 
chance to ask questions if this were to happen.
UNIVERSITY STATEMENT CONCERNING RESEARCH RISKS 
The University is committed to providing participants in its research all rights due them under State and 
federal law. You give up none of your legal rights by signing this consent form or by participating in the 
research project. This research has been reviewed and approved by the Institutional Review Board (IRB).  
Please call the Institutional Review Board (IRB) if you have questions about your rights as a research 
participant.
The research described in this consent form has been classified as minimal risk by the IRB of the 
University of Maryland, Baltimore (UMB). The IRB is a group of scientists, physicians, experts, and 
other persons. The IRB’s membership includes persons who are not affiliated with UMB and persons who 
do not conduct research projects.  The IRB’s decision that the research is minimal risk does not mean that 
the research is risk-free. You are assuming risks of injury because of research participation, as discussed 
in the consent form.
If you are harmed as a result of the negligence of a researcher, you can make a claim for compensation.  If 
you have questions, concerns, complaints, or believe you have been harmed through participation in this 
research study as a result of researcher negligence, you can contact members of the IRB or the staff of the 
Human Research Protections Office (HRPO) to ask questions, discuss problems or concerns, obtain 
information, or offer input about your rights as a research participant.  The contact information for the 
IRB and the HRPO is:
University of Maryland 
Human Research Protections Office 
620 W. Lexington St, Second Floor
Baltimore, MD 21201
410-706-5037
Signing this consent form indicates that you have read this consent form (or have had it read to you), that 
your questions have been answered to your satisfaction, and that you voluntarily agree for your child to 
participate in this research study.  You will receive a copy of this signed consent form.
If you agree for your child to participate in this study, please sign your name below.
___________________________________
Signature of Parent/Guardian
Relationship:________________________
HP-00049647 UM IRB Approval Date 10/20/2019
Do Not Sign this Form after this Date 10/19/2020


Page 5 of 5
Date:______________________________
___________________________________
Investigator or Designee Obtaining Consent 
Signature
Date:______________________________
___________________________________
Witness*
Date:______________________________
*Witness is optional unless parent is 
illiterate or unable to sign.
HP-00049647 UM IRB Approval Date 10/20/2019
Do Not Sign this Form after this Date 10/19/2020