L. Reuteri for Pediatric Diarrhea in Peru: Growth, Enteropathy, and Microbiota
Phase: PHASE1
Conditions: Healthy
Sponsor: Tulane University School of Medicine

PRIDEC-Peru 
Protocol # 1: Safety of Lactobacillus reuteri in healthy children aged 2-24 months 
Version 2.0  
11 Jan 2019 
 

PRIDEC-Peru 
Protocol # 1: Safety of Lactobacillus reuteri in healthy children aged 2-24 months 
Version 2.0  
11 Jan 2019 
 
 
 
 
Safety of Lactobacillus reuteri in healthy children aged 2-24 months 
 
 
NIDDK Funding Mechanism: U01 DK105849 
 
Pharmaceutical Support Provided by: Biogaia 
Other Identifying Number:   
JHU IRB 6724  
Tulane IRB (JHU reliant)  
INS resolution 412-2018-OGITT/INS 
 
 
 
Draft or Version Number: 
 
                 2.0 
Day Month Year 
11 Jan 2019 
 

PRIDEC-Peru 
Protocol # 1: Safety of Lactobacillus reuteri in healthy children aged 2-24 months 
Version 2.0  
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STATEMENT OF COMPLIANCE 
 
 
The study will be carried out in accordance with Good Clinical Practice (GCP) as 
required by ICH E6; 62 Federal Register 25691 (May 9, 1997) and laws governing the 
conduct of clinical trials in Peru under the authority of Peru’s National Institute of Health 
(INS) in accordance with XV article 28 of law 26842 and the oversight of the National 
Institutes of Health of Peru (INS) as specified by articles 4 and 5 of Decree. 017-2006-
SA in accordance to current guidelines described in the Guidelines for Clinical Trials in 
Peru, 20 August 2011 (Reglamento de Ensayos Clinicos en el Peru, Lima 2011, 
integra: El DS 0-17-2006-SA, El DS 006-2007-SA y El DS 011-2007-SA). 
 
 
All key personnel and persons involved in human subjects contact, contact with CRFs 
and source documents and data management for the project have completed Human 
Subjects Protection Training. 

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TABLE OF CONTENTS 
Contents 
Safety of Lactobacillus reuteri  in healthy children aged 2-24 months ................................. i 
STATEMENT OF COMPLIANCE ...................................................................................... ii 
TABLE OF CONTENTS .................................................................................................... iii 
LIST OF ABBREVIATIONS .............................................................................................. vi 
PROTOCOL SUMMARY ................................................................................................... 1 
1 
KEY ROLES .............................................................................................................. 1 
2 
INTRODUCTION: BACKGROUND INFORMATION AND SCIENTIFIC RATIONALE 3 
2.1 Background Information ............................................................................................. 3 
2.2 Rationale ................................................................................................................... 3 
2.3 Potential Risks and Benefits ...................................................................................... 4 
2.3.1 Potential Risks ................................................................................................ 5 
2.3.2 Known Potential Benefits ................................................................................ 5 
3 
OBJECTIVES ............................................................................................................ 6 
3.1 Study Objectives ....................................................................................................... 6 
3.2 Study Outcome Measures ......................................................................................... 6 
3.2.1 Primary Outcome Measures ........................................................................... 6 
3.2.2 Secondary Outcome Measures ....................................................................... 8 
4 
STUDY DESIGN ........................................................................................................ 8 
5 
STUDY ENROLLMENT AND WITHDRAWAL .......................................................... 11 
5.1 Subject Inclusion Criteria ......................................................................................... 12 
5.2 Subject Exclusion Criteria ........................................................................................ 13 
5.3 Strategies for Recruitment and Retention ................................................................ 14 
5.4 Treatment Assignment Procedures ......................................................................... 14 
5.4.1 Randomization Procedures ........................................................................... 14 
5.4.2 Masking Procedures ..................................................................................... 14 
5.4.3 Reasons for Withdrawal ................................................................................ 14 
5.4.4 Handling of Withdrawals ............................................................................... 15 
5.4.5 Termination of Study ..................................................................................... 15 
6 
STUDY INTERVENTION/INVESTIGATIONAL PRODUCT ...................................... 17 
6.1 Study Product Description ....................................................................................... 17 
6.1.1 Acquisition .................................................................................................... 17 
6.1.2 Formulation, Packaging, and Labeling .......................................................... 17 
6.1.3 Product Storage and Stability ........................................................................ 17 
6.2 Dosage, Preparation and Administration of Study Intervention/Investigational 

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Product .................................................................................................................... 18 
6.3 Modification of Study Intervention/Investigational Product for a Subject .................. 19 
6.4 Accountability Procedures for the Study Intervention/Investigational Product(s) ..... 19 
6.5 Assessment of Subject Compliance with Study Intervention/Investigational Product
 ................................................................................................................................ 19 
6.6 Concomitant Medications/Treatments ...................................................................... 19 
7 
STUDY SCHEDULE ................................................................................................ 21 
7.1 Recruitment process ............................................................................................... 21 
7.2 Consent  Process and Documentation .................................................................... 21 
7.3 Screening ................................................................................................................ 21 
7.4 Product  Administration period ................................................................................. 23 
7.5 Follow-up ................................................................................................................. 25 
7.6 Final Study Visit ....................................................................................................... 26 
7.7 Early Termination Visit ............................................................................................. 27 
7.8 Unscheduled Visit .................................................................................................... 27 
8 
STUDY PROCEDURES/EVALUATIONS ................................................................. 28 
8.1 Clinical Evaluations.................................................................................................. 28 
8.2 Laboratory Evaluations ............................................................................................ 28 
8.2.1 Clinical Laboratory Evaluations ..................................................................... 28 
8.2.2 Special Assays or Procedures ...................................................................... 29 
9 
ASSESSMENT OF SAFETY ................................................................................... 30 
9.1 Specification of Safety Parameters .......................................................................... 30 
9.2 Methods and Timing for Assessing, Recording, and Analyzing Safety Parameters . 30 
9.2.1 Adverse Events ............................................................................................. 30 
9.2.2 Serious Adverse Events (SAE) ..................................................................... 33 
9.2.3 Unanticipated Problems ................................................................................ 34 
9.2.4 Procedures to be Followed in the Event of Abnormal Laboratory Test Values 
or Abnormal Clinical Findings ....................................................................... 35 
9.3 Reporting Procedures .............................................................................................. 37 
9.3.1 Serious Adverse Events ................................................................................ 38 
9.3.2 Regulatory Reporting for Studies Not Conducted Under NIDDK-Sponsored 
IND 
39 
9.4 Type and Duration of Follow-up of Subjects after Adverse Events .......................... 39 
9.5 Halting Rules ........................................................................................................... 39 
9.6 Safety Oversight ...................................................................................................... 40 
10 
CLINICAL MONITORING ........................................................................................ 41 
10.1 Site Monitoring Plan ................................................................................................ 41 
11 
STATISTICAL CONSIDERATIONS ......................................................................... 42 
11.1 Study Hypotheses and Analysis ............................................................................... 42 

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11.2 Sample Size Considerations .................................................................................... 44 
11.3 Planned Interim Analyses (if applicable) .................................................................. 46 
11.3.1 Safety Review ............................................................................................... 46 
12 
SOURCE DOCUMENTS AND ACCESS TO SOURCE DATA/DOCUMENTS ......... 48 
13 
QUALITY CONTROL AND QUALITY ASSURANCE .............................................. 49 
14 
ETHICS/PROTECTION OF HUMAN SUBJECTS .................................................... 51 
14.1 Ethical Standard ...................................................................................................... 51 
14.2 Institutional Review Board ....................................................................................... 51 
14.3 Informed Consent Process ...................................................................................... 52 
14.3.1 Informed Consent/Assent Process (in Case of a Minor) ................................ 52 
14.4 Exclusion of Women, Minorities, and Children (Special Populations) ...................... 52 
14.5 Subject Confidentiality ............................................................................................. 52 
14.6 Study Discontinuation .............................................................................................. 53 
14.7 Future Use of Stored Specimens ............................................................................. 53 
15 
DATA HANDLING AND RECORD KEEPING .......................................................... 54 
15.1 Data Management Responsibilities ......................................................................... 54 
15.2 Data Capture Methods............................................................................................. 55 
15.3 Timing/Reports ........................................................................................................ 56 
15.4 Study Records Retention ......................................................................................... 56 
15.5 Protocol Deviations .................................................................................................. 57 
16 
STUDY ADMINISTRATION ..................................................................................... 58 
16.1 STUDY LEADERSHIP AND GOVERNANCE .......................................................... 58 
16.2 SUBCOMMITTEES ................................................................................................. 59 
17 
LITERATURE REFERENCES ................................................................................. 60 
APPENDIX A:STUDY PROCEDURES ........................................................................... 63 
APPENDIX A.1: Planned laboratory tests ....................................................................... 64 
Appendix B.  Definitions for Adverse Events Monitoring ................................................. 65 
APPENDIX C: Clinical adverse events to be monitored : Symptoms .............................. 66 
APPENDIX D:  LABORATORY VALUES ........................................................................ 67 
APPENDIX E.   FEVER SEVERITY GRADING .............................................................. 69 
 

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Protocol # 1: Safety of Lactobacillus reuteri in healthy children aged 2-24 months 
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LIST OF ABBREVIATIONS 
 
AE 
Adverse Event 
CFR 
Code of Federal Regulations 
CIOMS 
Council for International Organizations of Medical Sciences 
CONSORT 
Consolidated Standards of Reporting Trials 
CRF 
Case Report Form 
DSMB 
Data and Safety Monitoring Board 
DSMP 
Data Safety Monitoring Plan 
EC 
Ethics Committee 
FWA 
Federal-Wide Assurance 
GCP 
Good Clinical Practice 
IATA 
International Air Transit Association 
ICH 
International Conference on Harmonisation 
IND 
Investigational New Drug Application 
INS 
Peruvian National Institute of Health (Instituto Nacional de Salud) 
IRB 
Institutional Review Board 
CITI 
Collaborative Institutional Training Initiative 
LR or Lr 
Lactobacillus reuteri 
MOP 
Manual of Procedures 
NIDDK 
National Institute of Diabetes and Digestive and Kidney Diseases 
NIH 
National Institutes of Health 
OHRP 
Office for Human Research Protections 
OHSR 
Office of Human Subjects Research 
PI 
Principal Investigator 
PID 
Personal Identifying Code 
PL 
Placebo 
QA 
Quality Assurance 
QC 
Quality Control 
SAE 
Serious Adverse Event 
SID 
Specimen identification code 
SOP 
Standard Operating Procedure 
WHO 
World Health Organization 
 

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Protocol # 1: Safety of Lactobacillus reuteri in healthy children aged 2-24 months 
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PROTOCOL SUMMARY 
 
 
 
Title: 
Safety of Lactobacillus reuteri in healthy children aged 2-24 
months 
Précis: 
Phase I double blinded randomized trial of the safety and 
tolerability of Lactobacillus reuteri DSM 17938 given for five 
successive days in healthy children.  Sixty children will receive 
study product at a treatment to placebo ratio of 2:1 and 
followed for 3 months for safety outcomes. 
Objectives: 
Primary: To assess the safety and tolerability of Lactobacillus 
reuteri (Lr) strain DSM 17938 in healthy infants and children.  
Safety will be assessed through the evaluation of adverse effects 
in both study arms for 90 days following product administration 
and by laboratory monitoring of CBC, AST, ALT, T. bilirubin, BUN 
and creatinine prior to product or placebo administration with 
follow-up assessment at 5 and 28 days.  Tolerability will be 
assessed based on parental report of subjective symptoms of 
well-being during product administration and thru D28 of the 
study. 
 
Secondary:   
 To evaluate the duration of shedding of Lactobacillus reuteri (Lr) 
strain DSM 17938 following administration.  This will be 
determined by and endpoint PCR assay for L. reuteri done on 
stool collected on D3, D5, D12, D15, D18, D24, D28, D36 on all 
participants. 
 

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Population: 
The sample size of sixty is expected to include an equal 
number of healthy boys and girls 2-24 months of age at the 
time of enrollment from a rural community in Loreto Peru 
where chronic undernutrition and diarrheal disease are both 
highly prevalent. 
Phase: 
I 
Number of Sites: 
Single site- Loreto, Peru 
Study Duration: 
7 months 
Subject Participation 
Duration: 
3 months 
Description of Agent or 
Intervention: 
1. Active Product:  Description Lactobacillus reuteri  17938 
suspended in sunflower oil, medium chain triglyceride oil, silicone 
dioxide.  Total viable count of L. reuteri  17938 1 x 108 CFU/5 drops 
per day for 5 consecutive days 
2.  Placebo:  Composition Sunflower oil, medium chain 
triglyceride oil and silicon dioxide.  Total viable count of L. reuteri is 
zero CFU/ 5 drops. 
Estimated Time to 
Complete Enrollment: 
4 months 

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Protocol # 1: Safety of Lactobacillus reuteri in healthy children aged 2-24 months 
Version 2.0  
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Schematic of Study Design: 
 
  
 
1 
KEY ROLES 
 
 
Individuals: 
Cesar Ramal Asayag, MD 
AB PRISMA- Iquitos (Branch office) 
Ramirez Hurtado 622 
Iquitos, Maynas Peru 
ramalasayag@yahoo.fr 
011-51-65-234250 
Peruvian PI 

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Protocol # 1: Safety of Lactobacillus reuteri in healthy children aged 2-24 months 
Version 2.0  
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Institutions: 
 
 
 
Johns Hopkins Bloomberg School of 
Public Health 
615 N Wolfe St E5545 
Margaret Kosek, MD 
Tel 410-955-9545 
mkosek@jhu.edu 
Pablo Penataro Yori, MPH 
pyori@jhu.edu 
Tel. 443-287-2750 
 
 
 
Tulane School of Public Health and Tropical Medicine 
 1440 Canal Street, Suite 2301 
New Orleans, LA 70112 
Richard Oberhelman, MD 
oberhel@tulane.edu 
Tel. 504-988-2511 
Co-PI 
 
AB PRISMA- Iquitos (Branch office) 
Ramirez Hurtado 622 
Iquitos, Maynas Peru 
Maribel Paredes Olortegui, BS 
mparedeso@prisma.org.pe 
011-51-65-234250 
Study Coordinator 
 
AB PRISMA-Lima (central office) 
Carlos Gonzales 251 
San Miguel, Lima, Peru 32 
Delia Haustein GinHoven,  
dhaustein@prisma.org.pe 
011-51-1- 6165500 
Executive Director 
 
 
 
 
 
 
 
 
 

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Protocol # 1: Safety of Lactobacillus reuteri in healthy children aged 2-24 months 
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1 
 
2 
2 
INTRODUCTION: BACKGROUND INFORMATION AND 
3 
SCIENTIFIC RATIONALE 
4 
 
5 
2.1 
Background Information 
6 
 
7 
 
8 
Probiotic therapy is intrinsically inexpensive and readily applicable in resource-poor 
9 
developing countries.  Because of already published trials showing efficacy in the 
10 
treatment of diarrheal diseases, probiotic organisms such as L. reuteri are already in 
11 
clinical use for the treatment of acute infectious diarrhea1,2, infantile colic,3,4 and atopic 
12 
disease.5  However, the avoidance of the formal IND process and FDA observed clinical 
13 
trials has compromised the credibility of many of these reports.  There is substantial 
14 
evidence that the quality of preparations in distribution is highly variable and labels do not 
15 
always reflect true contents of marketed materials.6-8   
16 
 
17 
Our long term goal is to develop Lactobacillus reuteri DSM 17398 as a safe and effective 
18 
therapy for acute infectious diarrhea in children. We have completed a Phase 1 trial in 
19 
adults9 ((NCT00774163) and are in the process of completing a Phase 1 study of children 
20 
ages 2-5 years with this product under IND ((NCT020124122). This study is designed to 
21 
provide evidence of the safety of the study product in well children in Peru under 
22 
demanding international quality standards.  The study will not be done under IND but will 
23 
be done with extensive oversight by the study team in collaboration with NIDDK and a 
24 
DSMB. If shown to be efficacious in phase II/III trials we believe this will allow for the 
25 
greater use and acceptance of this product as part of the public health response to the 
26 
management of acute infectious diarrhea in children. 
27 
 
28 
 
29 
2.2 
Rationale 
30 
 
31 
 
32 
We hypothesize that L reuteri given at a dose of 1 x 108 CFU/ day is safe in infants in 
33 
children and likely to be beneficial in shortening the duration of episodes of diarrhea in 
34 
children in resource constrained settings.   
35 
 
36 
BioGaia has chosen the dose level of 1x108 CFU/day for L. reuteri in man as a safe 
37 
and efficacious dose. The same dose is recommended for all ages.  
38 
Unlike pharmaceuticals, where doses are absorbed and metabolized, Lactobacillus reuteri 
39 
consists of live cells that become active and multiply along the entire digestive tract. 
40 
Whether in adults, children or infants, Lactobacillus reuteri grows from the initial dose to 
41 
colonize the available gut mucosa and thus, even though the surface area of the GI tract 
42 
differs with age, the substrates for multiplication are not limiting and thus the same dose is 
43 
effective.   
44 

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Protocol # 1: Safety of Lactobacillus reuteri in healthy children aged 2-24 months 
Version 2.0  
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1 
The recommended daily dose of 1x108 CFU has shown efficacy in 10 studies on 
2 
prevention or treatment of symptoms.3,10-13 
3 
 
4 
Doses in the range of 108 – 109 CFU/day have shown efficacy in studies by: Eom 2005 
5 
(2x108),14 Valeur 2004 (4x108),15 Ouwehand 2002 (7x108).16  
6 
 
7 
Some early studies in humans used higher doses up to 1010-11 CFU/day,1,2,17 primarily in 
8 
safety studies in young children, adults and immunocompromised adults (HIV-positive). 
9 
Blood, urinary, fecal and clinical parameters showed such higher levels to be safe. The 
10 
only possible side effect observed was a mild, and temporary, increase in flatulence. 
11 
 
12 
Studies performed with lower than the recommended 1x108 CFU dose demonstrated gut 
13 
colonization, but some of studies demonstrated less optimal effect for prevention or 
14 
treatment of diarrhea.  The 1x105 and 1x107 dosages both colonized the gut and had 
15 
significant effect on watery stools in newborns.  1x107 CFU/day had significant effect on 
16 
frequency of diarrhea and close to significant effect on duration of watery diarrhea in 
17 
rotavirus gastroenteritis.2 
18 
 
19 
The five day treatment period is based on clinical studies that generally showed biologic 
20 
response with 24-48 hours.  Shorter duration of therapy is preferred as it is more 
21 
practical to implement and limits cost per treatment making this treatment a feasible 
22 
option for a common important public health problem. 
23 
 
24 
2.3 
Potential Risks and Benefits 
25 
 
26 
Potential Risks 
27 
There are slightly greater than minimal risks associated with study participation.  Blood 
28 
draws have a slight risk of bruising or infection.  Health workers are trained in phlebotomy. 
29 
The prospective surveillance portion involves the limited inconvenience of daily interviews 
30 
to obtain symptom and illness histories.  There also exists a small risk that the information 
31 
collected may not remain confidential, however, the data collected is not of a sensitive 
32 
nature and would be unlikely to have important social or legal consequences for the 
33 
participant and his or her family.  Each enrollment in the study involves the risk associated 
34 
with the possible consumption of the probiotic Lactobacillus reuteri.  Although there is 
35 
extensive worldwide use of these products, it is possible that their ingestion could lead to 
36 
complications, including sepsis.18,19  As we have cited numerous studies where these 
37 
probiotics have been used for both treatment and prophylaxis of diarrhea in children 
38 
without serious adverse effects, we believe the risk is very small.  We will monitor closely 
39 
for adverse events and address them promptly with appropriate medical interventions, 
40 
which will be supplied by the study. 
41 
 
42 
Benefits 
43 
It is not believed that subjects will receive any health benefit related to participation in the 
44 
study.  Parents may only perceive that they are contributing to the evaluation of a 
45 
treatment of diarrhea that may be important in the future.
46 

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Protocol # 1: Safety of Lactobacillus reuteri in healthy children aged 2-24 months 
Version 2.0  
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1 
2.3.1 
Potential Risks 
2 
 
3 
The major potential risk associated with the administration of probiotics is the rare 
4 
disseminated bloodstream or deep tissue infection with the probiotic strain or the 
5 
contamination of the probiotic product with a pathogen or toxin.  Bloodstream infection, 
6 
liver abscesses, and joint infections have occurred with probiotic bacteria, predominantly 
7 
Lactobacillus rhamnosus GG, although there have been reports of L. acidophilus 
8 
bacteremia associated with product ingestion.18,20-23    Additionally recently intestinal 
9 
mucormycosis caused by the contamination of  probiotic preparation ABC Dophilus 
10 
powder which contains B. lactis, S. thermophilus, and L. rhamnosus  was found to be 
11 
contaminated with Rhizopus oryzae which lead to the death of one infant in Connecticut in 
12 
2014.24 
13 
 
14 
To mitigate this risk we have taken several precautions.  We have chosen a product that 
15 
was isolated by the current provider who can trace the origin and any changes in the 
16 
product, a standard almost no other product with demonstrated efficacy in treating acute 
17 
enteric infections can meet.  This product was derived from a strain isolated in human 
18 
breastmilk in Peru by scientists at BioGaia who have held this reference strain since that 
19 
time.25  We have also chosen a product that has no documented cases in Pubmed 
20 
(Reviewed Feb 20th, 2015) of disseminated bloodstream infections or deep tissue 
21 
infections despite the distribution of the product in the US and Europe since 2007.  The 
22 
derivative strain Lactobacillus reuteri 55730 (different from the product strain only because 
23 
it had additional plasmids that contained antibiotic resistance genes) was previously 
24 
distributed for 10 years and taken together millions of doses of these two highly related 
25 
products have been sold with no reports in the literature of bacteremia or deep tissue 
26 
infection and no product contamination concerns.  This product has been evaluated in 
27 
several clinical trials26,27 including ones done  in infants, with no safety concerns, including 
28 
studies that gave it at the study dose daily to prevent necrotizing enterocolitis in low birth 
29 
weight newborns,28,29 and in infants with colic.30-35  Furthermore, as detailed in our product 
30 
assurance plan, we will rigorously run independent assessments of the viability and 
31 
identity of the product provided by our probiotic supplier which will further mitigate risk to 
32 
subjects in compliance with CBER guidance on early clinical trials and provide a high 
33 
standard for the evaluation of probiotic products by other investigators. 
34 
 
35 
Evidence on the safety and history of the product strain are available in Appendix F. 
36 
 
37 
 
38 
2.3.2 
Known Potential Benefits 
39 
 
40 
 
41 
There are no direct benefits to subjects who participate in this study.  Potential benefits of 
42 
this product in other studies have shown it efficacious in the treatment of infantile colic,4 
43 
the prevention and treatment of acute infectious diarrhea,2 and the prevention of 
44 
necrotizing enterocolitis in low birth weight babies.29 
45 

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1 
 
2 
3 
OBJECTIVES 
3 
 
4 
3.1 
Study Objectives 
5 
 
6 
Conduct a Phase 1 study to document safety and tolerability of Lactobacillus reuteri 
7 
17938 in children 2-24 months of age.   
8 
 
9 
3.2 
Study Outcome Measures 
10 
 
11 
 
12 
3.2.1 
Primary Outcome Measures 
13 
 
14 
There are three major areas of safety and tolerability that will be assessed in this phase I 
15 
trial.  The outcome measurements will be compared between the two treatment groups 
16 
(Intervention v placebo) with intent to treat analysis.   
17 
 
18 
1. Evidence of invasive infection resulting from the administration of L. reuteri DSM 
19 
17938.  Baseline and daily temperature measurements will be made during the study 
20 
period. Complete hemogram with differential count obtained at baseline, on day 5, and 
21 
on day 28.  Vital signs will be monitored daily during the administration of the treatment 
22 
(days 1-5).  The evaluation of all febrile episodes will be done and include a physician 
23 
exam and the evaluation of blood smears for malaria and blood cultures for the 
24 
evaluation of possible bacteremia. 
25 
 
26 
Specific outcome parameters in this category include: 
27 
 
28 
a. Mean daily temperature 
29 
b. Incidence of fever on study days 1-5 
30 
c. Frequency of temperature readings categorized as fever (adjusted for number of 
31 
subjects per group) 
32 
d. Number of episodes of L. reuteri bacteremia 
33 
 
34 
2. Clinical tolerance.  The inclusion of a placebo group will allow for comparisons of daily 
35 
symptom measurements to be compared between the treatment and placebo group.  
36 
The symptoms will be monitored for at household visits, and will be graded as 
37 
adverse events according to defined criteria.  Symptoms monitored as AEs include 
38 
fever, nausea/upset stomach, vomiting, diarrhea, irritability, rash, pruritis, and 
39 
wheezing or bronchospasm.  Diagnostic microbiology will be performed on all stool 
40 
specimens from subjects who develop diarrhea during the 90 days following initiation 
41 
of therapy.   
42 
 
43 
Specific outcome parameters in this category include: 
44 
 
45 

PRIDEC-Peru 
Protocol # 1: Safety of Lactobacillus reuteri in healthy children aged 2-24 months 
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a. Frequency and severity of symptoms monitored as AEs (adjusted for number of 
1 
subjects per group) 
2 
b. Frequency of all AEs (adjusted for number of subjects per group) 
3 
c. Frequency of SAEs (adjusted for number of subjects per group) 
4 
 
5 
  
6 
3.  Evidence of toxicity resulting from the L. reuteri DSM 17938 preparation. 
7 
Participants will have baseline, day 5, and day 28 assessment of hematologic status and 
8 
renal and hepatic function.  Assessment of renal function will include blood urea nitrogen 
9 
(BUN) and creatinine (Cr).  Assessment of hepatic toxicity will include transaminases 
10 
(AST, ALT) and total bilirubin.  Hematologic status will be determined by CBC to measure 
11 
hemoglobin and absolute leucocyte count, as mentioned above. 
12 
 
13 
Specific outcome parameters in this category include: 
14 
 
15 
a. Mean values for laboratory tests monitored (adjusted for number of subjects per 
16 
group).  These are listed in Appendix D.   
17 
b. Frequency of out of range values for laboratory tests monitored (adjusted for number 
18 
of subjects per group).  These are listed in Appendix D.  
19 
 
20 
 
21 
 
22 
 
23 
 
24 

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1 
3.2.2 
Secondary Outcome Measures 
2 
 
3 
 
4 
Secondary outcomes are restricted to:  
5 
1) The assessment of the duration of shedding of Lactobacillus reuteri (Lr) strain DSM 
6 
17938 following administration.  This will be determined by and endpoint PCR assay for L. 
7 
reuteri done on stool collected on D3, D5, D12, D15, D18, D24, D28, D36 on all 
8 
participants. 
9 
 
10 
 
11 
4 
STUDY DESIGN 
12 
 
13 
The study is a single center Phase 1 double blind placebo controlled trial of 
14 
Lactobacillus reuteri DSM 17938 in children aged 2-24 months.  The study population is 
15 
based in a community with a registered clinical trial center in rural Peru where this 
16 
protocol can be done in a highly controlled setting.   The study is intended to enroll 
17 
healthy children living in a resource constrained setting where chronic undernutrition 
18 
and diarrheal disease is common, as this is the population that is most likely to receive 
19 
benefit from the use of this and or other probiotic products for the treatment of acute 
20 
infectious diarrhea and environmental enteropathy. 
21 
 
22 
Up to 100 subjects will be enrolled in order to obtain the randomization of 60 subjects, 
23 
who will receive study product or placebo at a ratio of 2:1.  Participants will be enrolled 
24 
between the ages of 8 weeks and 24 months.  There will be no age stratification.  
25 
Participants will be screened with a baseline history and physical examination and 
26 
laboratory tests to determine the HIV status of the child, as well baseline laboratory 
27 
tests to evaluate occult infections, hepatic function, and renal function.  Upon 
28 
randomization subjects will be screened and randomized to a treatment arm.  Product 
29 
will be administered under the direct supervision of study staff daily for 5 days. The 
30 
primary outcome is the safety and tolerability of the product at this dose and formulation.  
31 
Safety will be assessed by the monitoring of baseline laboratory tests at 5 and 28 days 
32 
following the initiation of therapy and by AE monitoring.  Tolerability will be evaluated by 
33 
analyzing 28 day diary cards that are distributed to all randomized subjects on which 
34 
parents record their child’s  symptoms  (specifically fever, anorexia/oral intake, vomiting, 
35 
diarrhea, irritability, rash, wheezing, or open fields which allows them to describe any 
36 
issue their child may experience and grade its severity) .   Secondary outcomes include:  
37 
1) The assessment of the duration of shedding of Lactobacillus reuteri (Lr) strain DSM 
38 
17938 following administration.  This will be determined by and endpoint PCR assay for 
39 
L. reuteri done on stool collected on D3, D5, D12, D15, D18, D24, D28, D36 on all 
40 
participants. 
41 
 
42 
The planned enrollment period is four months and the participation of individual subjects 
43 
is for 3 months.  The study should be completed in 7 months.  Safety monitoring by 
44 
treatment group, i.e., infants assigned to active or placebo study product will be 
45 
conducted by NIDDK appointed DSMB under the direction of the NIDDK Program 
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Official. 
1 
 
2 
 
3 
 
4 

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1 

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1 
 
2 
5 
STUDY ENROLLMENT AND WITHDRAWAL
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1 
5.1 
Subject Inclusion Criteria 
2 
 
3 
 
4 
In order to be eligible to participate in this study, a subject must meet all of the following 
5 
criteria: 
6 
• 
Have a parental permission form signed by both parents 
7 
• 
Be between  8 weeks to 24 months of age with no preexisting exclusion criteria 
8 
• 
Have parents who are willing to comply with all planned study procedures and be 
9 
available for planned study visits for 3 months. 
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1 
5.2 
Subject Exclusion Criteria 
2 
 
3 
1) No enrollment of family members in households where any of the following are 
4 
present: 
5 
 
6 
a.  Another study participant in the household  
7 
 
8 
 
9 
b.  Presence of immune suppressed individuals or use of immunosuppressive 
10 
agents (including but not limited to corticosteroids, methotrexate, etc.) by any 
11 
household member 
12 
 
13 
c.  Presence of a serious congenital anomaly or chronic medical condition that in 
14 
the opinion of the investigators would contraindicate participation in any household 
15 
member, including history of gastrointestinal surgery, chronic gastrointestinal 
16 
illness, abnormal intestinal anatomy, or abnormal bowel functionality 
17 
 
18 
The following risk factors are at the level of the individual child: 
19 
 
20 
2)  
Allergy to penicillin, cephalosporins, clindamycin or gentamicin 
21 
3) 
History of antibiotic use in the last 30 days 
22 
4) 
Use of probiotic products within the past 30 days, including masato (local 
23 
product with fermenting bacteria) and yogurt products containing live 
24 
bacterial cultures. 
25 
5)   
History of diarrheal illness within the past 30 days (See definition in Protocol 
26 
Appendix B) 
27 
6)  
Presence of fever or a pre-existing adverse event monitored in the study 
28 
(See Protocol Appendix B Definitions of AEs for specific adverse events 
29 
monitored in the study) 
30 
7)  
Positive results on serum diagnostic tests for antibodies to HIV. 
31 
 
32 
 
33 
8) 
Presence of severe anemia, defined as serum hemoglobin < 7 gm/dL 
34 
 
35 
9) 
Out of range laboratory values for total leucocyte count, BUN, Creatinine, 
36 
AST, ALT, and total bilirubin monitored as potential adverse events, as 
37 
described in Appendix E.   
38 
10) 
Pre-enrollment stool sample (collected within 14 days of day 1 of the study) 
39 
is positive for L. reuteri by PCR. 
40 

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1 
5.3 
Strategies for Recruitment and Retention 
2 
 
3 
The community where recruitment will occur has been a research site for the current 
4 
group for the last fifteen years.  During this time we have had successful recruitment 
5 
and retention for intensive birth cohort studies and have recruited and trained nurses 
6 
and health care workers and a study team from the area.  The community is 
7 
experienced in research participation which aids recruitment and retention of study 
8 
subjects.9,36-42 
9 
 
10 
5.4 
Treatment Assignment Procedures 
11 
 
12 
Treatment assignment is based on the order in which subjects complete screening and 
13 
are assigned a study code.  This study code is the same as the treatment vial ID 
14 
number.  
15 
 
16 
5.4.1 
Randomization Procedures 
17 
 
18 
Randomization.  Upon satisfactory completion of screening, participants will be assigned 
19 
a study code (different from the PID that accompanied screening data) that is associated 
20 
with a list completed by the project biostatistician and shared with the individual at 
21 
BioGaia preparing the treatment vials.  This sequential code is associated with treatment 
22 
vials randomized to one of two treatment groups (T or P) on a 2:1 (T:P) basis with the first 
23 
randomized subject receiving the first study vial, etc.   All vials are identical (see labelling 
24 
section below) and the Treatment ID on the vial matches the study code number. 
25 
 
26 
5.4.2 
Masking Procedures 
27 
 
28 
This is a double masked trial.  Only the project biostatistician has the table linking 
29 
treatment assignment to randomization code.  Unmasking of the rest of the study team 
30 
will occur only when the database is locked for final analysis or if a clinical situation 
31 
arises in which the PI feels unmasking is necessary for the treatment of a SAE. 
32 
 
33 
 
34 
5.4.3 
Reasons for Withdrawal 
35 
 
36 
A study subject will be discontinued from participation in the study prior to completing 5 
37 
days of study product administration if: 
38 
 
39 
 
40 
A subject may be discontinued from the study for the following medical or 
41 
administrative reasons: 
42 

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1) Occurrence of an AE, which in the judgment of the investigator suggests an 
1 
unacceptable risk to the subject to receive a test product (The investigator will 
2 
follow the subject until satisfactory resolution of the AE or the AE is determined 
3 
to be stable.)  A likely example in this community might be the development of 
4 
severe falciparum malaria during the product administration period. 
5 
2) Development of ANY condition fulfilling one of the exclusion criteria (except for 
6 
out of range laboratory values); 
7 
3) Subject/parental request. 
8 
 
9 
 
10 
5.4.4 
Handling of Withdrawals 
11 
 
12 
 
13 
The study is voluntary and allows for the withdrawal of participation at any point in the 
14 
study.  Despite this, we have a very low attrition rate in our studies.  In general 
15 
withdrawal from the study is partial- most frequently related to blood draws.  In all 
16 
cases, the reason for the withdrawal of consent will be discussed at length with 
17 
parents.  We will ask that the parents of the participant to continue to allow visits to 
18 
monitor vital signs and illness history at the least.  In most cases our experience 
19 
informs us that parents will allow stool collection to continue.  Blood draws are the most 
20 
problematic- we will allow the family to opt for a fingerstick sample if the venous blood 
21 
draw experience is that which is underlying the withdrawal.  We also would allow for 
22 
extended windows (i.e., blood draw at 35 days rather than 28 if the parent feels more 
23 
time between blood draws would allow for their continued complete monitoring, 
24 
although in this case a protocol deviation form would be included with the values.  In 
25 
short, every attempt will be made to obtain the most complete safety and tolerability 
26 
data possible while respecting parental autonomy and their right to cease participation 
27 
in a voluntary study.  No replacement of subjects is planned, and we expect the 
28 
number of consent withdrawals and missed study visits or missed study procedures to 
29 
be low. 
30 
 
31 
5.4.5 
Termination of Study 
32 
 
33 
 
34 
This study may be prematurely terminated if, in the opinion of the PI or sponsor there is 
35 
sufficient reasonable cause for termination. An example of a reason underlying 
36 
termination would be NIDDK concurrence with DSMB recommendation to end study 
37 
early, new findings that alter the safety profile of the product, discontinuation of study 
38 
product for active and matching placebo production or impending market 
39 
unavailability, the discovery of important protocol violations that may undermine the 
40 
integrity and conclusions of the study.   
41 
If the study is prematurely terminated or suspended, the sponsor will promptly inform 
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the investigators/institutions so that the investigators can inform the U.S. and Peruvian 
1 
regulatory authority(ies) of the termination or suspension and communicate the 
2 
reason(s) for the termination or suspension. The IRB/IEC will also be informed 
3 
promptly and provided the reason(s) for the termination or suspension by the sponsor 
4 
or by the investigator/institution. 
5 
 
6 

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1 
 
2 
6 
STUDY INTERVENTION/INVESTIGATIONAL PRODUCT 
3 
 
4 
 
5 
6.1 
Study Product Description 
6 
 
7 
 
8 
6.1.1 
Acquisition 
9 
The product will be obtained from BioGaia (Stockholm,Sweden).  It is likely that a single 
10 
shipment will be sufficient to cover the needs of the phase 1 infant trial as both product 
11 
and placebo have a shelf life of 18 months.  If unanticipated circumstances delay the start 
12 
of  phase 1 infant trial , a repeat shipment will be requested no later than 4 months prior to 
13 
the expiration date of the product due to expire.  It is possible that subsidiaries or agencies 
14 
contracted by BioGaia packaging and preparation of product or placebo.  
15 
 
16 
6.1.2 
Formulation, Packaging, and Labeling 
17 
 
18 
Formulation of test product and placebo are the following: 
19 
 
20 
1. Active Product:  Description Lactobacillus reuteri  17938 suspended in sunflower oil, 
21 
medium chain triglyceride oil, silicone dioxide.  Total viable count of L. reuteri  17938 1 x 
22 
108 CFU/5 drops.  Study products supplied generally contain 2-8 x 108 CFU/5 drops from 
23 
the manufacturer, in order to guarantee a potency of at least 1 x 108 CFU/5 drops by the 
24 
product expiration date. 
25 
2.  Placebo:  Composition Sunflower oil, medium chain triglyceride oil and silicon dioxide.  
26 
Total viable count of L. reuteri is zero CFU/ 5 drops. 
27 
 
28 
The product is packaged in a single multidose amber glass vial per participant at a volume 
29 
several times that required for the 5 day dose.  The individual vials are packed in 
30 
reinforced boxes for shipping with a temperature logging. 
31 
 
32 
The label contains the following information 
33 
Name of Study: Safety of Lactobacillus reuteri in healthy children ages 2-24 months in 
34 
Peru 
35 
BioGaia Drops  Dose: 5 drops per dose   Store at 2-8C  Shake well before using 
36 
Test article code: xxxxxxx  Discard 10 days after opening  Exp date:  yyyy  Batch No:  
37 
zzzz 
38 
Responsible MD: Margaret Kosek, Johns Hopkins University 
39 
Manufactured for BioGaia AB, Stockholm Sweden 
40 
Caution: New Drug- Limited to investigational use 
41 
 
42 
 
43 
6.1.3 
Product Storage and Stability 
44 

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1 
The study product is to be stored at 2-8ºC in multidose subject specific amber vials.   
2 
 
3 
Once opened, the contents of the vial should be dispensed in 10 days. 
4 
 
5 
The product is administered in the clinical trial center by a member of the study staff in 
6 
a designated product administration space.  This space is adjacent to the dedicated 
7 
product storage space in the clinic.  The dosing is done once daily for 5 days and 
8 
programmed based on participant and staff convenience.  Details of product 
9 
prescription and vial treatment confirmation upon each dose administration are 
10 
described in the Manual of Procedures. 
11 
 
12 
6.2 
Dosage, Preparation and Administration of Study 
13 
Intervention/Investigational Product 
14 
 
15 
The individual treatment vial is prepared and labelled by BioGaia.  No reconstitution or 
16 
dilution is needed.  The vial will be inverted gently 5 times to assure homogenization and 
17 
five drops of the suspension will be administered orally to the study subject by study staff 
18 
that is blinded as to the treatment status of the individual.
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1 
6.3 
Modification of Study Intervention/Investigational Product for a 
2 
Subject 
3 
If the child vomits or spits up within 20 minutes of administration, the dose will be re-
4 
administered.  If a subject has a SAE during the 5 day treatment window or if the child 
5 
has blood cultures that are positive for L. reuteri product administration will be 
6 
discontinued in that individual.  All safety and planned protocol follow-up will continue. 
7 
 
8 
6.4 
Accountability Procedures for the Study 
9 
Intervention/Investigational Product(s) 
10 
The study PI is accountable for the investigational product and the appropriate conduct 
11 
of the clinical trial in compliance with ICH GCP guidelines and Peruvian laws and 
12 
regulations. 
13 
Details on product accountability, including initial certificate of analysis, product 
14 
accountability including the documentation of international cold chain transport and 
15 
evaluation of product viability will be held onsite for evaluation by external monitors.  
16 
The trial product will be shipped from the provider BioGaia and or suppliers by World 
17 
Courier to Peru with temperature monitoring.  Procedures for the temperature 
18 
controlled movement and tracking of study product between the laboratory and the 
19 
clinic is outlined in the MOP.   Tests for product viability and the detection of possible 
20 
contaminants are detailed in laboratory SOPs and detailed in the product assurance 
21 
plan.  At the end of the study all unused product will be autoclaved prior to disposal.   
22 
 
23 
6.5 
Assessment of Subject Compliance with Study 
24 
Intervention/Investigational Product 
25 
   All treatments are directly administered by study staff.  Missed dosages will be 
26 
documented.  In the case that a subject is not available on a study day a 1 day window will 
27 
be tolerated for the administration of a single missing dose.  The daily dose is restricted to 
28 
a single dose per day, so in this case, the dosing interval would occur over a 6 day period.  
29 
Follow-up schedule would not be altered from the programmed plan.  No more than a 
30 
single dose will be given this window- if a second dose were to be missed, it would be 
31 
documented in a protocol deviation form and analyzed per group treatment assignment.  
32 
Subsequent doses are done at 24 hour intervals from the last dose, but visit scheduling 
33 
cannot be altered after the first dose is administered. 
34 
 
35 
6.6 
Concomitant Medications/Treatments 
36 
 
37 
 
38 
All concomitant medications, including herbal or natural therapies are documented at 
39 

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baseline or that are taken by the subject during the follow-up period will be documented 
1 
on CRFs.  Information on concomitant medications is solicited at baseline, upon each 
2 
dosage with study product (D1-D5), D8, D12, D15, D18, D24, and D28. 
3 
 
4 
Treatment for sepsis will be standard weight based doses of ceftriaxone and gentamicin.  
5 
If the project clinician suspects that the product is a likely source of the sepsis (no source 
6 
of lower respiratory tract infection, urinary tract infection, or diarrheal illness noted) 
7 
clindamycin will be added to the treatment.  Treatment guidelines are reviewed with study 
8 
physicians and treatment is available on site to facilitate the rapid treatment of sepsis.
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1 
 
2 
7 
STUDY SCHEDULE 
3 
 
4 
7.1 
Recruitment process 
5 
 
6 
 
7 
A town meeting will be held to explain the research project to the community.  Volunteers 
8 
will be registered at this meeting or by contacting community health workers.  A consent 
9 
form, approved by the institutional review boards of Tulane, Johns Hopkins School of 
10 
Public Health (JHSPH), and AB PRISMA as well as the Peru Instituto Nacional de Salud 
11 
(INS; Peruvian government agency responsible for regulating clinical research) and 
12 
written at the 8th grade level will be read aloud in Spanish to parents of potential 
13 
participants after an informal explanation of the project by CITI trained study staff as per 
14 
GCP guidelines.  Parents of potential participants who agree to participate will then be 
15 
asked to a private consent meeting where the process is repeated and they are given 
16 
individualized attention in an environment that allows them to ask questions more freely 
17 
by study staff, all of whom are CITI certified and have experience in conducting research 
18 
in this community.  They will then be asked to sign the form and be given a copy of the 
19 
consent form with information that allows them to contact the Principal Investigator and 
20 
the director of the IRB at AB PRISMA should the need arise prior to or during their child’s 
21 
participation in the study. 
22 
 
23 
 
24 
7.2 
Consent  Process and Documentation 
25 
 
26 
Consent will be obtained from CITI certified employees of PRISMA with experience in 
27 
biomedical research and community based research.  Both parents must sign the consent 
28 
form for children of this age to participate in clinical trials in Peru.  The discussion will 
29 
begin in a public forum where interested parties are invited by public loudspeaker to a 
30 
community center.  Individuals who retain interest in having their child participate after this 
31 
forum will be invited to the study center where the process will be repeated in a private 
32 
setting. 
33 
 
34 
The study involves young children.  To ensure the parental permission to have the child 
35 
participate is valid, the study will be explained in detail to ensure that parents understand 
36 
the risks and benefits for their particular child.  The study team has extensive experience 
37 
working with young children and their parents and the stress of study participation to the 
38 
child participants will be minimized in part by clinical rapport gained from this experience.  
39 
 
40 
A standard signed consent document written in Spanish at the 8th grade reading level will 
41 
be used.  Children of guardians lacking signatory capacity will not be allowed to 
42 
participate. 
43 
 
44 
7.3 
Screening 
45 

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1 
1) The screening period may take up to 14 days, all days are categorized as D0 (day 0).  
2 
repeat measures of biochemistry and hematology will be allowed in a one month time 
3 
frame. 
4 
 
5 
2) If the participant meets all criteria that do not require blood tests and both parents sign 
6 
the informed consent, basic demographic data and clinical data, including use of 
7 
medications and use of herbal or homeopathic treatments, will be recorded.  A stool 
8 
specimen will then be collected to test for presence of L. reuteri by PCR per Laboratory 
9 
SOP (xxx).   Potential participants must not currently be experiencing any of the monitored 
10 
clinical adverse events as specified in Protocol Appendix C (Clinical adverse events to be 
11 
monitored).  
12 
 
13 
3) Following this step, blood will be collected for baseline (day 0) CBC, AST, ALT, total 
14 
bilirubin, creatinine and BUN. Blood collection may be done by fingerstick, heelstick, or 
15 
venipuncture, with method based on subject and parent/guardian preference and 
16 
technical considerations.  A total volume of 1.5 milliliters of blood is the minimum required 
17 
volume to perform all of the assays listed above and a sample volume of 3.0 ml is the goal 
18 
to ensure specimen adequacy for primary safety outcomes. Due to the time required for 
19 
testing, reporting, and review of stool PCR and lab tests that would potentially exclude a 
20 
participant, results of stool PCR and blood tests listed above from specimens collected 
21 
within 14 days of initiating study product will be used as Day 0 blood and stool results.  
22 
Out of range laboratory values detected by Day 0 blood test results will be documented on 
23 
a CRF, and potential subjects will be excluded if an out of range laboratory value for tests 
24 
monitored as potential adverse events is found, as described in Toxicity Table (Protocol 
25 
Appendix E, based on Day 0 blood test results.  However, subjects will only be excluded 
26 
based on serum hemoglobin if they meet exclusion criterion 8. 
27 
 
28 
4) Participants with no laboratory exclusion criteria (from L reuteri stool PCR, HIV, 
29 
leucocyte count, severe anemia, AST, ALT, T. bili, BUN, creatinine) will have a 
30 
standardized history and physical examination performed by a physician.  Results of 
31 
serum hemoglobin (exclusion criterion #8) and HIV serum antibody (exclusion criterion #7) 
32 
will be reported as promptly as possible, usually within 48 hours, to determine if 
33 
participants meet these exclusion criteria.  If the stool specimen is positive for L. reuteri, 
34 
the child will be excluded from the study (exclusion criterion #10).  Subjects with negative 
35 
stool PCR results for L. reuteri, negative HIV serology and adequate hemoglobin levels 
36 
(per inclusion/exclusion criteria) will progress to the initial medical examination.   The 
37 
screening will take place in a nationally certified (INS- certified) clinical trial center in the 
38 
community of Santa Clara. Subjects with positive results on HIV serology or inadequate 
39 
hemoglobin levels will receive appropriate counseling and referral to medical services 
40 
available in the community for patients with these conditions.  HIV seropositive children 
41 
will be referred to the only HIV referral (Hospital Regional de Loreto) directly by the study 
42 
MD.   
43 
 
44 
 
5) Subjects not excluded on the basis of laboratory tests will receive a baseline 
45 
medical exam. A baseline assessment for presence of symptoms monitored for as AEs 
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will be done on Day 1 prior to randomization and receipt of initial dosing of study product.  
1 
Subjects who have developed an exclusionary clinical adverse event since onset of the 
2 
screening process (e.g. diarrhea, bronchospasm) will not be allowed to be randomized, 
3 
but may be re-evaluated for inclusion at a later date by restarting the screening process 
4 
for a period of thirty days following the onset of the AE.   
5 
 
6 
7.4 
Product  Administration period 
7 
 
8 
On days 1-5 participants will attend the study clinic to receive study product. The health 
9 
worker will administer LR or PL and this treatment will be continued once daily for a total 
10 
of five days.  Participants will be monitored for 20 minutes.  Any dose vomited within 20 
11 
minutes of administration will be repeated.  Vital signs are monitored and symptoms are 
12 
recorded. 
13 
 
14 
Study participants will have daily visit at the community-based study clinic on study days 
15 
1-5 to detect symptoms, adverse events, and to administer the test article (LR or PL) 
16 
under directly observed therapy.  Parents of subjects will be instructed to in the use of a 
17 
diary card, provided by the study personnel, to record any symptoms the infant 
18 
experienced during the treatment phase and the first 28 day follow-up period.   Parents of 
19 
subjects will be instructed to record both solicited AEs (clinical symptoms listed as 
20 
monitored AEs in Protocol Appendices C and E below) and unsolicited AEs on the diary 
21 
cards. Field workers will verify information recorded, determine and document the severity 
22 
of each AE recorded on the form, and assist with completing the card as needed. In the 
23 
event that the AEs in question are assessed to be of a more severe intensity than what is 
24 
recorded by the subject’s parents, the diary card may be revised to reflect this 
25 
change.  However, field workers will not “downgrade” any solicited or unsolicited adverse 
26 
events recorded by the parents.  Parents of subjects will be instructed to contact the field 
27 
worker or study physician immediately if the enrolled child develops fever or becomes ill.  
28 
Subjects with AEs will be referred to the study physician, who evaluate the subject, 
29 
generate an AE report, and make an initial determination of association with study product 
30 
or not.  AEs not listed in Appendices C, D, and E will be reported but not graded for 
31 
severity.   
32 
  
33 
On day 5, when the final dose is administered, study personnel will collect a 3 ml. blood 
34 
sample for CBC, AST, ALT, total bilirubin, creatinine, and BUN.  Out of range laboratory 
35 
values will be documented on the appropriate CRFs for laboratory values.   
36 
 
37 
Temperature will be documented daily while receiving study intervention, and participants 
38 
with new onset of Grade 3 or 4 fever following initiation of intervention therapy (defined as 
39 
oral, axillary, or temporal temperature > 39.0 degrees C) and participants who appear 
40 
especially ill or toxic to the field worker will have a physician evaluation.  In these cases, 
41 
blood will be examined in for malaria parasites by thick smear and a blood culture will be 
42 
performed.   In the event of positive blood cultures, presumptive antibiotic treatment and 
43 
hospitalization (if deemed necessary by the attending physician) will be facilitated by 
44 
project personnel.  Antibiotic therapy will be the standard pediatric doses of Ceftriaxone 
45 
and Gentamicin (for common causes of bacteremia from urinary and pulmonary sources) 
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with the addition of clindamycin if the study product is the suspected origin. Further details 
1 
on procedures for evaluating and empirically treating subjects with fever are found in the 
2 
section on Adverse Events, Bacteremia.  Serious Adverse Events (SAEs) are defined in 
3 
Section 9.2.2, and any participant who develops an SAE, regardless of cause, will 
4 
discontinue receiving study product if this is still in progress, but will continue with study 
5 
visits, data collection, and safety monitoring for the 3 month follow-up period of the study.   
6 
 
7 
In order to compensate for the time and inconvenience of multiple visits, upon completing 
8 
enrollment screening we will give participants an improved water storage container and a 
9 
small food supply basket (containing milk, rice, oil, and tuna) and a small toy for your 
10 
child.  Following the 5th dose of study product we will offer  another toy for their child.  On 
11 
study day 28 we will give a second food basket and a third toy that their child can choose 
12 
for successful completion of the study. 
13 

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1 
7.5 
Follow-up 
2 
 
3 
A.  First 28 Days.  The first 28 day follow-up period includes intense monitoring for 
4 
adverse effects, starting on the day when the first dose of probiotic or placebo is 
5 
administered (day 0), and continuing past the end of the treatment phase on day 5 and 
6 
ending on day 36, or 36 days after the first dose was administered.  Symptom data for AE 
7 
monitoring will be completed daily by the participant’s parent or guardian for 6 weeks and 
8 
verified by project staff during home visits on days 8, 12, 15, 18, 24, and 28.  Information 
9 
on symptoms and medication use will be recorded by the parents of participant on diary 
10 
cards provided by the study.  Temperature will be recorded on these visits by project staff.  
11 
Diary cards used will be similar to those used in the Phase One study for children ages 2-
12 
5 years (NCT020124122).  On day 28, field workers will collect diary cards and document 
13 
data on clinical symptoms, vital signs, and repeat blood tests (as on day 5).    
14 
 
15 
Subjects with fever will be referred to the study physician for a full physical exam and 
16 
collection of specimens for fever evaluation (malaria smear, blood culture).  Subjects with 
17 
serum hemoglobin less than 11 gm/dL at baseline or at day 5 (or Hb less than 9.5 gm/dl at 
18 
baseline or at day 5 for infants 5 months of age or younger) will be treated with iron 
19 
supplementation in the form of iron sulfate drops or chewable iron tablets, according to 
20 
American Academy of Pediatrics guidelines [Clinical Report—Diagnosis and Prevention of 
21 
Iron Deficiency and Iron-Deficiency Anemia in Infants and Young Children (0–3 Years of 
22 
Age) Baker RD, Greer FR, The AAP committee on Nutrition; Pediatr 126 (5):  1-11, 2010] 
23 
(PMID:20923825).  When indicated, iron supplementation will be provided starting at least 
24 
5 days after the last dose of study product to prevent confounding the assessment of 
25 
study related adverse events with the common side effects of supplemental iron therapy 
26 
(gastrointestinal upset, etc), an administration of iron supplementation will be controlled for 
27 
in analysis of adverse event data (clinical and laboratory test outcomes by study group at 
28 
days 5 and 28).   Treatment will consist of an oral liquid formulation dosed at 3-6 mg 
29 
elemental iron/kg/24 hours.  Iron supplementation will be continued for 2 months after 
30 
initiation, and serum hemoglobin will be measured using blood collected by fingerstick 
31 
tested in a HemoCue device at the end of the 2 months treatment period.  Children with 
32 
serum hemoglobin less than 11 gm/dL at this 2 month point (or less than 9.5 gm/dl at 
33 
baseline or at day 5 for infants 5 months of age or younger) will be referred to a local 
34 
pediatrician for further evaluation and individualized treatment if needed.  Individuals with 
35 
helminth infection will be treated with albendazole therapy based on cumulative testing 
36 
results on study day 36.  For ascaris and hookworm this consists of one dose of 
37 
albendazole, whereas doses will be given for 3 days for trichuris and strongyloides 
38 
infections. 
39 
 
40 
Stool samples will be obtained on day 3, 5, 12, 15, 18, 24, 28, and 36 for the presence of 
41 
Lactobacillus reuteri by PCR. 
42 
 
43 
B.  Day 28 to 3 month endpoint.  After day 28, parents of the subjects will be instructed 
44 
to report all episodes of febrile illness occurring during the 3 month follow-up period, and 
45 
to present to the study clinic for fever evaluation whenever the parents of the subject 
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suspects they are febrile or a new febrile episode is documented.  Febrile episodes 
1 
detected by participant presentation during the follow-up period will be handled in the 
2 
same way as febrile episodes detected by active surveillance during the treatment phase.   
3 
Study personnel will contact parents of participants 3 months after starting study treatment 
4 
to document new onset of AEs and SAEs not previously recorded, onset of new medical 
5 
conditions, hospitalizations, and use of new medications and herbal or homeopathic 
6 
treatments not previously documented.   
7 
 
8 
In all, 13 study contacts are expected with study participants (See Appendix A).  The study 
9 
is expected to be conducted in a 7 month period. 
10 
 
11 
 
12 
7.6 
Final Study Visit 
13 
 
14 
On the final visit, the CRF for study completion or non-continuation form is filled out to 
15 
allow for CRF and laboratory specimen form missingness to be tracked.
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1 
 
2 
7.7 
Early Termination Visit 
3 
 
4 
All subjects who do not complete the full 3 months of study participation for any reason 
5 
will have a termination visit that documents the reason and date of non-continuation with 
6 
all study procedures.  If the participant agrees to continue with study visits, data 
7 
collection, and partial study tests or procedures (ie no blood draws) this is documented 
8 
only on protocol deviation forms for the procedures that the participants parent 
9 
specifically decline. 
10 
 
11 
7.8 
Unscheduled Visit 
12 
 
13 
Unscheduled visits to the clinic will be documented on an AE form.  This would both 
14 
document encounters initiated by the patient coming to the clinic, or the laboratory 
15 
seeking out the patient because of an unexpected laboratory finding (positive malaria 
16 
smear, for example). 
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1 
 
2 
8 
STUDY PROCEDURES/EVALUATIONS 
3 
 
4 
 
5 
8.1 
Clinical Evaluations 
6 
Clinical evaluations include a standardized medical history and physical 
7 
examconducted by a physician in an interview as part of the screening process.  No 
8 
personal health records will be sought and medical history will be based on parental 
9 
interview.  A standardized physical examination will also be done that will include 
10 
length, weight, temperature, heart rate, respiratory rate, ENT exam, cardiac, 
11 
pulmonary, and abdominal exams at the time of screening only.  Targeted physical 
12 
examinations will be done with all AE investigations, and vital signs (temperature, 
13 
pulse, respiratory rate) reported in each instance. 
14 
 
15 
8.2 
Laboratory Evaluations 
16 
 
17 
8.2.1 
Clinical Laboratory Evaluations
18 

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• 
Hematology: hemoglobin, white blood cells (WBC) with differential count, 
1 
platelet count done during screening and on D5 and D28. 
2 
• 
Biochemistry: creatinine, blood urea nitrogen, total bilirubin, alanine 
3 
aminotransferase (ALT), aspartate aminotransferase (AST) done on screening and 
4 
D5 and D28. 
5 
• 
Immunology: HIV test to be done as part of screening process. 
6 
 
7 
8.2.2 
Special Assays or Procedures 
8 
 
9 
Quantification of viable count of probiotic will be done per existing laboratory SOP.  
10 
Detection of Lactobacillus reuteri will be done following DNA extraction with a soil MoBio 
11 
kit with bead-beating per existing laboratory protocol.  Specimen reception, barcoding and 
12 
handling will be done as per laboratory SOP.  Sample preparation for international 
13 
shipping will be done by IATA certified staff and shipped under relevant DHHS CDC 
14 
permits and in compliance with national and international laws. 
15 
. 
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1 
 
2 
9 
ASSESSMENT OF SAFETY 
3 
 
4 
9.1 
Specification of Safety Parameters 
5 
 
6 
1.  Evidence of invasive infection resulting from the administration of L. reuteri DSM 
7 
17938.  Baseline and daily temperature measurements will be made during the study 
8 
period. Complete hemogram with differential count obtained at baseline, on day 5, and on 
9 
day 28.  Vital signs will be monitored daily during the administration of the treatment (days 
10 
1-5).  The evaluation of all febrile episodes will be done and include a physician exam and 
11 
the evaluation of blood smears for malaria and blood cultures for the evaluation of possible 
12 
bacteremia. 
13 
 
14 
2.  Clinical tolerance.  The inclusion of a placebo group will allow for comparisons of daily 
15 
symptom measurements to be compared between the treatment and placebo group.  The 
16 
symptoms will be monitored for at household visits, and will be graded as adverse events 
17 
according to defined criteria.   
18 
 
19 
Symptoms monitored as AEs include fever, vomiting, diarrhea, irritability, rash, pruritis, 
20 
and wheezing or brochospasm.  Diagnostic microbiology will be performed on all stool 
21 
specimens from subjects who develop diarrhea during the 90 days following initiation of 
22 
therapy.   
23 
 
24 
3.  Evidence of toxicity resulting from the L. reuteri DSM 17938 preparation. 
25 
Participants will have baseline, day 5, and day 28 assessment of hematologic status and 
26 
renal and hepatic function.  Assessment of renal function will include blood urea nitrogen 
27 
(BUN) and creatinine (Cr).  Assessment of hepatic toxicity will include transaminases 
28 
(AST, ALT) and total bilirubin.  Hematologic status will be determined by CBC to measure 
29 
hemoglobin and absolute leucocyte count. 
30 
 
31 
 
32 
9.2 
Methods and Timing for Assessing, Recording, and Analyzing 
33 
Safety Parameters 
34 
 
35 
9.2.1 
Adverse Events 
36 
 
37 
Adverse Event:  ICH E6 defines an AE as any untoward medical occurrence in a 
38 
patient or clinical investigation subject administered a pharmaceutical product 
39 
regardless of its causal relationship to the study treatment. An AE can therefore be any 
40 
unfavorable and unintended sign (including an abnormal laboratory finding), symptom, 
41 
or disease temporally associated with the use of study product including active or 
42 
placebo. 
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1 
 
2 
According to the U.S. DHHS Common Rule an AE does include any: 
3 
• 
exacerbation of a pre-existing illness, such as asthma. 
4 
• 
increase in frequency or intensity of a pre-existing episodic event or condition. 
5 
• 
condition detected or diagnosed after study product administration even though 
6 
it may have been present prior to the start of the study. 
7 
• 
continuous persistent disease or symptoms present at baseline that worsen 
8 
following the start of the study. 
9 
• 
symptoms associated with disease not previously reported by the subject will be 
10 
recorded as an AE. 
11 
 
12 
According to the U.S. DHHS Common Rule 
13 
(http://www.hhs.gov/ohrp/policy/advevntguid.html) an AE does not include a/an:   
14 
• 
medical or surgical procedure (eg, surgery, circumcision, transfusion); the 
15 
condition that leads to the procedure is an AE. 
16 
• 
pre-existing diseases or conditions present or detected at the start of the study 
17 
that do not worsen. 
18 
• 
situations where an untoward medical occurrence has not occurred (eg, 
19 
hospitalization for elective surgery, social and/or convenience admissions). 
20 
 
21 
All AEs including local and systemic reactions not meeting the criteria for “serious 
22 
adverse events” (See Section 9.2.2) will be captured on the ADVERSE EVENT CRF.  
23 
Information to be collected includes event description, time of onset, clinician’s (MD) 
24 
assessment of severity, relationship to study product and time of 
25 
resolution/stabilization of the event.  All AEs occurring while on study product or within 
26 
90 days of follow-up must be documented appropriately regardless of relationship to the 
27 
study product.  All AEs will be followed to adequate resolution. 
28 
 
29 
Any medical condition that is present at the time that the subject is screened will be 
30 
considered as baseline and not reported as an AE. However, if it deteriorates at any 
31 
time during the study, it will be recorded as an AE. 
32 
 
33 
Specific AEs that will be monitored in this project: 
34 
 
35 
During probiotic therapy, study field workers will monitor for other potential adverse effects 
36 
during their home visits, based on parental interview and information recorded on a study 
37 
log.  Expected AEs that are common in this study population will include fever, 
38 
nausea/upset stomach, vomiting, diarrhea, irritability, rash, pruritis, and wheezing or 
39 
bronchospasm.  Diarrhea episodes will be characterized as watery or dysenteric.  Space 
40 
will also be provided for the open end documentation of any other symptom which could 
41 

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potentially be related to probiotic use.  Appendix B and D details expected AEs and how 
1 
they will be graded by severity.   Grading of AEs will be confirmed by the project 
2 
physician.  AEs not listed as Expected AEs in the Toxicity Table will be reported but not 
3 
graded for severity.  Responses to AEs encountered will be at the discretion of the project 
4 
physician and study monitor, based on type of AE encountered and its severity.  
5 
Severity of Event:  Expected AEs will be assessed by the clinician using a protocol 
6 
defined grading system.  For events not included in the protocol defined grading 
7 
system, the following guidelines will be used to quantify intensity. 
8 
 
9 
Level 1 = Mild (awareness of a symptom but the symptom is easily tolerated) 
10 
 
11 
Level 2 = Moderate (discomfort enough to cause interference with usual activity) 
12 
 
13 
Level 3 = Severe (incapacitating; unable to perform usual activities; requires change in 
14 
intensity of caregiver activity or bed rest) 
15 
 
16 
Level 4 =   Life threatening 
17 
 
18 
Changes in the severity of an AE will be documented to allow an assessment of the 
19 
duration of the event at each level of intensity to be performed. Adverse events 
20 
characterized as intermittent require documentation of onset and duration of each 
21 
episode. 
22 
             
23 
Method, Frequency, and Time Period for Detecting AEs  
24 
At each visit, after the parent of the subject has had an opportunity to spontaneously 
25 
mention any problems, the investigator will inquire about AEs by asking the following 
26 
standard questions of the parent: 
27 
"Has your child had any (other) medical problems since your last visit/assessment?" 
28 
"Has your child taken any new medicines, other than those given to you in this study, 
29 
since your last visit/assessment?" 
30 
 
31 
Relationship to Study Products:  AEs will be assessed for relationship to study product. 
32 
The clinician’s assessment of an AE's relationship to test article (study drug) is part of 
33 
the documentation process, but it is not a factor in determining what is or is not reported 
34 
in the study.  If there is any doubt as to whether a clinical observation is an AE, the event 
35 
will be reported. All AEs must have their relationship to study product assessed using 
36 
the terms: associated or not associated. 
37 
 
38 
In a clinical trial, the study product must always be suspect. To help assess, the 
39 
following guidelines will be used. 
40 
• 
Associated – The event is temporally related to the administration of the study 
41 

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product and no other etiology explains the event. 
1 
• 
Not Associated – The event is temporally independent of study product and/or the 
2 
event appears to be explained by another etiology. 
3 
 
4 
 
5 
9.2.2 
Serious Adverse Events (SAE) 
6 
 
7 
Definition of a Serious Adverse Experience (SAE): 
8 
 
9 
Serious adverse event (SAE):  A Serious Adverse Event is defined as an AE meeting one 
10 
of the following conditions 
11 
 
12 
• Death during the 3 month period of protocol defined surveillance 
13 
 
14 
• Life Threatening Event (defined as a subject at immediate risk of death at the time 
15 
of the event) 
16 
 
17 
• An event requiring inpatient hospitalization or prolongation of existing 
18 
hospitalization during the period of protocol defined surveillance 
19 
 
20 
• Results in persistent or significant disability/incapacity 
21 
 
22 
• Any episode of bacteremia due to Lactobacillus reuteri, the study probiotic 
23 
 
24 
• Any other important medical event that may not result in death, be life threatening, 
25 
or require hospitalization, may be considered a serious adverse experience when, 
26 
based upon appropriate medical judgment, the event may jeopardize the subject 
27 
and may require medical or surgical intervention to prevent one of the outcomes 
28 
listed above.  Examples of such medical events include allergic bronchospasm 
29 
requiring intensive treatment in an emergency room or convulsions that do not 
30 
result in inpatient hospitalization. 
31 
 
32 
• Any monitored lab value meeting the Grade 3 or Grade 4 toxicity criteria 
33 
 
34 
Bacteremia:   Any episode of bacteremia due to Lactobacillus reuteri, the study probiotic, 
35 
will be considered a Serious Adverse Event.  Subjects started on probiotic/placebo 
36 
therapy will be monitored for adverse events during the 5 days of probiotic therapy and at 
37 
2-5 day intervals (when symptoms are recorded and stools are collected) following 
38 
discontinuation of therapy.  Temperature will be documented daily while receiving study 
39 
intervention and at follow-up visits which include AE monitoring, and participants with new 
40 
onset of fever following initiation of intervention therapy and participants who appear 
41 
especially ill or toxic to the field worker will have a physician evaluation.  Subjects will also 
42 
be instructed to present to study personnel for evaluation whenever they suspect they are 
43 
febrile or a new febrile episode is documented.  In cases where fever is grade 3 or 4, or 
44 
when the subject appears particularly ill or toxic in the estimation of the field worker or 
45 

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physician, blood will be examined in for malaria parasites by thick smear (a standard daily 
1 
diagnostic procedure in our laboratory) within 8 hours of collection, and antimalarial 
2 
treatment provided by the Ministry of Health will be administered to subjects with fever and 
3 
parasitemia.  Subjects evaluated in this manner will have a blood culture specimen taken 
4 
and close monitoring.  Presumptive medical therapy will be started at the discretion of the 
5 
study physician.  In the event of positive blood cultures, presumptive antibiotic treatment 
6 
and hospitalization (if deemed necessary by the attending physician) will be facilitated by 
7 
project personnel (including procurement).  Antibiotic therapy will be the standard pediatric 
8 
doses of Ceftriaxone and gentamicin for presumed or confirmed common causes of 
9 
sepsis in this population (E. coli, S. pneumonia) and clindamycin will be used if study 
10 
product associated bacteremia is suspected (no source of lower respiratory infection, 
11 
urinary tract infection, or diarrheal illness noted) or supported by microbiologic data (gram 
12 
positive rods on initial gram stain). 
13 
 
14 
Documenting SAEs 
15 
Any AE and SAE occurring during the study must be documented in the SAE CRF and on 
16 
form FDA 3500 MedWatch.  A separate MedWatch form should be used for each SAE.  
17 
However, if at the time of initial reporting, multiple SAEs are present, they may be reported 
18 
on the same “SAE” page.  If a subject dies during participation in the study or in the 85 
19 
days following the administration, NIDDK will be provided with a copy of any post-mortem 
20 
findings, including histopathology.   
21 
 
 
 
 
 
 
22 
Post-study AEs and SAEs 
23 
Investigators will not actively seek AEs or SAEs in study participants beyond day 28.  
24 
However, if the investigator learns of any AE at any time after active surveillance ends on 
25 
day 28 or a subject has completed the study at 3 months, and such event(s) is (are) 
26 
reasonably related to the study participation, the investigator will promptly notify NIDDK 
27 
and document the event in as much detail as can be ascertained. 
28 
 
29 
 
30 
9.2.3 
Unanticipated Problems 
31 
 
32 
The Office for Human Research Protections (OHRP) Common Rule 45 CFR §46.103 
33 
considers unanticipated problems involving risks to subjects or others to include, in 
34 
general, any incident, experience, or outcome that meets all of the following criteria: 
35 
• 
unexpected (in terms of nature, severity, or frequency) given (a) the research 
36 
procedures that are described in the protocol-related documents, such as the IRB- 
37 
approved research protocol and informed consent document; and (b) the 
38 
characteristics of the subject population being studied; 
39 
• 
related or possibly related to participation in the research (in the guidance document, 
40 
possibly related means there is a reasonable possibility that the incident, experience, 
41 
or outcome may have been caused by the procedures involved in the research); and 
42 
• 
suggests that the research places subjects or others at a greater risk of harm 
43 
(including physical, psychological, economic, or social harm) than was previously 
44 
known or recognized. 
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1 
An incident, experience, or outcome that meets any of the three criteria above may 
2 
warrant consideration of substantive changes in the trial in order to protect the safety, 
3 
welfare, or rights of subjects or others.  Examples of corrective actions or substantive 
4 
changes that might need to be considered in response to an unanticipated problem 
5 
include: 
6 
• 
changes to the research protocol initiated by the investigator prior to obtaining IRB 
7 
approval to eliminate apparent immediate hazards to subjects 
8 
• 
modification of inclusion or exclusion criteria to mitigate the newly identified risks 
9 
• 
implementation of additional procedures for monitoring subjects 
10 
• 
suspension of enrollment of new subjects 
11 
• 
suspension of research procedures in currently enrolled subjects 
12 
• 
modification of informed consent documents to include a description of newly 
13 
recognized risks 
14 
• 
provision of additional information about newly recognized risks to previously 
15 
enrolled subjects. 
16 
 
17 
Some incidents, experiences, and outcomes that occur during the conduct of human 
18 
subjects research represent unanticipated problems but are not considered adverse 
19 
events.  These may involve social or economic harm instead of the physical or 
20 
psychological harm associated with adverse events.  In other cases, unanticipated 
21 
problems place subjects or others at increased risk of harm, but no harm occurs. 
22 
Unanticipated problems must be reported, even if they do not involve adverse events. 
23 
However, adverse events that do not meet the criteria to be considered unanticipated 
24 
problems are not subject to these reporting requirements. 
25 
 
26 
Incidents or events that meet the OHRP criteria for unanticipated problems involving risks 
27 
to subjects or others require the completion of an AE report form.   
28 
 
29 
Additional reporting requirements.  Abuse or illegal activities will be reported to Peruvian 
30 
authorities in a manner consistent with local laws.  A written note, documenting the 
31 
concern, and witnessed of reported behavior giving rise to the concern, is issued to the 
32 
Peruvian community designated “Defensor de Pueblo” the local legal authority that 
33 
investigates cases of child abuse and exploitation.  
34 
 
35 
 
36 
 
37 
9.2.4 
Procedures to be Followed in the Event of Abnormal 
38 
Laboratory Test Values or Abnormal Clinical Findings 
39 
 
40 
Baseline lab tests will be reviewed by the study physician as part of the screening 
41 
process.  In order to be eligible to participate in the study, all monitored baseline 
42 
laboratory values must be within the normal range for the reference laboratory.  
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Laboratory values provided by the clinical laboratory that are not among the group of 
1 
monitored laboratory tests do not have to be within the normal range in order for a 
2 
consented subject to become a participant, but these reportedly abnormal values will 
3 
be noted on the FORM FOR EVALUTION OF LABORATORY VALUES.  These non-
4 
monitored laboratory values provided as part of the complete blood count (CBC) 
5 
include platelet count and percentage of various leukocyte categories in the differential.    
6 
Monitored laboratory values are: 
7 
 
8 
• Hemoglobin 
9 
• Total leukocyte count 
10 
• Total serum bilirubin 
11 
• Blood urea nitrogen (BUN) 
12 
• Creatinine 
13 
• Alanine transaminase (ALT) 
14 
• Aspartate aminotransferase (AST) 
15 
 
16 
Monitored laboratory values will be reviewed by the study physician within 48 hours of 
17 
receiving the report from the reference laboratory, and values will be recorded on the 
18 
appropriate CRF (FORM FOR EVALUATION OF LABORATORY VALUES).   Abnormal 
19 
values meeting toxicity level 1 or 2 which do not warrant further clinical investigation will 
20 
be noted as such on the laboratory values CRF along with an assessment of 
21 
relationship to the study product.  These abnormal values will be reported to the 
22 
oversight agencies as AEs according to procedures described below.    Abnormal 
23 
values meeting toxicity level 1 or 2 which may, in the judgment of the study physician, 
24 
warrant further investigation will result in a clinic visit for full evaluation recorded on the 
25 
ADVERSE EVENT CRF and will be followed to completion and resolution as with other 
26 
AEs.     Abnormal values meeting toxicity levels 3 or 4 will be considered SAEs and will 
27 
be evaluated, monitored, and reported according to procedures described for SAEs.    
28 
 
29 
Documenting AEs 
30 
Any AE occurring during the study must be documented in the subject’s medical records 
31 
and on the appropriate AE CRF, i.e. the AE Report Form.  Information to be collected on 
32 
the AE Report Form includes event description, time of onset, investigator assessment of 
33 
severity, and relationship to the study product, time of resolution of the event, seriousness, 
34 
and outcome.  All adverse events occurring during the study must be documented 
35 
appropriately regardless to the relationship of the event with study participation. 
36 
 
37 
The investigator or study physician should attempt to establish a diagnosis of the event 
38 
based on signs, symptoms, and/or other clinical information.  A diagnosis, if known, or 
39 
clinical signs and symptoms if diagnosis is unknown, rather than the clinically significant 
40 
abnormal laboratory finding, should be completed on AE CRF form.  If no diagnosis is 
41 
known and clinical signs and symptoms are not present, then the abnormal finding should 
42 
be recorded.  The laboratory data should either be recorded on the AE forms with the 
43 
reference range and baseline value(s) or copies of the laboratory reports and reference 
44 
ranges should be sent with the CRF pages. 
45 
 
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1 
 
2 
9.3 
Reporting Procedures 
3 
 
4 
AE Reporting: 
5 
 
6 
AEs.  This study will report AEs to the DSMB and NIDDK quarterly.  Any medical 
7 
condition that is present at the time that the patient is screened should be considered as 
8 
baseline and not reported as an AE.  However, if it deteriorates at any time during the 
9 
study it should be recorded as an AE. 
10 
    
11 
AEs will be reported annually to the following institutional representatives at the fax and 
12 
email addresses provided: 
13 
 
14 
1. 
Tulane University SPHTM.    
Ms. Roxanne Johnson 
15 
 
 
 
 
 
 
Director, Tulane University Biomedical IRB  
16 
 
 
 
 
 
 
rjohnso@tulane.edu 
17 
 
 
 
 
 
 
Fax 504-988-4766 
18 
 
 
 
 
 
 
Tel. 504-988-2665 
19 
 
20 
2. 
Johns Hopkins Bloomberg 
21 
School of  
 
 
 
Ms. Joan Petit, IRB Office Director 
22 
 
Public Health  
 
 
irboffice@jhu.edu 
23 
 
 
 
 
 
 
Fax: 410-502-0584 
24 
 
 
 
 
 
 
Tel. 410-502-1999  
25 
 
26 
3. 
Asociación Benéfica PRISMA    Salomon Zavala Sarrio, IRB Chairman 
27 
 
 
 
 
  
 
szavalas@gmail.com 
28 
 
 
 
 
 
 
Fax 011-511-6165501 
29 
Tel. 011-511-6165500 Ext 246 
30 
 
31 
4. 
Instituto Nacional de Salud :  Contact information to be provided 
32 
 
(Lima, Peru) 
33 
 
34 
5. 
NIDDK 
 
  
 
Jose Serrano, MD PhD 
35 
Program Official for PRIDEC-Peru 
36 
Division of Digestive Diseases and Nutrition 
37 
National Institute of Diabetes and Digestive and 
38 
Kidney Diseases 
39 
National Institutes of Health 
40 
serranoj@mail.nih.gov 
41 
Tel.  301-594-8871 
42 
Fax:  301-480-8300 
43 
 
44 
 
45 
7. 
BioGaia AB  
 
 
Karin B Diderot 
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BioGaia AB 
1 
P.O. Box 966 
2 
SE-220 09 Lund 
3 
SWEDEN 
4 
Phone: +46 46 311 903 
5 
Mobile: +46 70 348 58 21 
6 
Fax: + 46 46 311 901 
7 
 
8 
8.        DSMB 
 
 
 
Rebecca J. Torrance  RN, MS 
9 
Clinical Trials Specialist 
10 
Division of Digestive Diseases and Nutrition 
11 
NIDDK 
12 
6707 Democracy Blvd, Rm 644 
13 
Bethesda, MD 20892-5450 
14 
Phone:  301 594 7024 
15 
Fax: 301 480 8300 
16 
Email: torrancer@niddk.nih.gov 
17 
 
18 
Ms. Torrance will be the NIDDK designate for communications with the DSMB. 
19 
 
20 
 
21 
9.3.1 
Serious Adverse Events 
22 
 
23 
Any AE considered serious by the study physician or which meets the aforementioned 
24 
criteria must be submitted on a SAE form to the appropriate IRBs, INS, DSMB designate 
25 
and the NIDDK.  The SAE form will be sent as an email attachment or fax to each 
26 
relevant institutional representative within the time frame below:   
27 
 
28 
   *  All deaths, whether related or unrelated, will be recorded on the Serious Event 
29 
Form and on MedWATCH FDA 3500 (2/13)  and sent by fax and email within 24 
30 
hours of the site awareness of the death. 
31 
 
32 
  *  Serious adverse events other than death, regardless of relationship, will be 
33 
reported via fax and email by the site within 24 hours of becoming aware of the 
34 
event. 
35 
 
36 
Other supporting documentation of the event may be provided upon request as soon as 
37 
possible. 
38 
 
39 
All serious adverse events will be: 
40 
              ·  recorded on the appropriate serious event case report form 
41 
              ·  followed through resolution by a study physician 
42 
                ·reviewed by a study physician 
43 
 
44 
All SAEs will be followed until satisfactory resolution of the event or until the study 
45 
physician deems the event to be chronic or the patient to be stable. 
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The investigator, or responsible person according to local requirements, must comply with 
1 
the applicable local regulatory requirements related to the reporting of SAEs to regulatory 
2 
authorities and the IRB/EC. 
3 
 
4 
SAEs will be reported to the institutional representatives at the fax and email 
5 
addresses shown in the section entitled Reporting of Adverse Events.   
6 
 
7 
 
8 
 
9 
9.3.2 
Regulatory Reporting for Studies Not Conducted Under 
10 
NIDDK-Sponsored IND 
11 
 
12 
Procedures for reporting AEs and SAEs are described in the corresponding sections on 
13 
AE and SAE reporting.   
14 
 
15 
 
16 
9.4 
Type and Duration of Follow-up of Subjects after Adverse 
17 
Events 
18 
 
19 
Follow-up of AEs 
20 
All AEs will be followed until resolution, until the condition stabilizes, until the event is 
21 
otherwise explained, or the subject is lost to follow-up.  The ADVERSE EVENT CRF 
22 
includes space for up to two follow-up visits for the same AE.   The investigators will 
23 
ensure that follow-up includes any supplemental investigations as may be indicated to 
24 
elucidate as completely as practical the nature and/or causality of the AE.  This may 
25 
include additional laboratory tests or investigations, histopathological examinations, or 
26 
consultation with other health care professionals.  New or updated information should be 
27 
recorded on the originally completed AE CRF with all changes signed and dated by the 
28 
investigator. 
29 
 
30 
9.5 
Halting Rules 
31 
 
32 
The study will be halted if two or more individuals develop similar SAEs for which a 
33 
relationship to the study product cannot be excluded upon study physician and medical 
34 
monitor review.   Participants who develop an SAE, regardless of cause, will discontinue 
35 
the study treatment if treatment is still ongoing. 
36 
 
37 
Subsequent review of serious, unexpected, and related AEs by the Medical Monitor, 
38 
IRB, or NIDDK may also result in suspension of further administration of study product. 
39 
NIDDK, typically in conjunction with DSMB recommendations, retains the authority to 
40 
suspend additional enrollment and study interventions/administration of study product 
41 
for the entire study, as applicable. 
42 
 
43 
Examples of findings that might trigger a safety review are the number of SAEs overall 
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and by treatment group during the closed DSMB sessions, the number of occurrences 
1 
of a particular type of SAE, severe AEs/reactions, or increased frequency of events. 
2 
 
3 
9.6 
Safety Oversight 
4 
 
5 
Safety oversight for this Phase 1 study Safety of Lactobacillus reuteri in healthy children 
6 
aged 2-24 months is outlined in the attached Data and Safety Monitoring Plan.  A medical 
7 
monitor has been contracted to evaluate AE reporting and they and NIDDK staff and study 
8 
staff will review open reports that do not specify treatment group when adverse events 
9 
and out of range values are summarized on a quarterly basis.  Additionally, an NIDDK 
10 
appointed DSMB will monitor safety data by treatment group for this Phase 1 study by 
11 
examining a closed report in which out of range laboratory values and adverse events are 
12 
reported by treatment group on a quarterly basis.  This closed report will be prepared 
13 
based on the final open report.  Our project biostatistician (unmasked) will communicate 
14 
with the project biostatistician who is not involved in any data entry or cleaning of the data 
15 
while the trial is ongoing and transfer the data and treatment assignment by PID to the 
16 
biostatistician to elaborate the same tables as those present in the DSMP, but with 
17 
treatment assignment.  This will be reviewed by unblinded members of the NIDDK as well 
18 
as the DSMB. 
19 
 
20 
 
21 
 
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1 
 
2 
10 
CLINICAL MONITORING 
3 
 
4 
Clinical monitoring will be carried out in two ways: 
5 
 
6 
1.  The investigators have outlined a detailed Data and Safety Monitoring Plan, 
7 
described in a separate DSMP document.   The clinical and statistical monitors 
8 
performing this function are listed in section 9.6 Safety Oversight.  Procedures for 
9 
and frequency of this clinical monitoring are described in the DSMP. 
10 
 
11 
2. NIDDK will provide regulatory and compliance monitoring through an NIDDK-
12 
appointed site monitor, contracted through a certified research monitoring agency 
13 
such as Westat.  Frequency and scope of this oversight will be determined by 
14 
NIDDK.    
15 
 
16 
 
17 
10.1 Site Monitoring Plan 
18 
 
19 
Site monitoring is conducted to ensure that the human subject protection, study 
20 
procedures, laboratory, study intervention administration, and data collection processes 
21 
are of high quality and meet the sponsor, ICH E6 and, when appropriate, regulatory 
22 
guidelines.   
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1 
 
2 
11 
STATISTICAL CONSIDERATIONS 
3 
 
4 
 
5 
Data will be collected, checked, and entered into and a data management system in a 
6 
Microsoft ACCESS database with identity and time stamping.  All patients will be 
7 
assigned a unique identifier (Personal Identification or PID) which will be the link for all 
8 
databases and appear on all derived biospecimens. We will use filters for the invalid 
9 
entries and the identification of missing and out-of-range values and a double data entry 
10 
system.  All inconsistencies between entries must be resolved with differences able to be 
11 
reviewed by supervisors and auditors.  Access to the database will be restricted 
12 
combined physical access restriction and password protection.  Analyses will be 
13 
conducted in STATA and SAS. 
14 
 
15 
11.1 Study Hypotheses and Analysis 
16 
 
17 
 
18 
A. The primary hypotheses are that L. reuteri  therapy at the specified dose is safe 
19 
and well-tolerated in children aged 2-24 months.  There are three areas of safety and 
20 
tolerability that will be assessed in this Phase 1 trial.  
21 
 
22 
1. Evidence of bacteremia or invasive infection resulting from the administration 
23 
of L. reuteri 17938.  Baseline and daily temperature measurements will be made 
24 
during the study period during the product administration phase and then on all 
25 
scheduled home visits.  Complete blood counts with differential counts will be 
26 
obtained at baseline, on day 5, and on day 28.  The evaluation of all documented 
27 
febrile episodes will include an examination by a physician, and thick smear for 
28 
malaria, and blood culture for the evaluation of bacteremia is the temperature is 
29 
greater than 39ºC using temporal artery probe measures. 
30 
 
31 
Specific outcome parameters in this category include: 
32 
 
33 
a. Mean daily temperature 
34 
b. Incidence of fever on study days 1-5 
35 
c. Frequency of temperature readings categorized as fever (adjusted for number of 
36 
subjects per group) 
37 
d. Number of episodes of L. reuteri bacteremia 
38 
 
39 
 
40 
2. Evidence of toxicity resulting from the L. reuteri DSM 17938 preparation. 
41 
Participants will have baseline, day 5, and day 28 assessment of hematologic status and 
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renal and hepatic function.  Assessment of renal function will include blood urea nitrogen 
1 
(BUN) and creatinine (Cr).  Assessment of hepatotoxicity will include transaminases 
2 
(AST, ALT) and total bilirubin.  Hematologic status will be determined by CBC to 
3 
measure hemoglobin count and absolute neutrophil count, as mentioned above. 
4 
 
5 
Specific outcome parameters in this category include: 
6 
 
7 
a. Mean values for laboratory tests monitored (adjusted for number of subjects per 
8 
group).  These are listed in Appendix D.   
9 
b. Frequency of out of range values for laboratory tests monitored (adjusted for number 
10 
of subjects per group).  These are listed in Appendix D.  
11 
 
12 
 
13 
2. Clinical tolerance.  The inclusion of a placebo group will allow for comparisons of the 
14 
prevalence of daily symptom measurements between treatment groups.  The 
15 
symptoms monitored on the daily diary card (fever, anorexia/decreased oral intake, 
16 
vomiting, diarrhea, irritability, rash, itching, wheezing) are graded by the parent(s) of 
17 
the participant as mild, moderate, severe, or very severe.  Parents report of severity 
18 
will be analyzed, except in the case that the physician or study team deems them to be 
19 
more severe than reported (the study team may upgrade, but NOT downgrade the 
20 
severity of the symptom). 
21 
 
22 
Specific outcome parameters in this category include: 
23 
 
24 
a. Frequency and severity of symptoms monitored as AEs (adjusted for number of 
25 
subjects per group) 
26 
b. Frequency of all AEs (adjusted for number of subjects per group) 
27 
c. Frequency of SAEs (adjusted for number of subjects per group) 
28 
 
29 
 All continuous variables will be summarized using descriptive statistics including number 
30 
(N), mean, standard deviation (SD), median, maximum, and minimum.  All categorical 
31 
variables will be summarized using frequency counts and percentages. 
32 
 
33 
Changes from baseline temperature and fever graded by defined toxicity categories will 
34 
be summarized using descriptive statistics by treatment group. 
35 
 
36 
Concomitant medications, including those taken prior to study drug administration, will be 
37 
listed by subject.  All concomitant medications taken after initiation of the study treatment 
38 
will be tabulated overall and by formulation. The number and percentage of subjects who 
39 
take at least 1 concomitant medication and the number and percent of subjects who take 
40 
each specific concomitant medication will be presented. 
41 
 
42 
Treatment-emergent AEs will be defined as any event with a start date occurring on or 
43 
after treatment Day 1 or, if pre-existing, worsening after treatment Day 1.    If a subject 
44 
reports the same AE more than once, then that subject will only be counted once for the 
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summary of that AE, using the most severe intensity.   
1 
 
2 
Treatment-emergent AEs will be summarized as follows to the DSMB during closed 
3 
sessions: 
4 
• 
All treatment-emergent AEs; 
5 
• 
All treatment-emergent AEs by intensity; 
6 
• 
SAEs;  
7 
• 
All treatment-emergent SAEs by relationship to study drug; and 
8 
• 
AEs that led to premature discontinuation of study drug. 
9 
All SAEs and AEs leading to premature withdrawal from the study will be listed.  
10 
 
11 
Hematology and blood chemistry parameters will be summarized at baseline and at the 
12 
end of the study.  Laboratory values and changes from baseline in laboratory values will 
13 
be summarized descriptively for all subjects and by treatment group.  A summary of shifts 
14 
from baseline to final evaluation will be given for each parameter for all subjects and by 
15 
treatment group.  The normal range for each parameter will be used to create categories 
16 
of low, normal, or high.  Any result higher than the upper limit normal (ULN) will be 
17 
categorized as high, any result lower than the lower limit of normal (LLN) will be 
18 
categorized as low, and any result within the LLN and ULN will be categorized as normal.  
19 
The number and percentage of subjects in each shift category from baseline to final 
20 
evaluation will be shown for each parameter.  Only the hematology and blood chemistry 
21 
tests with a numeric normal range and at least one follow-up value will be analyzed for 
22 
shifts from baseline.  The number and percentage of subjects with clinically significant 
23 
values will be presented. 
24 
 
25 
 
26 
B. The secondary outcome measurements are 
27 
 
28 
1. To determine the duration of shedding of L. reuteri DSM 17938 following controlled 
29 
administration.   
30 
 
31 
Since the longitudinal nature of the repeated PCR assays results in correlated outcomes, 
32 
generalized estimating equations (GEE) will be used to model the binary response 
33 
whether subjects exhibited a positive PCR result during the various period of study. The 
34 
duration of shedding of L. reuteri will be assessed using Kaplan Meier Product limit 
35 
estimates.  
36 
 
37 
 
38 
 
39 
11.2 Sample Size Considerations 
40 

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1 
 
2 
The sample size for this Phase 1 safety trial in healthy children aged 2-24 months is 
3 
determined by historical precedent for safety in human use. 
4 
 
5 
Enrollment for the prior Phase 1 studies in adults (Safety of LR in Healthy volunteers 
6 
#NCT00774163) and children (Safety of LR in healthy children 2 to 5 years of age in Peru 
7 
#NCT02124122) have been able to recruit and retain >95 subject (Phase 1 child study in 
8 
progress).  We have high and similar rates of retention in our other studies with more 
9 
complexity and participant burden, even among our birth cohorts.37 
10 
 
11 
Missing data will not be imputed.  Data will be analyzed per treatment group assignment 
12 
after the administration of a single dose of product for all safety and tolerability analyses.  
13 
Data regarding the duration of carriage will be restricted to those individuals who complete 
14 
5 days of therapy.   
15 
 
16 
The amount of subjects in this study will likely be underpowered to detect differences in 
17 
most secondary outcomes.  This is in part due to the lack of information on key 
18 
parameters needed for sample size calculations.  For example, in this age and context, to 
19 
calculate the duration of L. reuteri carriage following administration it is not known how 
20 
many subjects will become positive for L. reuteri by PCR following administration.  
21 
Similarly, it is not known how frequent intermittent passage of L reuteri positive stool is in 
22 
individuals in this community, although about 10% of individuals in this age range test 
23 
positive. This data collected as part of this Phase 1 study will provide important 
24 
information of use in subsequent phase 2 trial [Efficacy of Lactobacillus reuteri in healthy children aged 
25 
2-24 months in the treatment of environmental enteropathy] . 
26 
 
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1 
 
2 
 
3 
11.3 Planned Interim Analyses (if applicable) 
4 
 
5 
No formal interim analyses are planned; however, accumulated phase 1 trial data will be 
6 
reviewed by treatment group every 3 months by the NIDDK and DSMB.   
7 
 
8 
 
9 
11.3.1 Safety Review 
10 
 
11 
 
12 
Subject accrual, status adherence data on visits and intervention, adverse events and 
13 
rates of adverse events will be reported to the DSMB every three months.  SAEs will be 
14 
reported within 24 hours of occurrence.  Details of the interval reports are specified in 
15 
the DSMP. 
16 
 
17 
The study would be halted if two or more subjects develop a similar SAE for which the 
18 
relationship to the study product cannot be excluded.  This would include, but not be 
19 
limited to bacteremia with the product strain. 
20 
 
21 
Individuals with a SAE during the period of probiotic administration will have the 
22 
intervention stopped regardless of the determined relationship to the study product, but 
23 
follow-up monitoring visits and testing would be completed to the greatest extent 
24 
possible, and the SAE would be managed until resolved. 
25 
 
26 
INS requests SAE reporting, annual reports, and study termination report.  Annual 
27 
reporting to IRB is planned for Tulane, Johns Hopkins University and AB PRISMA. 
28 
 
29 
 
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1 
 
2 
 
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1 
 
2 
12 
SOURCE DOCUMENTS AND ACCESS TO SOURCE 
3 
DATA/DOCUMENTS 
4 
 
5 
All participants will have a chart composed of a copy of the signed consent document, all 
6 
CRFs, clinical laboratory values (CBC, AST, ALT, BUN, Creatinine, and total bilirubin 
7 
and screening day 0 L. reuteri PCR status, but not subsequent Lr PCR tests), diagnostic 
8 
testing results performed as part of adverse event evaluations, and copies of hospital 
9 
records in the case of a SAE.  These charts will be available for review by study 
10 
personnel, the Medical Monitor, the INS, the regional directorate of Health of Loreto, 
11 
authorized representatives of NIDDK, OHRP, and other Department of Health and 
12 
Human Services agencies, as applicable; the Ethics committee of AB PRISMA, and the 
13 
Institutional Review Boards of Johns Hopkins University and Tulane University.  Study 
14 
source documents are stored in locked cabinets within locked rooms of private research 
15 
space when not being reviewed.  Databases are present in restricted areas of these 
16 
buildings and are password protected. 
17 
 
18 
The study product control, including the review of temperature and shipping logs, viability 
19 
tests, and product tracking logs are available for review by auditors from the agencies 
20 
mentioned above. 
21 
 
22 
Tests related to PCR carriage are generated in experimental files.  These results are 
23 
then matched to SID and transferred to the database (in the case of ELISA results) or 
24 
transferred via a access file in which the technician manually reads the photo file and 
25 
inputs the result by SID which is then added to the database. All experimental files are 
26 
available for review. 
27 
 
28 
All source documents, with the exception of microbiome data, will have primary storage 
29 
at the laboratory and field sites in Iquitos Peru.  Microbiome data will be stored in in the 
30 
absence of identifiers in commercial laboratories in the United States and on servers 
31 
located at Johns Hopkins University.   The Laboratory and offices at AB PRISMA are 
32 
used for long term storage of physical documents and the secure storage of electronic 
33 
versions of the data on a server.  The building has 24hr staff presence and is a private 
34 
facility with limited access. 
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1 
 
2 
13 QUALITY CONTROL AND QUALITY ASSURANCE 
3 
 
4 
 
5 
 
6 
Quality Assurance 
7 
To ensure the quality and integrity of laboratory and clinical data, detailed SOPs will be 
8 
written to clarify correct procedures and handing of data and specimens that extends from 
9 
field activities to data management.  Staff will be trained in CITI and the SOP portions that 
10 
correspond to their responsibilities in the study.  Study responsibilities will be specified in 
11 
the Site personnel and signature log.  Training will be documented and documentation will 
12 
consist of a training duration, specific areas covered in training, and register of 
13 
participants.  This training, which will occur prior to the study and reinforced during the 
14 
study, will be documented in the study file for review by study monitors. 
15 
 
16 
Laboratory protocols 
17 
Laboratory protocols for the collection, registration and barcoding of all biologic samples 
18 
will be detailed.   
19 
 
20 
 
21 
Product Assurance 
22 
The Certification of Assurance of the study product, obtained by the product supplier, will 
23 
contain the following information. 
24 
 
25 
Identity 
26 
Identity of the test product will be confirmed by standard carbohydrate utilization tests (API 
27 
50CH test (BioMerieux) pattern and the plasmid profile of the strain will be documented.   
28 
 
29 
 
30 
Purity 
31 
 Information on the content of Salmonella, E. coli, yeast and mold, and total aerobic count 
32 
per USP <61> .43Evaluation for contamination with Shigella will be by FDA protocol BAM 
33 
Chapter 6.).  The release limit of Salmonella, Shigella and E.coli is absent in 10g of 
34 
product, yeast and mold < 100 CFU/g and total aerobic count < 1000 CFU/g. 
35 
 
36 
Viable counts of probiotic product strain 
37 
Existing laboratory protocols will be employed, with modifications as necessary, for 
38 
quantification of the viable count of product strain (name protocol).  Viable counts will be 
39 
performed prior to study enrollment and every month following study initiation until the last 
40 
dose of product is administered.  Tolerance for variability will be + 1 log CFU count.  If the 
41 
results are not within this range, the product will be re-analyzed from two vials from the 
42 
same stock.  If again not within range, this stock will be discarded and replaced with new 
43 
study product provided by BioGaia, the product manufacturer.   
44 
 
45 
All study personnel engaged in study activities must have training documented in the form 
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of signatures on training logs.  For laboratory scientists, this includes practicums 
1 
demonstrating test proficiency in assay performance and the correct storage and naming 
2 
of experimental files.  Upon completion of testing, they sign the SOP to acknowledge 
3 
proficiency certification and compliance with the protocol SOP. 
4 
 
5 
 
6 
Quality Control 
7 
CRFs and source documents will be reviewed for completeness by a field or data entry 
8 
personnel daily and forms will be reviewed on site by the Study Coordinator, the study 
9 
physician, and /or overseeing investigators.  Upon completion, forms are further evaluated 
10 
for missing or incomplete data by data management personnel prior to data entry.   
11 
 
12 
 
13 
 
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1 
 
2 
14 
ETHICS/PROTECTION OF HUMAN SUBJECTS 
3 
 
4 
 
5 
14.1 Ethical Standard 
6 
 
7 
The investigators will ensure that this study is planned and conducted in full conformity 
8 
with the principles set forth in the Declaration of Helsinki, CIOMS, International Ethical 
9 
Guidelines for Biomedical Research Involving Human Subjects (2002), and in 
10 
accordance to laws governing the conduct of clinical trials in Peru (XV article 28 of law 
11 
26842). 
12 
 
13 
14.2 Institutional Review Board 
14 
 
15 
The relevant IRB and oversight committees are the following, which must approve of the 
16 
protocol, parental permission documents, and any amendments to the protocols.  They also must 
17 
be made aware of protocol deviations affecting the safety of participants, product concerns, or 
18 
any new information that may affect the balance of risk and benefit to participants. 
19 
 
20 
1. 
Johns Hopkins School of  
Joan Pettit, IRB Director 
21 
 
Hygiene & Public Health 
jhsph.irboffice@jhu.edu 
22 
 
 
 
 
 
Fax: 410-502-0584 
23 
 
 
 
 
 
Tel. 410-502-1999 
24 
 
25 
2. 
Tulane University SPHTM       Ms. Roxanne Johnson 
26 
 
 
 
 
 
Director, Tulane University Biomedical IRB  
27 
 
 
 
 
 
rjohnso@tulane.edu 
28 
 
 
 
 
 
Fax 504-988-4766 
29 
 
 
 
 
 
Tel. 504-988-2665 
30 
 
31 
 
32 
 
33 
3. 
Asociación Benéfica PRISMA  Salomon Zavala Sarrio, IRB Chairman 
34 
 
 
 
 
  
szavalas@gmail.com 
35 
 
 
 
 
 
Fax 011-511-6165501 
36 
Tel. 011-511-6165500 Ext 246 
37 
 
38 
4. 
Instituto Nacional de Salud 
   Contact information to be provided 
39 
 
(INS; Lima, Peru) 
40 
 
41 
 
42 
 
43 
 
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14.3 Informed Consent Process 
1 
 
2 
Consent will be obtained by CITI certified employees of PRISMA with experience in 
3 
biomedical research and community based research.  Both parents must sign the 
4 
consent form for children of this age to participate in clinical trials in Peru.  The 
5 
discussion will begin in a public forum where interested parties are invited by public 
6 
loudspeaker to a community center.  Individuals who retain interest in having their 
7 
children participate after this forum will be invited to the study center where the process 
8 
will be repeated in a private setting. 
9 
 
10 
The study involves young children.  To ensure the parental permission to have the child 
11 
participate is valid, the study will be explained in detail to ensure that parents understand 
12 
the risks and benefits for their particular child.  The study team has extensive experience 
13 
working with young children and their parents and the stress of study participation to the 
14 
child participants will be minimized in part by clinical rapport gained from this experience.  
15 
 
16 
A standard signed consent document will be used.  Children of guardians lacking 
17 
signatory capacity will not be allowed to participate.  Study participants will be given a 
18 
copy of the consent document to ensure their understanding (by giving them 
19 
opportunities to present and discuss with 3rd parties, and to provide necessary contact 
20 
information with study personnel or local or international IRBs overseeing the study. 
21 
 
22 
 
23 
14.3.1 Informed Consent/Assent Process (in Case of a Minor) 
24 
 
25 
All subjects are under the age of 2 years and will participate with parental permission. 
26 
 
27 
14.4 Exclusion of Women, Minorities, and Children (Special 
28 
Populations) 
29 
Not relevant. 
30 
 
31 
14.5 Subject Confidentiality 
32 
 
33 
The confidentiality of research subjects is strictly maintained by the study team.  
34 
Although the study monitors and agencies may access study information in the oversight 
35 
of the clinical trial to ensure quality measures are being met or that human subjects are 
36 
being treated appropriately.  Data regarding subjects will not be released to other third 
37 
parties. 
38 
 
39 
The prolonged storage of source documents with identifiers is done at a secure space 
40 
with limited access and rigorous security. Digital records of the study and nearly all 
41 
source documents are identified by PID only to diminish risk of the dissemination of 
42 
information regarding the identity or characteristics of subjects. 
43 

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1 
Databases lack identifiers and are further protected by password.  All study personnel, 
2 
including data entry personnel, receive specific training on their obligations regarding the 
3 
privacy of participants and their families. 
4 
 
5 
14.6 Study Discontinuation 
6 
 
7 
In the case that the study is discontinued based on safety concerns, follow-up will be 
8 
maintained to document all relevant safety outcomes. 
9 
 
10 
14.7 Future Use of Stored Specimens 
11 
 
12 
 
13 
Data collected will be stored in servers at AB PRISMA and Johns Hopkins University 
14 
Bloomberg School of Public Health.  Specimens will be stored at the laboratories of AB 
15 
PRIMSA in Iquitos Peru, with the option of secure storage at Johns Hopkins University.  In 
16 
the parental permission form, there is a provision for the use of residual sample for long 
17 
term use for research related to the discovery and evaluation of new tests for enteric 
18 
infections, microbiome research, testing for biomarkers of undernutrition, and 
19 
gastrointestinal function.  Subjects can withdraw their consent to have their samples in 
20 
this biorepository and still participate in the study.  Subjects may withdraw their consent to 
21 
have their samples stored at a date no later than 2 years after the completion of the study.  
22 
De- identified samples will be shared with third parties only under a materials and transfer 
23 
agreement assuring compliance with restriction (No human DNA analysis, not for profit, 
24 
not to be used to derive patents) are upheld. 
25 
 
26 

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1 
 
2 
15 
DATA HANDLING AND RECORD KEEPING 
3 
 
4 
Data collected in paper format will be transferred to a data unit at the research center 
5 
and entered into a data management system.  Records will be checked by a study 
6 
physician or coordinator prior to being transferred for data entry.  
7 
 
8 
Personal identifiers (such as names, birthdates, addresses) will be collected and held 
9 
on a small number of forms that will not be entered into the electronic database. 
10 
 
11 
All data from the study will be held in secure space locally to permit validation of the 
12 
electronic database by auditors (including the Medical monitors, study PIs, INS staff, or 
13 
other trial auditors from or designated by NIDDK). 
14 
 
15 
15.1 Data Management Responsibilities 
16 
 
17 
All study data must be held in secure areas with limited access.  Transfer of physical 
18 
files and electronic data will be kept to a minimum.  Transfer of files and backup files 
19 
will all be done on password protected files.  Transfer of study files will always be done 
20 
by a study team member.  The site data manager has responsibility for the security and 
21 
integrity of the physical files, the existence and safety of back up files, and the 
22 
protection of digitally stored information.  Data is held on a server in Iquitos Peru with a 
23 
mirror copy at Johns Hopkins in Baltimore Maryland. 
24 
 
25 
All records and laboratory reports must be reviewed by the study physician, PI, study 
26 
coordinator or designee before being transferred to the data unit.  All corrections made 
27 
on CRFs will be made by striking thru the original text with a single line and by writing 
28 
the correct data above, beside or below the original entry.  The correction must be 
29 
accompanied by the initials of the individual making the change and the data of the 
30 
change.  The original entry must not be obscured. 
31 
 
32 
If the form is stamped as being already entered in the electronic database, it is the 
33 
responsibility of the individual initialing the change on the paper form to contact the data 
34 
center to ensure entry of the change in the study database.   
35 
 
36 
The data management team in Iquitos will ensure records from participants are 
37 
adequate and complete.  They will discuss active issues regarding incomplete or data 
38 
that appears to have unexpected values with the Study Coordinator to minimize 
39 
recurrence.  
40 
 
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15.2 Data Capture Methods 
1 
 
2 
Data captured on case report forms will be documented on paper forms.  Clinical 
3 
laboratory data will be entered directly from the source document.  These forms will 
4 
be entered into the study base on an ongoing basis.  Once forms are entered, the 
5 
forms are stamped to communicate this state to the study team.  Details of data 
6 
management are included in a separate data management SOP. 
7 
 
8 
Data will be entered in a MS ACCESS with filters for invalid entries and missing 
9 
values by project personnel at the data unit and time and date stamping.  Complete 
10 
double data entry with verification will be performed.  Validation of the DDE system 
11 
will be performed using STATA or a similar program.  All inconsistencies will be 
12 
resolved by the data manager in communication with the Study Coordinator or 
13 
project PI.  These files will be saved and available for review as part of the audit 
14 
trail to document data integrity.    
15 

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1 
 
2 
15.3 Timing/Reports 
3 
 
4 
The report content and timing is specified in the DSMP. 
5 
 
6 
15.4 Study Records Retention 
7 
 
8 
Study documents will be retained for a minimum of 7 years after the data is accrued in 
9 
accordance with regulations regarding the conduct of clinical trials in Peru.  They will be 
10 
secured in locked furniture in the AB PRISMA offices in Iquitos.
11 

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1 
 
2 
15.5 Protocol Deviations 
3 
 
4 
A protocol deviation is the con-compliance with the specified clinical trial protocol, GCP, 
5 
MOP, or laboratory SOP procedures.  The non-compliance can be the result of logistics, 
6 
the investigator, study site staff, or the subject.  It is however, always the responsibility of 
7 
the site PI to identify protocol deviations, characterize the nature, extent, and reason for 
8 
the deviation, and report them as appropriate to the INS, the IRBs of AB PRISMA, 
9 
Tulane, Johns Hopkins University, and the sponsor in a timely manner.   
10 
 
11 
 
12 
Protocol deviations are recorded on a form and become part of the study file reported to 
13 
the IRB annually if the deviation is characterized as one warranting reporting.  All protocol 
14 
deviations should be included in the reports to the DSMB as well. 
15 
 
16 
 
17 
Study deviations that are recorded that are purely the result of study compliance (such as 
18 
missing stool specimens, or specimens collected 3 days late) are reviewed by the study 
19 
staff but not reported to IRBs.
20 

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1 
 
2 
16 
STUDY ADMINISTRATION 
3 
16.1 STUDY LEADERSHIP AND GOVERNANCE 
4 
 
5 
16.1.1 Steering Committee 
6 
 
7 
The Steering Committee will govern the conduct of the study. The Steering Committee 
8 
will be composed of the study PI and co-PI and representatives of the sponsoring NIH 
9 
Institute (NIDDK), the study coordinator (AB PRIMSA), the local PI (Cesar Ramal 
10 
Asayag) and the PI of the laboratory of AB Prisma in Iquitos.  The Steering Committee 
11 
will oversee protocol and MOP development and meet by phone once prior to study 
12 
initiation for final review of study materials.  The steering committee members will receive 
13 
the reports of the medical monitor and meet by teleconference on an ad hoc basis. 
14 
 
15 
16.1.2 Executive Committee 
16 
 
17 
An executive committee is not required to coordinate and oversee a small single site study 
18 
that already has DSMB oversight 
19 
. 
20 
16.1.3 Conflict of Interest Policy 
21 
 
22 
The study product is supplied free of charge by BioGaia, (or with a minimal charge to 
23 
cover product labelling and shipping).  BioGaia does not participate in study design, 
24 
data analysis, or sponsor this trial with additional funds.  No member of the 
25 
investigative staff owns stock in or receives any compensation from BioGaia, nor have 
26 
they in the past.  BioGaia makes no determinations regarding the study outcomes, nor 
27 
do they have any capacity to influence data analysis or the dissemination of results.
28 

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1 
 
2 
16.2 SUBCOMMITTEES 
3 
 
4 
No subcommittees are planned for this small single site study.  Prior to enrollment the 
5 
trial will be registered at clinicaltrials.gov.  All resulting publications will be submitted to 
6 
the digital archive at Pubmed Central as per NIH public access regulations 
7 
(https://grants.nih.gov/grants/NIH-Public_Access-Plan.pdf) and preferentially submitted 
8 
to journals that have open access options. 
9 
 
10 
 
11 

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17 
LITERATURE REFERENCES 
 
 
1. 
Shornikova AV, Casas IA, Isolauri E, Mykkanen H, Vesikari T. Lactobacillus reuteri as a therapeutic 
agent in acute diarrhea in young children. J Pediatr Gastroenterol Nutr 1997;24:399-404. 
2. 
Shornikova AV, Casas IA, Mykkanen H, Salo E, Vesikari T. Bacteriotherapy with Lactobacillus reuteri 
in rotavirus gastroenteritis. Pediatr Infect Dis J 1997;16:1103-7. 
3. 
Savino F, Pelle E, Palumeri E, Oggero R, Miniero R. Lactobacillus reuteri (American Type Culture 
Collection Strain 55730) versus simethicone in the treatment of infantile colic: a prospective randomized 
study. Pediatrics 2007;119:e124-e30. 
4. 
Savino F, Cordisco L, Tarasco V, et al. Lactobacillus reuteri DSM 17938 in infantile colic: a 
randomized, double-blind, placebo-controlled trial. Pediatrics 2010;126:e526-e33. 
5. 
Rosenfeldt V, Benfeldt E, Nielsen SD, et al. Effect of probiotic Lactobacillus strains in children with 
atopic dermatitis. JAllergy ClinImmunol 2003;111:389-95. 
6. 
Arnold C. The pros and cons of probiotics. Lancet Infect Dis 2013;13:571-2. 
7. 
Hamilton-Miller JM, Shah S, Winkler JT. Public health issues arising from microbiological and 
labelling quality of foods and supplements containing probiotic microorganisms. Public health nutrition 
1999;2:223-9. 
8. 
Hamilton-Miller JM, Shah S. Deficiencies in microbiological quality and labelling of probiotic 
supplements. Int J Food Microbiol 2002;72:175-6. 
9. 
Oberhelman RA, Kosek MN, Penataro-Yori P, Paredes-Olortegui M, Connolly E. A phase one safety 
study of Lactobacillus reuteri conducted in the Peruvian Amazon: Observations from the field. The 
American journal of tropical medicine and hygiene 2014;90:777-80. 
10. 
Abrahamsson TR, Jakobsson T, Bottcher MF, et al. Probiotics in prevention of IgE-associated 
eczema: a double-blind, randomized, placebo-controlled trial. JAllergy ClinImmunol 2007;119:1174-80. 
11. 
Lionetti E, Miniello VL, Castellaneta SP, et al. Lactobacillus reuteri therapy to reduce side-effects 
during anti-Helicobacter pylori treatment in children: a randomized placebo controlled trial. 
AlimentPharmacolTher 2006;24:1461-8. 
12. 
Tubelius P, Stan V, Zachrisson A. Increasing work-place healthiness with the probiotic Lactobacillus 
reuteri: a randomised, double-blind placebo-controlled study. EnvironHealth 2005;4:25. 
13. 
Weizman Z, Asli G, Alsheikh A. Effect of a probiotic infant formula on infections in child care 
centers: comparison of two probiotic agents. Pediatrics 2005;115:5-9. 
14. 
Eom TH, Oh EY, Kim YH, et al. The therapeutic effect of Lactobacillus reuteri in acute diarrhea in 
infants and toddlers. Korean J Ped 2005;48:986-9. 
15. 
Valeur N, Engel P, Carbajal N, Connolly E, Ladefoged K. Colonization and immunomodulation by 
Lactobacillus reuteri ATCC 55730 in the human gastrointestinal tract. ApplEnvironMicrobiol 2004;70:1176-
81. 
16. 
Ouwehand AC, Lagstrom H, Suomalainen T, Salminen S. Effect of probiotics on constipation, fecal 
azoreductase activity and fecal mucin content in the elderly. AnnNutrMetab 2002;46:159-62. 
17. 
Wolf BW, Wheeler KB, Ataya DG, Garleb KA. Safety and tolerance of Lactobacillus reuteri 
supplementation to a population infected with the human immunodeficiency virus. Food ChemToxicol 
1998;36:1085-94. 
18. 
De Groote MA, Frank DN, Dowell E, Glode MP, Pace NR. Lactobacillus rhamnosus GG bacteremia 
associated with probiotic use in a child with short gut syndrome. Pediatr Infect Dis J 2005;24:278-80. 
19. 
Franko B, Vaillant M, Recule C, Vautrin E, Brion JP, Pavese P. Lactobacillus paracasei endocarditis in 

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a consumer of probiotics. Med Mal Infect 2013;43:171-3. 
20. 
Kunz AN, Noel JM, Fairchok MP. Two cases of Lactobacillus bacteremia during probiotic treatment 
of short gut syndrome. J PediatrGastroenterolNutr 2004;38:457-8. 
21. 
Land MH, Rouster-Stevens K, Woods CR, Cannon ML, Cnota J, Shetty AK. Lactobacillus sepsis 
associated with probiotic therapy. Pediatrics 2005;115:178-81. 
22. 
Ledoux D, Labombardi VJ, Karter D. Lactobacillus acidophilus bacteraemia after use of a probiotic 
in a patient with AIDS and Hodgkin's disease. Int J STD AIDS 2006;17:280-2. 
23. 
Mehta A, Rangarajan S, Borate U. A cautionary tale for probiotic use in hematopoietic SCT patients-
Lactobacillus acidophilus sepsis in a patient with mantle cell lymphoma undergoing hematopoietic SCT. 
Bone Marrow Transplant 2013;48:461-2. 
24. 
Vallabhaneni S, Walker TA, Lockhart SR, et al. Notes from the field: Fatal gastrointestinal 
mucormycosis in a premature infant associated with a contaminated dietary supplement--Connecticut, 
2014. MMWR Morb Mortal Wkly Rep 2015;64:155-6. 
25. 
Rosander A, Connolly E, Roos S. Removal of antibiotic resistance gene-carrying plasmids from 
Lactobacillus reuteri ATCC 55730 and characterization of the resulting daughter strain, L. reuteri DSM 
17938. ApplEnvironMicrobiol 2008;74:6032-40. 
26. 
Cimperman L, Bayless G, Best K, et al. A randomized, double-blind, placebo-controlled pilot study 
of Lactobacillus reuteri ATCC 55730 for the prevention of antibiotic-associated diarrhea in hospitalized 
adults. JClinGastroenterol 2011;45:785-9. 
27. 
Mangalat N, Liu Y, Fatheree NY, et al. Safety and tolerability of Lactobacillus reuteri DSM 17938 
and effects on biomarkers in healthy adults: results from a randomized masked trial. PLoSOne 
2012;7:e43910. 
28. 
Oncel MY, Sari FN, Arayici S, et al. Lactobacillus Reuteri for the prevention of necrotising 
enterocolitis in very low birthweight infants: a randomised controlled trial. ArchDisChild Fetal Neonatal Ed 
2013. 
29. 
Hunter C, Dimaguila MA, Gal P, et al. Effect of routine probiotic, Lactobacillus reuteri DSM 17938, 
use on rates of necrotizing enterocolitis in neonates with birthweight < 1000 grams: a sequential analysis. 
BMCPediatr 2012;12:142. 
30. 
Indrio F, Di Mauro A, Riezzo G, et al. Prophylactic Use of a Probiotic in the Prevention of Colic, 
Regurgitation, and Functional Constipation: A Randomized Clinical Trial. JAMA pediatrics 2014. 
31. 
Indrio F, Riezzo G, Raimondi F, Bisceglia M, Cavallo L, Francavilla R. The effects of probiotics on 
feeding tolerance, bowel habits, and gastrointestinal motility in preterm newborns. JPediatr 2008;152:801-
6. 
32. 
Indrio F, Riezzo G, Raimondi F, et al. Lactobacillus reuteri accelerates gastric emptying and 
improves regurgitation in infants. EurJClinInvest 2011;41:417-22. 
33. 
Cabana MD. Lactobacillus reuteri DSM 17938 appears to be effective in reducing crying time for 
colic. JPediatr 2011;158:516-7. 
34. 
Brown N. In infants with 'colic'/persistent crying, administration of daily Lactobacillus reuteri DSM 
17938 was associated with reduced crying time at 1, 2 and 3 weeks. ArchDisChild EducPractEd 
2011;96:239. 
35. 
Szajewska H, Gyrczuk E, Horvath A. Lactobacillus reuteri DSM 17938 for the management of 
infantile colic in breastfed infants: a randomized, double-blind, placebo-controlled trial. JPediatr 
2013;162:257-62. 
36. 
Fakhry C, Marks MA, Gilman RH, et al. Comparison of the immune microenvironment of the oral 
cavity and cervix in healthy women. Cytokine 2013;64:597-604. 
37. 
Kosek M, Haque R, Lima A, et al. Fecal markers of intestinal inflammation and permeability 
associated with the subsequent acquisition of linear growth deficits in infants. The American journal of 
tropical medicine and hygiene 2013;88:390-6. 

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38. 
Kosek M, Yori PP, Pan WK, et al. Epidemiology of Highly Endemic Multiply Antibiotic-Resistant 
Shigellosis in Children in the Peruvian Amazon. Pediatrics 2008. 
39. 
Lee G, Pan W, Penataro Yori P, et al. Symptomatic and asymptomatic Campylobacter infections 
associated with reduced growth in Peruvian children. PLoS neglected tropical diseases 2013;7:e2036. 
40. 
Lee G, Yori P, Olortegui MP, et al. Comparative effects of vivax malaria, fever and diarrhoea on 
child growth. IntJEpidemiol 2012. 
41. 
Peterson KM, Buss J, Easley R, et al. REG1B as a predictor of childhood stunting in Bangladesh and 
Peru. Am J Clin Nutr 2013;97:1129-33. 
42. 
Platts-Mills J, Liu,J., Gratz, J., Mduma,E., Amour,C., Swai,N, Taniuchi,M,m Begum,S., Penataro 
Yori,P., Tilley,D.H., Lee,G., Shen,Z, Whary,M.T., McGrath,M.,Kosek,M., Haque,R.,Houpt,ER. Campylobacter 
detection and significance in stool by culture,immunoassay, and PCR in developing country settings. J Clin 
Microbiol 2014;In press. 
43. 
Conventions USP. Microbial Examination of nonsterile products: Microbial Enumeration Tests. 

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APPENDIX A.1: Planned laboratory tests 
Hematology – Hemoglobin, WBC and differential count, platelet count. Biochemistry 
– BUN, creatinine, total bilirubin, AST, ALT. 
Blood samples have a target volume of 3ml, a minimum of 1.5 ml. 
 
Immunology – HIV rapid test done as part of screening 
Probiotic carriage- PCR is done on stool samples to detect L. reuteri per laboratory 
SOP. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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Appendix B.  Definitions for Adverse Events Monitoring 
 
Safety of Lactobacillus reuteri in healthy children ages 2-24 months in Peru 
 
 
 
Estimating Clinical Severity: 
 
 
Grade 1 (mild):  Symptoms causing no or minimal interference with usual activities 
 
 
 
Grade 2 (moderate): Symptoms causing greater than minimal interference with usual 
activities 
 
 
Grade 3 (severe): Symptoms causing inability to perform usual activities 
 
 
Grade 4 (potentially life-threatening):  Symptoms causing inability to perform basic self-
care functions, or if medical intervention is needed to prevent permanent impairment or death 
 
 

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APPENDIX C: Clinical adverse events to be monitored : Symptoms* 
  
 
 
*  Based on NIAID Division of AIDS Table for Grading the Severity of Adult and Pediatric Adverse 
Events, December 2004 
(http://www.niaid.nih.gov/labsandresources/resources/daidsclinrsrch/documents/daidsaegradingta
ble.pdf) 
 
 
 
Systemic  
(General) 
Mild  
(Grade 1) 
Moderate  
(Grade 2) 
Severe  
(Grade 3) 
Potentially Life 
Threatening (Grade 
4) 
1. Altered 
feeding/Anorexia 
Mild 
Moderate—
decreased oral 
intake 
Severe—little oral 
intake 
Unable to ingest 
food or fluid for 
more than 24 hours 
2. Vomiting 
1 episode/24 hours 2 -3episodes/24 
hours  
4-6 episodes/24 
hours  
> 6 episodes/24 
hours or 
hypotensive shock 
2. Diarrhea 
 3 liquid stools per 
24 hours 
4 liquid stools per 
24 hours 
Liquid stools, 5-8 
per 24 hours 
Liquid stools, > 8 
per 24 hours, or 
requires 
hospitalization 
4. Irritability 
Slightly irritable, no 
interference with 
activity 
Moderately 
irritable, some 
interference with 
activity 
Significant; 
prevents daily 
activity or seeks 
medical attention 
Incapacitating or 
requires 
hospitalization 
5. Rash 
Localized macular 
rash 
Diffuse macular, 
maculopapular, or 
morbilliform rash 
Grade 2 rash with 
vesicles, bullae, 
ulcerations, target 
lesions, or 
angioedema 
Severe generalized 
rashes (e.g. Stevens 
Johnson syndrome, 
toxic epidermal 
necrolysis 
6. Pruritis 
Localized itching, 
relieved 
spontaneously or in 
> 48 hours 
Localized itching 
lasting 48 hours or 
longer 
Generalized itching Generalized, severe 
itching or 
generalized itching 
lasting > 48 hours 
7. Bronchospasm or 
wheezing 
Mild wheezing, or 
minimal increase in 
respiratory rate 
Wheezing with 
nasal flaring, 
intercostal 
retractions, or pulse 
oximetry 90-95% 
Wheezing with 
dyspnea at rest, 
inability to perform 
normal activities, or 
pulse oximetry 
<90% 
Respiratory failure 
or anaphylaxis 

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APPENDIX D:  LABORATORY VALUES* 
 
 
 
* Based on NIAID Division of AIDS Table for Grading the Severity of Adult and Pediatric Adverse 
Events, December 2004 
(http://www.niaid.nih.gov/labsandresources/resources/daidsclinrsrch/documents/daidsaegradingta
ble.pdf), adapted for normal reference values for age for our reference laboratory (Asociacion Civil 
Selva Amazonica: Iquitos, Peru;  www.selvaamazonica.org) 
 
Serum Chemistries 
(normal range for 0-2 year old 
children at our reference lab) 
Mild  
(Grade 1) 
Moderate  
(Grade 2) 
Severe  
(Grade 3)  
Potentially Life 
Threatening  
(Grade 4)**  
8.  Bilirubin, mg/dL (< 1.0) 
1.1-1.9 
2.0-2.9 
3.0-7.5 
>7.5 
9. Blood Urea Nitrogen  
    BUN mg/dL (< 23) 
24-60 
61-80 
 >81 
Requires dialysis  
10. Creatinine, mg/dL (< 0.4) 
0.5 – 1.0  
1.1 – 1.6  
1.7 – 2.0  
> 2.0 or requires 
dialysis  
11. Liver Function Tests –ALT (< 52) 
units per liter (U/L)     
53-200 
201-400 
401-600 
> 600 
12. Liver Function Tests –AST (< 39) 
units per liter (U/L)     
40-200 
201-400 
401-600 
> 600 
Hematology   
 
Mild  
(Grade 1)  
Moderate  
(Grade 2)  
Severe  
(Grade 3)  
Potentially Life 
Threatening  
(Grade 4)  
13. Children 6-24m : Hemoglobin 
gm/dL (> 11) 
10.0 – 10.9  
 
7.0 – 9.9  
< 7.0 
Cardiac 
insufficiency due to 
anemia 
13a.Children 2-5 m: Hemoglobin 
gm/dL (>9.5) 
9.4-8.5 
6.5-8.4 
<6.5 
Cardiac 
insufficiency due to 
anemia 
14. Total WBC Count - cell/mm3 
(6,000-17,500) 
17,501 – 
20,000  
20,001 – 
25,000  
25,001 – 28, 
000  
> 28,000  

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APPENDIX E.   FEVER SEVERITY GRADING 
  
 
 
 
APPENDIX F.  PRODUCT INFORMATION 
 
Downloaded from www.biogaia.con May 8 2015 
 
Lactobacillus reuteri 
Lactobacillus reuteri is one of the most well researched probiotic species, especially in the very 
young.  
To date 137 clinical studies using BioGaia’s human strains of Lactobacillus reuteri have been 
performed in more than 11 700 individuals of all ages. Results are published in 108 articles in 
scientific journals (February 2015). 
In this section you will learn about the different aspects of Lactobacillus reuteri, such as the history 
of this probiotic, its natural occurrence and about Reuterin, the antimicrobial substance that 
Lactobacillus reuteri produces. 
- See more at: http://www.biogaia.com/lactobacillus-reuteri#sthash.N1KjzKol.dpuf 
History of Lactobacillus reuteri 
Lactobacillus reuteri is a Gram-positive bacterium whose natural habitat is the digestive tract 
of mammals and birds. 
Body Temperature 
Mild  
(Grade 1) 
Moderate  
(Grade 2) 
Severe  
(Grade 3) 
Potentially Life 
Threatening  
(Grade 4)  
15. Fever (°C)  
            
38.0 – 38.4  
 
38.5 – 38.9  
 
39.0 – 40  
 
> 40  
 

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The species Lactobacillus reuteri was recognized and recorded in scientific classifications of 
lactic acid bacteria, as early as the turn of the 20th century. At this time it was mistakenly 
grouped as a member of Lactobacillus fermentum. In the 1960s, further work by German 
microbiologist Gerhard Reuter could distinguish Lactobacillus reuteri from Lactobacillus 
fermentum. Reuter reclassified the species as "Lactobacillus fermentum biotype II". 
Lactobacillus reuteri was eventually identified as 
a distinct species in 1980 by Kandler et al. They 
chose the species name "reuteri", after discoverer 
Gerhard Reuter, and Lactobacillus reuteri has 
since been recognized as a separate species within 
the Lactobacillus genus. 
The first strain of Lactobacillus reuteri for human 
use was isolated in 1990 from the breast milk of a 
Peruvian mother living in the Andes. This strain 
was deposited at the American Type Culture 
Collection (ATCC) as Lactobacillus reuteri SD 
2112 (SD = safety deposit), and was later given the 
number ATCC 55730. 
In 2007 Lactobacillus reuteri ATCC 55730 was 
replaced by the “daughter strain” Lactobacillus 
reuteri DSM 17938. The only difference between 
the strains is the loss of two plasmids of ATCC 
55730 that carried resistance to tetracycline and 
lincomycin, respectively. 
The new strain Lactobacillus reuteri DSM 17938 
can be considered comparable to Lactobacillus 
reuteri ATCC 55730 in all aspects of probiotic 
function: genetic sequence information has shown 
the irrelevance of the genes of the plasmids to the 
functions of the strain, and also confirmed that all 
tested probiotic-related functions of Lactobacillus 
reuteri DSM 17938 are retained from those 
of Lactobacillus reuteri ATCC 55730. 
The commercial name of Lactobacillus 
reuteri DSM 17938 is Lactobacillus 
reuteri Protectis. Other human strains that are 
used commercially are Lactobacillus 
reuteri ATCC PTA 5289 and ATCC PTA 6475. 
- See more at: http://www.biogaia.com/history-
lactobacillus-reuteri#sthash.zJI8di1z.dpuf 

PRIDEC-Peru 
Protocol # 1: Safety of Lactobacillus reuteri in healthy children aged 2-24 months 
Version 2.0  
11 Jan 2019 
71 
 
 
Natural occurrence 
Lactobacillus reuteri is one of very few probiotic species whose natural habitat is the digestive tract 
and it is therefore especially well adapted to thrive in this kind of environment. It is among the first 
bacterial species to become naturally established in the normal microbiota of the newborn. Most 
other probiotic strains are only temporary or transient inhabitants of the gastrointestinal tract, 
derived from the intake of food. 
In humans Lactobacillus reuteri has been isolated from:  
• 
Breast milk 
• 
The mouth 
• 
The stomach 
• 
The small intestine (duodenum and ileum) 
• 
The colon (large intestine) 
• 
Faeces 
• 
The vagina 
- See more at: http://www.biogaia.com/natural-occurrence#sthash.d26hjspa.dpuf