Exploratory Pilot Studies to Demonstrate Mechanisms of Preventing Antibiotic-Associated Diarrhea and the Role for Probiotics
Phase: EARLY_PHASE1
Conditions: Antibiotic-associated Diarrhea
Sponsor: Georgetown University

IND # 13691 Merenstein/Fraser
Clinical Research Protocol
CLINICAL RESEARCH PROTOCOL
Exploratory Pilot Studies to Demonstrate Mechanisms of Preventing
Antibiotic-Associated Diarrhea and the Role for Probiotics (R33)
Principal Investigators:
Daniel J. Merenstein, MD (contact)
Director of Research Programs
Georgetown University Medical Center
Claire M. Fraser, PhD
Director, Institute for Genomic Sciences
University of Maryland Baltimore
Supported by:
The National Center for Complementary and Integrative Health
4 R33 AT009622-03
Study Intervention Provided by:
The Pennsylvania State University
Department of Food Science
Sponsor of IND:
Daniel J. Merenstein, MD
Georgetown University Medical Center
IND # 13691
IRB ID: STUDY00002431
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Protocol Revision History
Version Number: 0.1
Version Date: April 6, 2020
Summary of Revisions Made: Initial draft.
Version Number: 1.0
Version Date: November 1, 2020
Summary of Revisions Made: Approved protocol.
●
At the request of the statistician, the number per group has been updated in order to balance
randomization over time (to 28/group and 12 in the antibiotic-only group).
●
Updated ingredient component in Table 5.
●
Global correction of typographical and administrative errors. Clarified text to align with the approved
R33 research plan.
Version Number: 1.1
Version Date: January 26, 2021
Summary of Revisions Made:
●
Addition of prior R61 study participation to the exclusion criteria in Section 4.4.
Version Number: 1.2
Version Date: May 12, 2021
Summary of Revisions Made:
●
Addition of Spanish to inclusion criteria in Section 4.2.
●
Updated précis timeline.
●
Corrected sample size typo in Table 8.
Version Number: 1.3
Version Date: June 14, 2021
Summary of Revision Made:
●
Updated Section 4.5 Study Enrollment Procedures to account for higher withdrawals prior to
randomization. Change from 150 to 200 enrollees to end with the final (original) sample size.
Version Number: 1.4
Version Date: January 25, 2022
Summary of Revision Made:
●
Diet recall data will be collected for one day at the day 30 follow-up, instead of two days during the
run-in period. The paragraph on diet recall data was removed from Section 6.5.2 Baseline
Assessments and added to Section 6.7.3 Completion/Final Evaluation. The précis and Table 8:
Participant Timeline were updated accordingly.
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TABLE OF CONTENTS
Page
CLINICAL RESEARCH PROTOCOL
1
Protocol Revision History
2
TABLE OF CONTENTS
3
STUDY TEAM ROSTER
6
PARTICIPATING STUDY SITES
6
PRÉCIS
7
1. STUDY OBJECTIVES
9
1.1 Primary Objective
9
1.2 Secondary Objectives
9
2. BACKGROUND AND RATIONALE
10
2.1 Background Information
10
2.2 Microbiota Play an Important Role in the Gastrointestinal Tract
10
2.3 General Bifidobacterium Information
10
2.4 Likely Mechanisms of BB-12 Health Effects
10
2.5 BB-12 Survival in Stools
11
2.6 Safety of Use of BB-12
11
2.7 Probiotics Treating and Preventing Antibiotic-Associated Diarrhea (AAD)
11
2.8 Functional Foods and Yogurt
12
2.9 Summary and Significance
12
2.10 Summary of Completed Studies
13
2.11 Rationale
16
2.12 Potential Risks and Benefits
16
3. STUDY DESIGN
17
3.1 Primary Outcome Measure
17
3.2 Secondary Outcome Measures
18
4. SELECTION AND ENROLLMENT OF PARTICIPANTS
19
4.1 Recruitment Strategy
19
4.2 Inclusion Criteria
19
4.3 Inclusion of Women and Minorities
19
4.4 Exclusion Criteria
19
4.5 Study Enrollment Procedures
20
5. STUDY INTERVENTIONS
21
5.1 Interventions, Administration, and Duration
21
5.2 Handling of Investigational Product
21
5.3 Concomitant Interventions
26
5.4 Adherence Assessment
27
6. STUDY PROCEDURES
28
6.1 Schedule of Evaluations
28
6.2 Description of Evaluations
28
6.3 Participant Stipends
31
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6.4 Screening Evaluation and Consenting Procedure
31
6.5 Enrollment, Baseline, and Randomization
32
6.6 Blinding
33
6.7 Follow-up Visits
34
7. SAFETY ASSESSMENTS
36
7.1 Specification of Safety Parameters
36
7.2 Methods and Timing for Assessing, Recording, and Analyzing Safety Parameters
36
7.3 Adverse Events and Serious Adverse Events
36
7.4 Characteristics of an Adverse Event
37
7.5 Expected Risks
39
7.6 Reporting Procedures
40
7.7 Follow-up for Adverse Events
40
7.8 Safety Monitoring
40
8. INTERVENTION DISCONTINUATION
42
9. STATISTICAL CONSIDERATIONS
43
9.1 General Design
43
9.2 Sample Size and Randomization
43
9.3 Definition of Populations
44
9.4 Interim Analyses and Stopping Rules
44
9.5 Outcomes
44
9.6 Anticipated Results
44
9.7 Data Analyses
44
9.8 Missing Data
45
10. DATA COLLECTION AND QUALITY ASSURANCE
46
10.1 Data Collection Forms
46
10.2 Data Management
46
10.3 Quality Assurance
47
11. PARTICIPANT RIGHTS AND CONFIDENTIALITY
49
11.1 Institutional Review Board (IRB) Review
49
11.2 Informed Consent Forms
49
11.3 Participant Confidentiality
49
11.4 Study Discontinuation
49
11.5 Ethical Standard
49
12. COMMITTEES
50
13. PUBLICATION OF RESEARCH FINDINGS
50
14. REFERENCES
51
15. SUPPLEMENTS/APPENDICES
56
I. Bristol Stool Chart
56
II. Summary of BB-12 Safety Articles
57
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Clinical Research Protocol
Signature Page
The signature below constitutes the approval of this protocol and the attachments, and provides the
necessary assurances that this trial will be conducted according to all stipulations of the protocol,
including all statements regarding confidentiality, and according to local legal and regulatory
requirements and applicable US federal regulations and International Conference on Harmonisation
guidelines.
Principal Investigator: Daniel Merenstein, MD
Signed:
Daniel Merenstein, MD
Principal Investigator
Date: 05/12/2021
Statement of Compliance
The study will be carried out in accordance with Good Clinical Practices (GCP) as required by the
following:
●
United States (US) Code of Federal Regulations (CFR) applicable to clinical studies (45 CFR Part 46;
21 CFR Part 50, 21 CFR Part 56, and 21 CFR Part 312)
●
ICH E6 (R2); 83 FR 8882
●
NIH Clinical Terms of Award
All key personnel (all individuals responsible for the design and conduct of this study) have completed
Human Subjects Research Protection Training.
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STUDY TEAM ROSTER
Recruitment Site
Principal Investigator:
Daniel J. Merenstein, MD
Professor
Director of Research Programs
Department of Family Medicine
Georgetown University Medical Center
4000 Reservoir Road, NW
Building D 240
Washington, DC 20007
P: 202-687-2745 F: 202-687-7244
djm23@georgetown.edu
Laboratories
Principal Investigator:
Claire M. Fraser, PhD
Institute for Genomic Sciences
University of Maryland Baltimore
Baltimore, MD
cmfraser@som.umaryland.edu
Co-Investigator:
Maureen A. Kane, PhD
School of Pharmacy
University of Maryland Baltimore
Baltimore, MD
mkane@rx.umaryland.edu
Biostatistician:
Frank D’Amico PhD
University of Pittsburgh Medical Center, St. Margaret
Duquesne University Department of Mathematics
212 Sweet Gum Road
Pittsburgh, PA 15238
412-820-9587
damicof@upmc.edu
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PRÉCIS
Study Title: Exploratory Pilot Studies to Demonstrate Mechanisms of Preventing Antibiotic-Associated
Diarrhea and the Role for Probiotics
Protocol Title: Observing and Understanding antibiotic-induced Reduction in SCFAs and time dependent
administration of BB-12 proBIOTIC (OURBIOTIC)
Phase: Mechanistic Study
Population: 124 healthy adults ages 18-65 years
Number of Sites: Single Center
Study Duration: R33 Phase: 36 months
Description of Study Design: Randomized, double-blinded, controlled clinical trial
National Clinical Trial (NCT) Number: NCT04414722
Georgetown University Institutional Review Number: STUDY00002431
Primary Objective:
To determine the ability of BB-12 to impact antibiotic-induced reduction in SCFA concentration, as
reflected by the levels of acetate. The effect size according to an adjusted Cohen’s d will be calculated at
day 7 and 30. SCFA produced by anaerobic gut microbiota will be quantified from human fecal samples
via liquid chromatography-tandem mass spectrometry (LC-MS/MS) and adjusted for each participant’s
own baseline value. Also, to determine the impact of concurrent versus delayed administration of BB-12
in relation to the antibiotics.
Secondary Objectives:
To determine the ability of BB-12 to impact antibiotic-induced disruption of the gut microbiota with 16S
rDNA profiling, with the addition of the time variable. This will be assessed at days 7 and 14. Changes in
absolute abundance of specific bacterial genera (with a particular emphasis on known butyrate
producers in the Firmicutes phylum) will also be assessed with qPCR. Also, to determine the role of
acetate produced by BB-12 in situ as cross-feeder to butyrate producers in the Firmicutes phylum
resulting in an up-regulation of butyrate biosynthetic pathways.
Design and Outcomes:
This is a randomized, placebo-controlled double-blinded, single center, mechanistic study to test the
mechanism of Bifidobacterium animalis subsp. lactis (B. lactis) strain BB-12 (BB-12) yogurt in AAD,
compared to yogurt without BB-12, in healthy adults.
Interventions and Duration:
The probiotic-supplemented yogurt is composed of the investigational agent, BB-12, and contains the
yogurt starter culture YF-L702 (a mixed culture of Streptococcus thermophilus and Lactobacillus
delbrueckii subsp. bulgaricus). The yogurt starter culture will be present and alive in the control yogurt.
Participants are to provide informed consent at Baseline and complete a 30-day run-in period, during
which they will refrain from all probiotics, and begin the antibiotic and yogurt interventions on Day 1.
Participants will be randomized into five groups in a 28:28:28:28:12 ratio with proper allocation
concealment (Figure 1).
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Group 1 (n=28): Probiotic BB-12 supplemented yogurt, 4-ounces (which delivers ≥1010 CFU of BB-12)
taken orally for 14 days concurrently with amoxicillin-clavulanate (AMC) 875/125 mg, twice daily, for 7
days.
Group 2 (n=28): Probiotic BB-12 supplemented yogurt, 4-ounces taken orally for 14 days. Yogurt is
consumed four hours after taking AMC 875/125 mg, twice daily, for 7 days.
Group 3 (n=28): Control yogurt, 4-ounces taken orally for 14 days concurrently with AMC 875/125 mg,
twice daily, for 7 days.
Group 4 (n=28): Control yogurt, 4-ounces taken orally for 14 days. Yogurt is consumed four hours after
taking AMC 875/125 mg, twice daily, for 7 days.
Group 5 (n=12): No Yogurt. AMC 875/125 mg, twice daily, for 7 days.
Figure 1.
Fecal samples will be collected at home (at baseline (pre and post run-in), days 7, 14, 21 and 30) for
SCFA and microbiome testing. Participants will be given detailed, written and illustrated instructions on
proper sample collection procedures, and research staff will review these protocols with participants during
enrollment.
Follow-up phone visits will be completed on Days 7, 14, and 30. Participants will keep a daily diary to track
number, shape and consistency of bowel movements, if/when antibiotic and/or yogurt was consumed,
Bristol Stool chart, general health, use of other medicines or products, adverse events, and the
Gastrointestinal Symptom Rating Scale (GSRS) - Irritable Bowel Syndrome (IBS) version.
Duration: Each participant will be in the study for approximately 8 weeks total. During the first 30 days,
the participant will refrain from antibiotics and probiotics (run-in period) and provide two baseline
samples. The participant will be taking the antibiotic intervention for 7 days and taking the yogurt
intervention for 14 days. There are two more data collection visits on day 21 and 30.
Pre run-in
Baseline
Run-in
(30 days)
Post run-in
(0/+2)
Day 1
Day 7
(±2)
Day 14
(±2)
Day 21
(±2)
Day 30
(±2)
Pre-screening
(prior to baseline)
Informed Consent
Inclusion/
exclusion
Enrollment
No
antibiotics
No
probiotics
Fecal
sample
Fecal sample
Post-run in
inclusion/
exclusion
Post run-in health
status
AMC start
Yogurt start
Diary start
●AMC end
●GSRS-IBS
●Fecal sample
●Follow-up
Yogurt end
Diary end
GSRS-IBS
Fecal sample
Follow-up
Fecal
sample
●Fecal
sample
●Follow-up
●Diet recall
(1 day)
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Demographics
Baseline health
GSRS-IBS
Fecal sample
Randomization
Sample Size and Population:
The population will be generally healthy adults of ages 18-65 years. This randomized controlled trial is
open to all eligible adults who meet the inclusion criteria. We are not proposing exclusion of any specific
sex/gender or racial/ethnic group. Approximately one hundred and fifty participants will be recruited from
the greater community and clinical sites in the Washington, DC metropolitan area, and one hundred and
twenty-four (n=124) participants will be randomized and commence the study. Based on the
demographics of the network and recruitment rates in previous studies at our site, we anticipate about
50% of participants will be women, 20% will be primary Spanish speakers, and 30-40% will self-identify
as one of the racial or ethnic minority populations.
1. STUDY OBJECTIVES
1.1
Primary Objective
To determine the ability of BB-12 to impact antibiotic-induced reduction in SCFA concentration as
reflected by the levels of acetate. on day 7. The R33 will also add a time variable to determine the impact
of concurrent versus delayed administration of BB-12 in relation to the antibiotics. SCFA produced by
anaerobic gut microbiota will be quantified from human fecal samples via liquid chromatography-tandem
mass spectrometry (LC-MS/MS) and adjusted for each participant’s own baseline value. As a secondary
analysis, we will also perform normal area under the curve (AUC) analysis of the percent change in
acetate from baseline.
Primary Hypothesis: Antibiotics will result in a reduction in fecal SCFA by day 7. BB-12 supplementation
will protect against antibiotic-induced SCFA reduction and/or be associated with a quicker return to
baseline SCFA levels, as compared to the control group. We hypothesize that concurrent administration
of the probiotic and antibiotic is not necessary for the probiotic impact on SCFA.
1.2
Secondary Objectives
To determine the ability of BB-12 to impact antibiotic-induced disruption of the gut microbiota with 16S
rDNA profiling. This will be assessed at days 7 and 14. Also, to determine the impact of concurrent
versus delayed administration of BB-12 in relation to the antibiotics. Changes in absolute abundance of
specific bacterial genera (with a particular emphasis on known butyrate producers in the Firmicutes
phylum) will also be assessed with qPCR.
Secondary Hypothesis: Antibiotics will result in a decrease in the overall number and diversity of bacterial
species present in the fecal microbiota. We hypothesize that concurrent administration of the probiotic
and antibiotic is not necessary for the probiotic effect on the composition of the gut microbiota. This will
be assessed at baseline and days 7, 14, 21 and 30, and we would expect a decrease in at least days 7
and 14. BB-12 supplementation will protect against antibiotic-induced shifts in the microbiota and/or will
be associated with a quicker return to a baseline microbiota composition, as compared to the control
group. Microbiota composition will be assessed with 16S rDNA profiling and qPCR.
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Tertiary Hypothesis and Aim: We hypothesize that acetate produced by BB-12 in situ will cross-feed
butyrate producers in the Firmicutes phylum resulting in an up-regulation of butyrate biosynthetic
pathways. We will longitudinally characterize the gut microbiota with high-throughput metatranscriptomics
in order to generate complementary information on the impact of antibiotics plus and minus BB-12 on
overall microbiome function.
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2.
BACKGROUND AND RATIONALE
2.1
Background Information
Probiotics are live microorganisms that, when administered in sufficient amounts, may improve health.(1)
Often, probiotics are ingested as supplements in powder, pill or liquid forms, designed specifically for
medicinal benefit. Such supplements have shown potential benefits in treatment and prevention of varied
diseases, including diarrhea, asthma, necrotizing enterocolitis and allergies.(2-6) As an alternative to
supplements, probiotics are also included as ingredients in fermented dairy products to produce
functional foods; that is foods providing health benefits beyond their nutritional value.(7-10) Yogurt, for
example, is a fermented milk product often considered a functional food. In fact, two-thirds of primary
care physicians who counsel patients about nutrition recommend consuming yogurt containing live active
cultures for the health benefits associated with this food.(11) Despite this common practice, however,
evidence for beneficial health outcomes is limited.
2.2
Microbiota Play an Important Role in the Gastrointestinal Tract
The gastrointestinal tract contains a complex commensal microbiota that contributes to homeostasis in
the gastrointestinal tract. The intestinal microbiota includes hundreds of species of facultative and
obligate anaerobes.(12) The human intestine is home to over 100 trillion microorganisms. When
functioning appropriately, this high-species diversity produces homeostasis of the gut.(13) This balance
is often disturbed by medical interventions such as antibiotics or viral diseases, causing a disturbance of
the fecal microbiota and resulting in, among other effects, decreased short chain fatty acid metabolism.
This causes the accumulation of luminal carbohydrate, subsequent pH changes, and water
absorption.(14) Supplemental probiotics may help regulate the microbiota when disturbed by outside
influences.(15)
2.3
General Bifidobacterium Information
Probiotics marketed as nutritional supplements and found in functional foods, such as yogurts, are
principally members of the genera Bifidobacterium and Lactobacillus. Members of these genera are
Gram-positive facultative anaerobes and are also classified as lactic acid bacteria (LAB). Bifidobacterium
species, particularly B. lactis BB-12, the principal focus of this study, can be found in the gastrointestinal
tract (GIT) as both autochthonous (native to a particular place) and allochthonous (derived from outside a
system) residents.(16) Newborns, especially those that are breast-fed, are colonized with bifidobacteria
within days after birth. Once the child is weaned, the population of these bacteria in the colon appears to
be relatively stable until advanced age when it appears to decline.(16, 17) Furthermore, B. lactis, unlike
the majority of anaerobic bifidobacteria, are moderately tolerant of oxygen. It is for this reason that B.
lactis is so extensively used for research and commercial application.(18)
2.4
Likely Mechanisms of BB-12 Health Effects
There are many potential mechanisms by which probiotics are believed to exert their effects. Possible
mechanisms of probiotic effect include: 1) production of antimicrobial substances; 2) binding to and
penetrating gastrointestinal receptors; 3) competition for nutrients; 4) enhancement of mucosal barrier
function; 5) altered immunoregulation, including both anti-inflammatory and immunostimulatory
responses; and, 6) ferment glucose, lactose, and fructose, thereby lowering fecal pH.(19-25) Due to the
immune system’s role in a multitude of disease states, many studies have concentrated on the
immunoregulatory effect of probiotics, examining probiotics’ role in diseases, such as eczema with
potential mechanisms involving T cell, B cell, epithelial cell, dendritic cell, macrophage, nature killer cell,
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antibody, and cytokine effects.(26-28) After providing infants who manifested atopic eczema (mean age
of 4.6 months) with a BB-12 infused formula, Isolauri, et al., found significant skin improvements in the
probiotic group compared to the control group (P=0.002).(28)
Additionally, many believe that inflammatory bowel disease (IBD) appears to be caused by immune
responses to commensal enteric bacteria in predisposed individuals.(24) A commercial blend of eight
different probiotic strains (Streptococcus thermophilus, three strains of Bifidobacterium species and four
strains of Lactobacillus species), VSL3, has been shown to limit epithelial inflammatory responses in vivo
and in vitro, and is often prescribed for individuals with IBD.(29-34)
Further support of an immunoregulatory response is a study conducted by Fukushima et al. on healthy
Japanese children between the ages of 15 and 31 months. The children were fed a follow-up formula
infused with 109 CFU BB-12 and were found to have a significant increase in total IgA and anti-poliovirus
IgA, respectively.(35) This suggests ingestion of the probiotic formula containing BB-12 and colonization
by the strain can trigger IgA production by the host. This type of data demonstrates that probiotics
positively influence the maturation process of immunity.
2.5
BB-12 Survival in Stools
The ability to isolate an orally administered probiotic in the stools is considered to be a good indicator
that the strain is able to reach key target sites throughout the intestinal tract. The ability of BB-12 to
survive intestinal transit has been documented in several studies.(36-39) Taken together, these studies
document the ability of BB-12 to survive gastrointestinal transit and persist for 1-3 weeks post feeding.
However, the degree of persistence is likely due to doses fed, delivery vehicle, host factors and the ability
of BB-12 to grow during colonization. Studies such as the one proposed here are needed to document
the success of probiotic survival in foods supplemented with probiotics.
2.6
Safety of Use of BB-12
Lactic acid bacteria (LAB) in foods have a long history of safe use. LAB and related organisms (with the
exception of enterococci) are seldom associated with infections, except in immunodeficient individuals or
individuals with severe underlying illness.(40) Although bifidobacteria are not naturally found in food, they
have been used as ingredients of dietary supplements and added to foods as functional ingredients for
decades.(16) The BB-12 strain has been used in numerous controlled human studies in pediatric and
adult populations with no adverse incidents reported. BB-12, in combination with a strain of S.
thermophilus, was the subject of a GRAS petition to the FDA March 2002 (Agency GRAS Notice GRN
000049), where the FDA did not object to use of these bacteria in infant formula.
Although transferable antibiotic resistances are not very common among LAB, they do occur. BB-12 was
evaluated for such resistance genes and was found to contain the tetracycline resistance gene tet(W).
Nestlé provided these data to the FDA in support of its view that the presence of tet(W) does not affect
the safety of BB-12 as an ingredient in infant formula. In November 2005, the FDA responded to this
additional information stating that:
“Based on the information provided by Nestlé, as well as other information available to FDA, the
agency has no questions at this time regarding Nestlé's conclusions that the presence of the
tet(W) gene in their B. lactis strain does not affect the safety of the intended use of B. lactis as
an ingredient in infant formula and that the discovery of the tet(W) gene in B. lactis does not
change their previous conclusion that B. lactis is GRAS for its intended use as an ingredient in
infant formula.”
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A summary of the BB-12 safety articles, using the identical strain as in this protocol, is included in the
Appendix. Consistent with this data the R61 demonstrated more proportional adverse events in the
control than active group. All adverse events were self-limiting and no serious adverse events occurred.
2.7
Probiotics Treating and Preventing Antibiotic-Associated Diarrhea (AAD)
Acute diarrhea is commonly caused by infections or antibiotics. Children and adults are often placed on
antibiotics and the rate of diarrhea associated with antibiotic usage is 20-35%, with 10-20% of the
diarrhea due to Clostridium difficile.(41-49)
Antimicrobial resistance, stemming from antibiotic use, is a multifactorial problem. Major elements of the
problem include over-prescription of antibiotics by physicians and poor compliance by patients.(50)
Although educating physicians and the lay public has led to dramatic decreases in antibiotic
prescriptions, approximately 25% of all visits for children under age five still results in a prescription for
antibiotics.(48, 49, 51-53) In fact, children ages three to 36 months average over two antibiotic
prescriptions per year, with nearly 30% receiving over four prescriptions per year.(49)
Probiotics have the potential to reduce the rate of AAD. Numerous meta-analyses have examined the
potential role of probiotics, with the positive studies showing an effect size of 25-35%.(54-59) In a recent
study, Correa et al. enrolled 80 children between six and 36 months of age who were receiving oral or
parental antibiotic therapy. In addition to the antibiotics, infants were given either a combination of 107 B.
lactis and 106 S. thermophilus or a placebo. In this study, 31% of the placebo group experienced diarrhea
compared to 16% of the probiotic group (p, 0.044).(60) In a similar study, Arvola et al. enrolled children
on antibiotics and gave them either a matching placebo or LGG at 2x1010 CFU/day. After two weeks, 5%
of the LGG group had diarrhea compared to 16% of the placebo group, with a treatment effect of -11%
(95% confidence intervals of -21%, 0%).(61)
2.8
Functional Foods and Yogurt
Consuming foods to provide health benefits beyond basic nutritional needs is a well-accepted practice in
the United States. For example, many parents feed their children cereal fortified with minerals and
nutrients. Another example is the addition of folic acid to many food products; this has greatly decreased
the incidence of neural tube defects in the U.S.(62, 63) Functional foods are enriched or enhanced foods
that provide health benefits beyond the inherent nutritional value of the food.(64) For example,
BENECOL® is a margarine type food proven in clinical trials to reduce LDL cholesterol.(65, 66) The
promise of using functional foods to mitigate disease and promote health is one of the major reasons so
many resources are being devoted to this exciting new field.(9, 67-70)
Yogurt is defined as milk fermented with two specific bacterial species, L. bulgaricus and S. thermophilus.
To differentiate yogurt containing live and active cultures and those pasteurized after fermentation, the
National Yogurt Association developed a seal, the Live Active Culture Seal. Any yogurt containing 10 8
CFU/g at the time of manufacture may display this seal. A dose of probiotics of 10 8-1010 cfu/day is the
amount that has generally been studied and has resulted in health benefits. However, several surveys
suggest the probiotic content of yogurt and probiotic supplements may be lower than labels would imply.
Hamilton-Miller et al. examined 52 European probiotic products, including 11 yogurts, and found that
none gave any indication of actual numbers and less than 50% accurately stated the bacterial species
contained in the product.(71) This study highlights the lack of useful information available to consumers
when selecting products containing probiotics.
Beniwal et al. studied the effect of yogurt containing L. acidophilus on AAD in an unblinded manner in
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hospitalized patients receiving oral or intravenous antibiotics. The 105 patients in the yogurt group
received two 8-ounce vanilla yogurt containers for eight days. Yogurt supplemented with L. acidophilus
decreased AAD from 24% to 12%.(72) However, the unblinded nature of this study makes these results
only suggestive.
2.9
Summary and Significance
Bifidobacterium lactis strain BB-12 is a commercially available probiotic strain that has been used in a
number of feeding and clinical trials.(73-84) BB-12 has been found to survive transit through the
stomach, small intestine and colon.(85-87) Additionally, long term consumption of probiotic-containing
formula at levels as high as 1 billion cfu/g (240 g serving) was found to be safe and well tolerated by
children.(88, 89)
Along with providing health benefits, BB-12 is a highly sought probiotic because it has a high resistance
to acids in fermented dairy products and is aerotolerant.(90) Importantly for our study, it is believed that
dairy products provide a protective environment allowing for probiotic bacteria to remain viable through
digestion.(90) An issue concerning probiotic delivery in dairy products is a decrease in viable cells by the
end of the product’s shelf-life. McBearty et al. manufactured a probiotic cheddar cheese containing 108
cfu/ml BB-12.(91) The levels of BB-12 remained constant throughout six months of ripening at a pH of
5.3. Fukushima et al. incorporated BB-12 into an unfermented, canned infant formula.(35) The levels of
viable BB-12 cells did not decrease for at least 18 months in the sealed can and for 30 days after the can
was opened. BB-12 appears to persist in dairy products; however, the persistence of BB-12 in yogurt,
where the pH is lower than 5.0, has not been as widely studied.
Nearly 190 million outpatient antibiotic prescriptions were given in the period of 1998-99. Nearly one
quarter (22%) of all pediatric visits resulted in antibiotic prescriptions. A study of 1992 data reported that
office visits for colds, upper respiratory tract infections, and bronchitis resulted in approximately 12 million
antibiotic prescriptions, accounting for 21% of antibiotic prescriptions.(92) Another study found that
antibiotics were prescribed for 78% of acute bronchitis episodes, 65% of acute pharyngitis episodes,
81% of acute sinusitis episodes, and 33% of nonspecific URI episodes, many of which are of viral
etiology.(93)
Noncompliance with prescriptions is often due to side effects, most commonly AAD.(94) Diarrhea is a
common and costly disease of children in the U.S. Children less than five years of age experience 20-35
million episodes of diarrhea per year. These episodes lead to 2-3.5 million physician visits (which
account for 10% of all visits by children), more than 200,000 hospitalizations (13% of hospital admissions
in children less than five years), and 325-425 deaths annually.(95-98) There have been many promising
studies demonstrating the potential benefits of probiotic supplementation in preventing diarrhea.
However, currently, probiotics are generally given in powder, pill or liquid forms and generally sold at
health food stores. Yogurt, which is more readily available and accepted, has the potential to greatly
influence health if efficacious probiotic dosages and species are available in the product.
This study will evaluate whether a well-defined, probiotic-containing yogurt can be used as an effective
delivery vehicle for probiotics. Our long-term goal is to create yogurt with sufficient probiotic dosages to
positively impact many different aspects of childhood and adult health. There is widespread acceptance
of yogurt with added cultures among the general population and this is one of the reasons we believe
yogurt will be more acceptable than supplements.
2.10
Summary of Completed Studies
I. Safety of BB-12 Supplemented Strawberry Yogurt For Healthy Adults on Antibiotics: The primary
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objective of this Phase 1 study was to establish safety of probiotic BB-12 supplemented yogurt
consumed daily for 10 consecutive days by adults concurrently taking antibiotics for an upper respiratory
infection. Forty subjects were randomized into two groups, BB-12 supplemented yogurt and control
yogurt without BB-12, using the random allocation rule. Forty participants were recruited through the
Capital Area Primary Care Research Network (CAPRICORN), a practice-based research network. The
participants in the study were healthy individuals between the ages of 18-65 years and who were
prescribed treatment with a penicillin class antibiotic regimen for an upper respiratory infection. A
respiratory infection was classified as any infection the physician designates as strep or non-strep
pharyngitis, otitis media, pneumonia, sinusitis or bronchitis that results in a 10-day prescription of
antibiotics. All reported adverse events were tabulated by type and treatment group (see Table 1). There
were no serious adverse events reported. No participants withdrew from the study due to adverse
events.
II. Safety of BB-12 Supplemented Strawberry Yogurt For Healthy Children: The primary objective of
our Phase 1 pediatric study was to assess the safety of strawberry flavored yogurt supplemented with
Bifidobacterium animalis subsp. lactis strain BB-12 when consumed daily for 10 consecutive days by
generally healthy children. The secondary objective was to assess the ability of BB-12 to survive gastric
transit. The participants in the study were healthy individuals between the ages of 1-5 years. Participants
were recruited through CAPRICORN. Twenty-nine participants received the BB-12 yogurt and 31
participants received the control yogurt. All reported adverse events were tabulated by type and
treatment group (Table 2). Three serious adverse events were reported (details in Table 2). No
participant deaths were reported and no participants withdrew from the study due to adverse events.
Both studies, Safety of BB-12 Supplemented Strawberry Yogurt For Healthy Adults on Antibiotics and
Safety of BB-12 Supplemented Strawberry Yogurt For Healthy Children were conducted under the same
IND.
Table 1. Phase I Adult Study Adverse Events
Event
Control Group (N=21)
BB-12 Group (N=19)
N
%
N
%
Abdominal pain
1
5
0
0
Acid reflux
0
0
3
16
Allergies (Seasonal, allergic rhinitis)
1
5
0
0
Back pain
0
0
1
5
Bloating
3
14
0
0
Bowel sounds
1
5
0
0
Breathing problems
3
14
0
0
Constipation
3
14
2
11
Cough
8
38
6
32
Decreased appetite
7
33
5
26
Diarrhea
2
10
2
11
Dizziness
0
0
1
5
Drug hypersensitivity
1
5
0
0
Ear aches
5
24
3
16
Fever
2
10
2
11
Gas
6
29
1
5
Headache
7
33
5
26
Irritability
1
5
1
5
Lethargy
8
38
4
21
Loose stool
9
43
4
21
Muscle pain
0
0
1
5
Nasal congestion
7
33
9
47
Nausea
1
5
0
0
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Runny nose
7
33
5
26
Sore throat
5
24
6
32
Stomach pain
8
38
3
16
Tonsil swelling
1
5
0
0
Vaginal discomfort
1
5
0
0
Vomiting
0
0
1
5
Yeast infection
0
0
2
11
Total events reported
98
 
67
 
Table 2. Phase I Pediatric Study Adverse Events
Days 0-10 (During Intervention)
Days 11-180 (Post-Intervention)
Event
Control
BB-12
Total
Control
BB-12
Total
Allergies (e.g. seasonal)
1
2
3
1
3
4
Broken finger
 
 
 
 
1
1
Bronchiolitis
 
 
 
 
1
1
Cold
2
 
2
2
1
3
Constipation
 
3
3
2
1
3
Cough
5
10
15
1
5
6
Croup
 
 
 
 
1
1
Cut finger
 
 
 
1
 
1
Diarrhea
2
2
4
2
5
7
Ear aches
 
 
 
 
1
1
Ear infection
 
 
 
2
1
3
Fever
 
2
2
2
3
5
Flatulence
2
2
4
1
1
2
Headache
 
 
 
1
 
1
Hives
 
1
1
 
 
 
Irritability
4
1
5
3
5
8
Laceration
 
 
 
1
 
1
Lack of/decreased appetite
2
2
4
3
1
4
Lethargy
1
5
6
 
 
 
Loose stool
2
6
8
3
3
6
Lump on back of head
 
 
 
1
 
1
Nasal congestion
4
6
10
 
2
2
Pain
2
1
3
2
 
2
Physical injury
 
 
 
1
 
1
Pink eye
 
 
 
 
1
1
Pneumonia
 
 
 
 
1
1
Rash
 
2
2
1
2
3
Runny nose
6
12
18
6
9
15
Skin infection
 
 
 
1
 
1
Sore foot
 
 
 
 
1
1
Sore throat
1
2
3
 
 
 
Strep throat
 
 
 
 
1
1
Umbilical hernia
1
 
1
 
 
 
Vomiting
 
2
2
1
2
3
Total
35
61
96
38
52
90
Serious adverse eventsa
 
1
1
 
2
2
a Serious adverse events included: 1) grade 4/potentially life-threatening fever reported on day 2, 2) grade
4/potentially life-threatening bronchiolitis reported at day 180 and 3) grade 4/potentially life-threatening pneumonia
reported at day 180. All serious adverse events were unrelated to the interventions and resolved.
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III. The Study to Investigate the Potential of Probiotics (SIPPY I and II): We have also conducted two
previous, non-IND but structure/function studies using the same investigational product,
strawberry-flavored BB-12 supplemented yogurt. Both were double-blinded, randomized,
placebo-controlled, allocation concealment clinical trials consisting of a combined 354 healthy children
between the ages of 1 and 5 years who attended daycare/school at least 3 days per week. The active
BB-12 and control products were the same as used in the two Phase I trials. The primary objective was
to determine if daily consumption of a probiotic-containing yogurt-based drink for 90 consecutive days
decreased absences from daycare for the children, a structure/function outcome. Thus, the total days of
BB-12 yogurt consumption was over 30,000 days. There were no significant differences in the days of
missed school per group. Importantly there were no differences in adverse events (see Table 3) among
BB-12 and control groups.
Table 3. SIPPY I and II Adverse Events
Control
BB-12
SIPPY I Events
N=95
N=87
Number of subjects with at least one adverse event
3
3
Diarrhea
3
2
Pyrexia
0
1
Dermatitis (Diaper)
3
0
Vomiting & Cough (1 participant had 2 adverse events at the same time)
3
0
Hordeolum (Stye)
1
0
Number of subjects with at least one serious adverse event
0
0
SIPPY II Events
N=81
N=91
Number of subjects with at least one adverse event
3
8
Diarrhea
0
2
Hyperactivity
1
0
Rash
0
1
Frequent Stools
1
1
Stomach Pain
2
0
Constipation
0
1
Diaper Rash
0
1
Loose Stools
0
3
Number of subjects with at least one serious adverse event
0
0
2.11
Rationale
The rationale for focusing on food as a vehicle for the transmission of probiotics is that it has the potential
to benefit a much larger public health population than using probiotics in a more medicinal manner, such
as pills or capsules. Compliance with most medicinal regimens is around 50%; although certain
interventions such as simplifying a regimen, collaboration with patients, different formulations, and
increased convenience improve compliance.(99-102) We believe a readily available drink containing a
high dose of probiotics has the potential to improve compliance through many of these mechanisms. This
product also has the potential to positively impact the health of children and adults around the world, as
yogurt will likely be more appealing to both children and their parents for long term consumption than
pharmaceutical-like preparations. In addition to the benefits associated with the consumption of
probiotics, there is an increased health benefit from consuming yogurt, a nutrient dense food.
2.12
Potential Risks and Benefits
2.12.1 Potential Risks
Yogurt: Potential risks and side effects related to the probiotic cultured yogurt include: allergic reaction to
drink ingredients, most likely strawberry, or digestive problems such as stomachaches or loose, watery
bowels due to lactose intolerance.
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Amoxicillin-clavulanate: nausea and/or vomiting, diarrhea, upset stomach and mild skin rash. The most
serious potential risk is diarrhea caused by a Clostridium difficile infection. Symptoms of C. difficile
infection include stomach pain or tenderness, bloody stools, fever, nausea, and diarrhea.
There may also be side effects, other than those listed above that we cannot predict. Many side effects
may go away shortly after the yogurt or antibiotic consumption is stopped, but in some cases side effects
can be serious, long lasting or permanent.
Stool Collection: There is no known risk to stool collection.  However, there may be some discomfort
with collecting stool samples.
2.12.2 Known Potential Benefits
BB-12: Bifidobacterium lactis strain BB-12 is a commercially available probiotic strain used in a number
of feeding and clinical trials. Such supplements have shown potential benefits in treatment and
prevention of various diseases including diarrhea, asthma, necrotizing enterocolitis and allergies. It has
also been found to significantly improve skin conditions in eczema patients.
Yogurt: Yogurt has its own known benefits coming from milk. For every 6-ounce serving, there are about
9 grams of animal protein, plus several other nutrients found in dairy foods, like calcium, vitamin B-2,
B-12, potassium, and magnesium. Yogurt with active cultures is found to help certain gastrointestinal
conditions including lactose intolerance, constipation, and diarrhea. Yogurt, which is more readily
available and accepted by the general public, has the potential to greatly influence health if efficacious
probiotic dosages and species are available in the product. Therefore, we believe that the benefits of the
BB-12 supplemented yogurt far outweigh the discomfort and small risks participants might experience.
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3. STUDY DESIGN
In order to determine the ability of BB-12 to impact antibiotic-induced perturbation and recovery, we will
conduct a randomized, double-blinded, controlled trial in 124 healthy participants, ages 18-65 years.
The full inclusion and exclusion criteria are listed in Section 4. All participants will be given a 7-day
prescription for 875/125 mg of amoxicillin-clavulanate (AMC), taken twice daily. In order to control for
confounding by disease state and specific antibiotic used, all participants randomized to a yogurt
intervention group will receive the same daily dose --a four-ounce (approximately 100 ml) serving of
probiotic (which delivers ≥1010 CFU of BB-12) or control yogurt-- and length of antibiotic treatment after a
30-day run-in period.
Participants are to provide informed consent on at baseline, complete the 30-day run-in --during which
they will refrain from all probiotics-- and begin the antibiotic and yogurt interventions on Day 1. As with
our previous studies, enrollment and informed consent will occur at a private location, either in Dr.
Merenstein’s laboratory at Georgetown University Medical Center, or at the participant’s home, as per
the participant’s preference. All interventions will be delivered in coolers to the participants’ homes prior
to day 1, at a time of their choosing.
This is a randomized, placebo-controlled double-blinded, single center, mechanistic study to test the
mechanism of Bifidobacterium animalis subsp. lactis (B. lactis) strain BB-12 (BB-12) yogurt in AAD,
compared to yogurt without BB-12, in healthy adults. 124 participants will be randomized into one of five
groups. One group (N=12) will receive only antibiotics and no other interventions. The other four groups
will receive either the active or control yogurt, or one of the control arms of the study. The active and
control yogurt groups will each have two groups that will take the yogurts at different times, and the
control non-yogurt arm will have one group, for a total of five groups. All participants will be given a 7-day
prescription for 875/125 mg of amoxicillin-clavulanate (AMC), taken twice daily. Participants in the two
active arms will be given BB-12 supplemented yogurt and instructed to consume the yogurt concurrently
with the antibiotics (N=28) or four hours after taking the antibiotics (N=28). Participants in the two control
yogurt arms will be given yogurt without the BB-12 supplement, and will be instructed to consume the
yogurt concurrently with the antibiotics (N=28) or four hours after taking the antibiotics (N=28).
Fecal samples will be collected at home (twice at baseline (pre-run-in and post-run-in), and on days 7,
14, 21 and 30) into a maximum of three (1 sample for SCFA, 1 sample for microbiome testing, and 1
sample for potential future analysis) sterile, screw-capped tubes and frozen within one hour of collection
in a home freezer. Participants will be given detailed, written and illustrated instructions on proper
sample collection procedures, using protocols from the Fraser laboratory and as described in previous
studies. Research staff will review these protocols with participants during enrollment. Immediately after
collection and freezing, a member of the research team will be alerted to retrieve and transport the
samples to the laboratory. The samples will be stored at -80°C until transport to The IGS. All staff will
receive certification on handling procedures.
Follow-up phone visits will be completed on Days 7, 14, and 30. Participants will keep a daily diary to
track number, shape and consistency of bowel movements, if/when antibiotic or yogurt was consumed,
Bristol Stool chart, general health, use of other medicines or products, adverse events, and the GSRS -
IBS instrument.
3.1
Primary Outcome Measure
The primary outcome is to determine the ability of BB-12 to impact antibiotic-induced reduction in SCFA
as reflected by the levels of acetate. The effect size according to an adjusted Cohen’s d will be calculated
at day 7 and 30. We will also explore the impact of concurrent versus delayed administration of BB-12 in
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relation to the antibiotics. SCFA produced by anaerobic gut microbiota will be quantified from human
fecal samples via liquid chromatography-tandem mass spectrometry (LC-MS/MS) and adjusted for each
participant’s own baseline value.  As a secondary analysis, we will also perform normal area under the
curve (AUC) analysis of the percent change in acetate from baseline.
Primary Hypothesis: Antibiotics will result in a reduction in fecal SCFA. BB-12 supplementation will
protect against antibiotic-induced SCFA reduction and/or be associated with a quicker return to baseline
SCFA levels, as compared to the control group. We also hypothesize that concurrent administration of
the probiotic and antibiotic is not necessary for the probiotic impact on SCFA.
3.2
Secondary Outcome Measures
The secondary aim is to determine the ability of BB-12 to impact antibiotic-induced disruption of the gut
microbiota using 16S rDNA profiling, and the impact of concurrent versus delayed administration of
BB-12 in relation to the antibiotics. This will be assessed at days 7 and 14. Changes in absolute
abundance of specific bacterial genera (with a particular emphasis on known butyrate producers in the
Firmicutes phylum) will also be assessed with qPCR.
Secondary Hypothesis: Antibiotics will result in a decrease in the overall number and diversity of bacterial
species present in the fecal microbiota. BB-12 supplementation will protect against antibiotic-induced
shifts in the microbiota and/or will be associated with a quicker return to a baseline microbiota
composition, as compared to the control group. We also hypothesize that concurrent administration of
the probiotic and antibiotic is not necessary for the probiotic effect on the composition of the gut
microbiota.
Tertiary Hypothesis and Aim: We hypothesize that acetate produced by BB-12 in situ will cross-feed
butyrate producers in the Firmicutes phylum resulting in an up-regulation of butyrate biosynthetic
pathways. We will longitudinally characterize the gut microbiota with high-throughput metatranscriptomics
in order to generate complementary information on the impact of antibiotics plus and minus BB-12 on
overall microbiome function.
The experiments in the R33 phase of the study will provide sequential samples from which data on both
microbial composition (based on 16S rDNA profiling) and total bacterial numbers (based on qPCR data)
can be generated. Longitudinal 16S rDNA profiling will provide insight into several distinct shifts in the
microbiota (e.g., overall decreases in bacterial numbers, loss of taxa, loss of overall community diversity)
that reflect the impact of antibiotics (control group) and antibiotics plus BB-12 (active yogurt group). We
will follow the extent and time course of recovery of the microbiota following cessation of antibiotic
therapy.
The following exploratory outcomes, while not adequately powered due to a relatively small sample size,
will be examined and listed on ClinicalTrials.gov.: diarrhea, loose stools, constipation, fever, flatulence,
lack of appetite, pain, rash, vomiting, allergic reaction, dyspepsia, and nausea. We will also examine
whether acetate changes are associated with any of the listed outcomes.
We anticipate that a subset of participants, conservatively 15%, will develop diarrhea, and we will
generate data on microbiota changes during the progression from the diarrheal state to the non-diarrheal
state. This will allow us to ask if changes in the relative abundance of specific members of the gut
microbiota (either alone or in combination) correlate with a clinical outcome.
The most likely outcomes from the study are:
1. +/- impact of BB-12 on fecal SCFA levels
2. +/- impact of BB-12 on gut microbiota composition (measured to quantify level of disruption achieved
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by antibiotics as a comparator within this study)
3. +/- impact of BB-12 on AAD (measured, but study not powered to detect clinical difference of AAD).
4. +/- impact of BB-12 influenced by the timing between AMC dose and BB-12 consumption.
4. SELECTION AND ENROLLMENT OF PARTICIPANTS
4.1
Recruitment Strategy
We will enroll and randomize 124 participants; 28 will be in the BB-12 yogurt concurrent consumption
group, 28 will be in the BB-12 yogurt delayed consumption group, 28 in the control yogurt concurrent
consumption group, 28 in the control yogurt delayed consumption group, and 12 in the no yogurt group.
Statistical analysis on the primary outcome will be performed using the intention-to-treat principle; all
subjects enrolled and randomized will be accounted for in the final analysis in their randomized group.
Flyers and posters will also be displayed in clinics, offices and community centers participating in the
study prior to initiation.
The CAPRICORN research network is specifically designed to help conduct clinical trials. Ideas for
studies either come from practicing physicians or University researchers. Regardless of where studies
originate, they are then reviewed and sent out to physicians for comment. Thus, not all physicians or
offices participate in each study; but are allowed to choose which studies that they have interest in and
work within their office flow. Because of this grassroots approach, CAPRICORN has been very
successful in fulfilling recruitment goals set forth a priori in studies. The network has helped the PI recruit
over 1,500 participants in eight randomized controlled trials.
4.2
Inclusion Criteria
Participants will be eligible if:
1.
S/he is between ages of 18-65 years
2.
S/he has the ability to read, speak and write English or Spanish
3.
S/he has refrigerator for proper storage of the intervention
4.
S/he has telephone access
4.3
Inclusion of Women and Minorities
This is an adult study and no one under age 18 will be included. All adults who meet the
inclusion/exclusion criteria, regardless of religion, sex, or ethnic background will be enrolled if they sign
the informed consent.
Based on rates of patients seen in our network, we anticipate about 50% of the potential participants will
be female. Many of our practices are based in the inner-city and have an over-representation of ethnic
minorities.
4.4
Exclusion Criteria
Participants will be ineligible if any of the following conditions are present:
1.
Diabetes or asthma that requires daily medication
2.
Allergy to strawberry
3.
Active diarrhea (three or more loose stools per day for two consecutive days)
4.
Any gastrointestinal medications, i.e. medicines for irritable bowel syndrome, gastroesophageal
reflux disease, inflammatory bowel disease, etc. (a full medication list will be reviewed by the PI
prior to enrollment)
5.
Lactose intolerance
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6.
History of heart disease, including valvulopathies or cardiac surgery, any implantable device or
prosthetic
7.
History of gastrointestinal surgery or disease
8.
Milk-protein allergy
9.
Allergy to any component of the product or the yogurt vehicle
10. Allergy to penicillin or cephalosporin class antibiotics
11. Allergy to any of the following medications:
a.
Penicillin
b.
Erythromycin
c.
Tetracycline
d.
Trimethoprim
e.
Ciprofloxacin
12. Women who are breastfeeding, pregnant, or planning to become pregnant during the study.
13. Prior participation in the “YOBIOTIC” Study (i.e. R61 protocol).
Participants must agree to refrain from all probiotics throughout the study, starting from the 30-day run-in
period through day 30.
4.5
Study Enrollment Procedures
Research staff will meet with area physicians via the CAPRICORN network and community members to
inform them about the study. Flyers and posters will also be displayed in clinics, offices, and community
centers participating in the study approximately three months prior to initiation of the study. Information
about the study procedures and criteria will be available on a public, study-specific website (content
pre-approved by the GU IRB).
Potential participants will be pre-screened over the phone or in-person using the Pre-screening Form
(PS). If they meet basic entry criteria, the potential participant will be offered the opportunity for study
participation and will be given a copy of the informed consent form and/or link to the website, and time to
review before making the choice about participation. If the participant agrees, an enrollment and
informed consent visit will be scheduled at a mutually agreed time and location. Information provided
during the consent process include, the purpose of the study, procedures, withdrawal procedures,
subject termination, risk/discomforts, benefits, costs, compensation, and alternatives to participation.
This study design is such that potential participants sign the informed consent about 45-60 days before
starting any interventions. About 1-3 weeks after signing the informed consent, the participants have at
least a 30-day wash out period where they refrain from consuming any probiotic products and antibiotics;
after which, they will start the intervention period. Due to the time lag between enrollment and the start of
the interventions, some participants decide to withdraw in the interim before starting any interventions.
Since this is a study to understand the mechanisms of preventing antibiotic-associated diarrhea using
probiotics, in order to accomplish the study aims, it is critical to have data collected from 124 participants
who have been randomized and commenced the interventions. As such, we may enroll approximately
200 individuals to account for the withdrawal rates, so that study is completed with a final sample size of
124 participants.
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5. STUDY INTERVENTIONS
5.1
Interventions, Administration, and Duration
The probiotic-supplemented yogurt is composed of the investigational agent, BB-12, and contains the
yogurt starter culture YF-L702 (a mixed culture of Streptococcus thermophilus and Lactobacillus
delbrueckii subsp. bulgaricus). The yogurt starter culture will be present and alive in the control yogurt.
Starter culture alone is unlikely to impact AAD, as it does not survive the acidic stomach environment
and is not recovered alive in the feces. Details about yogurt preparation, and enumeration and PCR
verification of BB-12 have been previously published and reviewed by the United States Food and Drug
Administration (FDA) Center for Biologics Evaluation and Research (CBER).
Participants in the four yogurt groups will take the same amount of either the BB-12 or control yogurt, as
determined by random assignment. Participants will each consume a four-ounce daily dose orally for 14
days while on amoxicillin-clavulanate for 7 days. Each four-ounce daily dose of the active yogurt will
deliver no less than 1010 CFU of BB-12 per day.
Study interventions
BB-12 yogurt and amoxicillin-clavulanate, or
Control yogurt and amoxicillin-clavulanate, or
No yogurt and amoxicillin-clavulanate
Administration
Oral, to individual participants
Dosing schedules
Amoxicillin-clavulanate 875/125 mg twice per day on days 1-7, and either:
●
4-ounces of BB-12 yogurt daily taken concurrently on days 1-14, or
●
4-ounces of BB-12 yogurt daily taken four hours after on days 1-14, or
●
4-ounces of control yogurt daily taken concurrently on days 1-14, or
●
4-ounces of control yogurt daily taken four hours after on days 1-14, or
●
no yogurt.
Possible adverse events
See Section 7.3
Setting
Community
Intervention delivery
Research personnel will provide interventions
Participants will self-administer
Use of appropriate supportive
care, medications or treatments
Not applicable
Dose escalation procedures
Not applicable
Instructions for modifications
Not applicable
Package insert information
See package insert for amoxicillin-clavulanate
5.2
Handling of Investigational Product
5.2.1
Study Product Description
5.2.1.1 Acquisition
The investigational agent, Bifidobacterium animalis subsp. lactis (B. lactis) strain BB-12 (BB-12), will be
supplied by manufacturer, Chr. Hansen, in Milwaukee, WI.
Chr. Hansen, Inc.
9015 West Maple Street
Milwaukee, WI  53214
The investigational agent will be shipped directly from the manufacturer to The Pennsylvania State
University where they will be added to the yogurts.
The Pennsylvania State University
Department of Food Science
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428 Food Science Building
University Park, PA  16802
5.2.1.2 Formulation, Packaging, and Labeling
The probiotic-supplemented yogurt is composed of the investigational agent, Bifidobacterium animalis
subsp. lactis (B. lactis) strain BB-12 (BB-12), and also contains the yogurt starter culture, YF-L702 (a
mixed culture of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus).
The Chr. Hansen Culture Collection (CHHC) numbers of the exact strains used in the manufacture of the
yogurt blend YF-L702 are Streptococcus thermophilus ST5389, Streptococcus thermophilus ST5086,
Streptococcus thermophilus ST4460, Lactobacillus delbrueckii subsp. bulgaricus LB4351, and
Lactobacillus delbrueckii subsp. bulgaricus LB2164.
Once manufactured, the probiotic-supplemented yogurt-drink will be packaged in pint-size plastic
containers, which will yield 4 servings of the 4-ounce daily dosage. Four containers of the yogurt drink
with their randomly assigned bin number will be provided to the participants after enrollment and the
4-week run-in period, prior to day 1, along with the plastic cups and markings that denote levels of 2, 4,
6, and 8 ounces. The daily dose is one serving of 4-ounces of yogurt each day. This will deliver no less
than 1010 CFU per day of BB-12.
Each plastic container will bear the label “Caution: New Drug-Limited by Federal (or United States) law to
investigational use” in addition to its manufactured date and time.
5.2.1.3 Yogurt Drink Manufacture
Yogurt drink manufacture is broken into two major activities, 1) preparation of the yogurt and 2) blending
with other ingredients to prepare the drink. To make the yogurt, a yogurt base is formulated to contain
9.0% milk solids non-fat (MSNF), 1.3% fat and 3.0% sugar using skim milk, cream, non-fat dry milk and
sugar. The wet ingredients (skim milk and cream) are mixed together and then blended with previously
weighed dry ingredients (sugar and non-fat dried milk). After blending the yogurt mix is pasteurized and
homogenized (2000 psi first stage, 500 psi second stage) at 83°C for 29 sec.  The pasteurized,
homogenized mix is pumped to the fermentation tank and given an additional heat treatment of 85°C for
30 min to denature whey proteins, then cooled to 42°C prior to inoculation with 0.02% of the yogurt
starter culture (YFL-720). The inoculated mix is agitated for 15 minutes and then allowed to incubate
quiescently until the pH reaches 4.6.
To prepare the yogurt drink the gel structure of the fermented yogurt is disrupted by agitation and
blended with a mixture of pectin, 36 DE corn syrup solids, sugar and water.  The pectin-containing
mixture is prepared by heating water, sugar, 36DE corn syrup solids, and pectin to 80°C, holding for 1
hour.  This mixture is then cooled to 40°C and added to the fermented white mass resulting in the
unflavored yogurt drink base.  To flavor the product, strawberry puree is then added to the yogurt drink.
The composition of the yogurt drink is shown in Table 4 and the ingredients used in manufacture are
listed in Table 5.  At this point, a portion of the yogurt drink is homogenized (1500 psi first stage, 500 psi
second stage) for a second time to create a drinkable consistency and packaged in plastic bottles. To
prepare the yogurt drink containing BB-12, an appropriate amount of (BB-12) is added to the yogurt
drink, allowed to mix for 15 minutes and then homogenized and packaged as described above. The
packaged yogurt is stored at 4°C.
Table 4. Composition of Strawberry Yogurt Drink
Component
Composition in Yogurt Drink
Fat
1.0%
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Milk Solids Non-Fat
6.8%
Sucrose
6.1%
Corn Syrup Solids
6.4%
Pectin
0.4%
Strawberry Puree
3.0%
Table 5. Ingredients for Strawberry Yogurt Drink
Component
Supplier
Specifications
Milk1
Land-O-Lakes
PMO2
Nonfat Dry MilkSolids3
Various approved vendors
COA4
Granulated Sugar3
Various approved vendors
COA
Corn Syrup Solids3 (42 DE)
Various approved vendors
COA
Pectin ADM 783
DuPont, New Century, KS
COA
Strawberry Puree #SN2000036366
Sensient Flavors, Amboy, IL
COA
YF-L702 (starter culture)
Chr. Hansen, Milwaukee, WI
COA
BB-12 (probiotic)
Chr. Hansen, Milwaukee, WI
COA
1 Milk is received on an as needed basis and is purchased from Land-O-Lakes.
2 Milk will be produced under and meet the requirements of the Pasteurized Milk Ordinance (PMO).
3 The supplier for nonfat dry milk, sucrose and corn syrup solids may change based on availability and price of
ingredient.
4 All ingredients will be food-grade and will require a Certificate of Analysis (COA) from the supplier.
5.2.1.4 Enumeration of BB-12 from the Yogurt
The viable count of BB-12 in the product is measured the day of manufacture (Day 0) and weekly until
Day 30 of shelf-life. The yogurt drink is diluted in sterile peptone water blanks (3M, St. Paul, MN) and
then appropriate dilutions of the product are cultured by pour plating on an MRS-based selective
bifidobacteria medium.(103) Modified MRS agar is made by adding 4.5 grams Agar (Difco, BD, Sparks,
MD) to 300 ml MRS broth (Difco, BD, Sparks, MD). After autoclaving, the agar is used immediately after
addition of three selective components, 1.5mL of 0.01% Dicloxacillin (Sigma-Aldrich, St. Louis, MO); 3.0
mL of 10% Lithium Choride (Sigma-Aldrich, St. Louis, MO); and 1.5 mL of 10% Cysteine Hydrochloride
(Sigma-Aldrich, St. Louis, MO). Plates are anaerobically incubated (VWR, West Chester, PA) at 37°C
and counted after 72 hours. The final yogurt drink also is evaluated for the presence of coliforms using
high sensitivity PetrifilmTM Coliform Count Plates (3MTM, Burlington, North Carolina) and PetrifilmTM
aerobic plate counts according to the 17th edition of Standard Methods for the Examination of Dairy
Products, #7.072 and #6.040, respectively.
5.2.1.5 PCR Verification of BB-12 Level in Finished Yogurt
To further verify the selectivity of the BB-12 agar, colonies of varying morphology are selected for
identification using B. lactis specific PCR primers according to the method of Ventura et al.(104) Cells are
lysed according to the microwave method of Bollet et al. as modified by Kullen et al.(105, 106) The
sequence of the forward primer, Bflact2, is 5’-GTGGAGACACGGTTTCCC-3’ and the reverse primer,
Bflact5, is 5’-CACACCACACAATCCAATAC-3’. Amplicons from PCR are electrophoresed through a 2%
agarose gel at 110 volts for 2 hours, stained with ethidium bromide (Promega, Madison, WI), destained
with distilled water and visualized using an Alpha-Imager (Alpha Innotech, San Leandro, CA). The
presence of amplicons of the appropriate size is taken as evidence the colonies are indeed B. lactis.
5.2.1.6 Acceptability Testing
We also completed two previous studies using the same BB-12 yogurt drink in a combined total of 354
children ages 1-4 years to determine the effects of BB-12 yogurt on daycare absences.  Participants
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consumed the same amount of yogurt we are proposing for this trial (four ounces daily) for 90
consecutive days. Only minimal adverse events were reported by 17 participants in over 30,000 total
days of product consumption; all were non-serious and self-limited. High rates of compliance, over 92%,
were reported in both trials, indicating the safety and tolerability of the BB-12 yogurt drink, at this
dosage.(107, 108)
5.2.1.7 Final Product
Table 6 contains information about the initial population of BB-12 in the product as well as the results of
viable count analysis conducted up through the end of shelf life for 42 separate yogurt-drink
manufacturing experiments.  The total solids of the product ranged from 20.53 to 22.11% with a mean of
21.2%. The fat content of the product ranged from 0.52 to 1.29% with a mean of 0.9%. The pH of the
product ranged from 4.42-4.7 with a mean of 4.42 on the day of manufacture and remained relatively
constant throughout shelf life.  The population of BB-12 declined slowly throughout the self-life but
remained within the target level (log 9 CFU/g).  Viable counts of BB-12 remained about the target value
in subsequent weeks (data not shown) but we chose to set the shelf life at 30 days. Thus a 100 ml
serving would supply no less than 1010 CFU per day of BB-12.
Table 6. pH and BB-12 Values for Yogurt Drink During Storage
Time
pH (± SD)
Log CFU/ml of BB 12 (± SD)
Day 0
4.41 (0.13)
8.86 (0.36)
Week 1
4.42 (0.12)
8.86 (0.32)
Week 2
4.39 (0.11)
8.65 (0.39)
Week 3
4.38 (0.12)
8.48 (0.47)
Week 4
4.42 (0.12)
8.49 (0.40)
The release criteria for the product will be similar to fresh dairy products. The proposed release criteria
are shown in Table 7.
Table 7. Release Criteria for finished product.
Item
Acceptable range
Method
Total Solids
20.5-22.5%
Microwave Drying1
Total Fat
0.5-1.5%
NMR1
pH
4.1-4.7
pH Meter
Coliforms
Negative by test
Method #7.0722
1Analysis obtained using CEM SMART Trac Fat and Moisture Analyzer, Matthews, NC
217th Edition of Standard Methods for the Examination of Dairy Products
A mock nutrition facts panel for the yogurt drink is shown in Figure 1, as well as a sample of the label,
which is placed on the final product when used as food (Figure 2).
Figure 1. Mock nutrition label for probiotic strawberry yogurt.
Nutrition Facts
Serving Size: 100g
Amount Per Serving
Calories 110
Calories from Fat 10
% Daily Value*
Total Fat 1.5g
2%
Saturated Fat 1g
5%
Trans Fat 0g
Cholesterol 5mg
2%
Sodium 55g
2%
Total Carbohydrate 23g
8%
Dietary Fiber 0g
0%
Sugars 19g
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Protein 3g
Vitamin A 0%            ●
Vitamin C 2%
Calcium 10%            ●
Iron 0%
*Percent Daily Values are based on a 2,000 calorie diet. Your daily
values may be higher or lower depending on your calorie needs:
Calories:
2,000
2,500
Total Fat
Less than 65g
80g
Sat Fat
Less than 20g
25g
Cholesterol
Less than 300mg
300mg
Sodium
Less than 2,400mg
2,400mg
Total Carbohydrate
300g
375g
Dietary Fiber
25g
30g
Calories per gram:
Fat 9 ● Carbohydrate 4 ● Protein 4
Ingredients: Cultured pasteurized skim milk, sugar, corn sweeteners, strawberries, cream, non-fat milk solids, pectin, modified
food starch (corn), natural flavors, sodium citrate, potassium sorbate (preservative), red 40, blue 1.
Figure 2. Mock Product Label
5.2.2
Dosage, Preparation and Administration of Investigational Product
Enrolled participants will be given a total 14-day supply of the yogurt following the 4-week washout
period. Participants will drink four (4) ounces of yogurt each day for 14 consecutive days. Yogurt will be
administered via mouth.
5.2.3
Accountability Procedures for the Investigational Product
Written records of receipt and storage of the investigational product, including date of receipt, quantity
received, amount distributed to participants, and final disposition will be maintained. Any known
discrepancies in the accountability will be documented. The investigator will not use the product in an
investigational manner other than that provided for in the protocol; however the product is also a yogurt
and the investigator requests the use of the surplus yogurt for teaching, charitable, and other educational
purposes.
5.2.4
Product Transportation, Handling and Storage
5.2.4.1 Transportation and Handling
The BB-12 and control products will be transported under refrigeration in coolers packed with ice from
the Penn State area via a van driven by Penn State personnel. A member of the study personnel will
meet the Penn State van at a designated meeting point halfway between the two campuses.  The Study
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personnel will then transport the products, still in its coolers with ice, the remainder of the trip to the
Washington, DC area.  While in transport, each cooler will be equipped with VWR® Digital
Refrigerator/Freezer Thermometer with Alarm, which will alert the study personnel by audible alarm if the
temperature of the coolers increased above the preset acceptable levels (between 32°F and 45°F) at any
time during transport.
5.2.4.2 Storage After Arriving in Washington, DC Area
Once the yogurt drinks arrive at Georgetown University in Washington, DC, they will immediately be
transferred to two refrigerators that are exclusively used for the study. Each refrigerator is equipped with
a lock and a thermometer.  Study personnel will take a daily temperature reading in each refrigerator to
ensure that the yogurt drinks are kept between 32°F and 45°F.  Each refrigerator is equipped with a
SENSAPHONE® 2.8K Weatherproof Temperature Sensor, which are connected to a SENSAPHONE® 400
Remote Monitoring and Alarm Notification System.  The sensor system will trigger an alarm when the
temperature falls below or rises above customizable limits (between 32°F and 45°F) or if there is a power
failure, and will contact up to four study personnel phone numbers.
BB-12 and control yogurt products expire one month from the date of manufacture. The date of
manufacture can be found printed on each bottle of the yogurt drink. Approximately every three weeks,
study personnel will make arrangements with Georgetown University Facilities Management to dispose
of the products before or on the date of expiration.
5.2.4.3 Transportation and Storage While Being Dispensed to Participants
The products will be transported to the participants’ homes in cooler bags. Prior to each delivery, the
study personnel will arrange and confirm a mutually convenient time with the participants to ensure that
they will be home to receive the drink.  This ensures that the participants can place the yogurt drinks into
their home refrigerators immediately upon receipt.
5.2.4.4 Duration of Storage
The products are stored in the study refrigerators at Georgetown University from the day they are
delivered from Penn State (same day delivery) until transported via coolers to study participants.  As
mentioned above, all bottles are labeled with the dates of manufacture and will thus be stored at
Georgetown University until expiration—one month after the manufacture date. Extra yogurt may be
delivered upon request by the participants due to spillage, spoilage or any other reasons as needed.
5.2.4.5 Instructions to Participants Regarding Storage and Dosing
BB-12 and control yogurt products expire one month from the date of manufacture.  The date of
manufacture can be found printed on each bottle of yogurt drink.  Since the drinks are only to be
consumed during a 14-day period and deliveries to participants will coincide with the arrival of fresh
product from Penn State, participants should always have enough unexpired product in their possession
to continue the study.  Participants will also be advised to dispose of any product that has been left out of
the refrigerator for longer than one hour. In such cases, they may contact study personnel for fresh
yogurt drinks to be delivered.
5.2.5
Assessment of Subject Compliance with Investigational Product
DNA extracted from stool samples collected at baseline and at day 7 will be amplified using B. lactis
specific PCR primers, in order to assess compliance. Participants will not be withdrawn from the study for
missed specimen collections but it will be noted in the study file.
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5.3
Concomitant Interventions
5.3.1
Allowed Interventions
Once enrolled, participants are allowed interventions or medications as needed, at the guidance of their
personal physician. Since the use of additional or concomitant interventions could bias results,
participants will be asked to report any of these additional treatments. Participants will be advised
multiple times before enrollment to avoid concomitants, but once enrolled they will be told of the
importance of articulating usage. All participants will be given a list of products they should avoid during
the study. Due to the intention-to-treat principle, participants will not be removed from the study but will
continue with all planned study interventions and data collection.
If the participant takes any antibiotics within the 30-day run-in period, they will repeat another 30-day
run-in upon completion of the antibiotics, prior to initiation of the interventions.
5.3.2
Required Interventions
Probiotic supplemented yogurt or control yogurt (if randomized to one of the four groups), and
amoxicillin-clavulanate (all groups) provided by the study.
5.3.3
Prohibited Interventions
Participants will be ineligible if they are taking the following medications at the time of enrollment:
●
Daily diabetes or asthma medication
●
Any gastrointestinal medications, i.e. medicines for irritable bowel syndrome, gastroesophageal
reflux disease, inflammatory bowel disease, etc. (a full medication list will be reviewed by the PI
prior to enrollment).
Participants must also avoid the following for the duration of the study:
●
Any probiotic not provided by the study.
●
Any antibiotic not provided by the study.
5.4
Adherence Assessment
We will assess adherence to the yogurt interventions in three distinct manners. First, we will assess
adherence to the antibiotic and yogurt interventions via the daily assessment diary. Second, participants
will take photos of the remaining antibiotics and yogurts bottles at the end of the intervention periods and
send them to the research assistant. Third, at all follow-up phone calls, participants will be asked if they
consumed the yogurt each day and how much was consumed.
Protocol compliance is defined as consuming 2 or more ounces of the assigned study yogurt per day, for
at least 11 of the 14 days. Participants will not be withdrawn from the study for missed interventions or
specimen collections, but these adherence assessments may be used for any per-protocol analysis.
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6. STUDY PROCEDURES
6.1
Schedule of Evaluations
All interventions will be completed by day 14 of the trial. At day 30, participants will provide the final
sample and be queried for changes in baseline health and other adverse events.
Table 8. Participant Timeline and Sample/Data Collection Schedule (N=124)
Assessment
Pre-
screen
Pre
Run-In
Baseline
30-day
Run-In
Post
Run-In
(0/+2)
1
7
(±2)
14
(±2)
21
(±2)
30
(±2)
Pre-Screening
X
Informed Consent Form & Process
X
Inclusion/Exclusion Criteria
X
X
Enrollment
Xa
Baseline Health Status
X
Demographic Information
X
Fecal Microbiome Collectiona
X
X
X
X
X
X
Automated Self-Administered 24-Hour
(ASA24®) Dietary Assessment Tool (1
day)
X
Run-in Period (30 days)
|→
→|
Post Run-In Health Status
X
Randomization
X
Amoxicillin-clavulanate Intervention (7
days)
|→
→|
Yogurt Intervention (14 days)
|→
→
→|
Follow-up Data Collection
X
X
X
Daily Assessment Diaryb (14 days)
|→
→
→|
Gastrointestinal Symptom Rating
Scale (GSRS-IBS)
X
X
X
SCFA Testing
X
X
X
X
X
X
16S rRNA Gene Sequences
X
X
X
X
X
X
Adverse Event Reporting
|→
→
→
→
→
→
→|
aEnrollment will occur a minimum of 30 days prior to starting the interventions. Participants will provide one stool sample
before the run-in period, refrain from antibiotics and probiotics during this period and provide 1 baseline sample after the
run-in period.
bDiary from days 1-14; information on yogurt consumption/dose, sample number, shape and consistency, health, use of
interventions, quality of life and adverse events.
X: collected on day; |→: starting from day; →|: through end day
6.2
Description of Evaluations
All laboratory and clinical measurements, as well as method of data collection, are listed in Table 9.
Table 9. R33 Phase Assessment
Collection Method
Impact of antibiotics on gut microbiota
Bacterial number: real-time qPCR;
Overall microbiota composition: 16S rDNA profiling
Impact of BB-12 on gut microbiota
Bacterial number: real-time qPCR;
Overall microbiota composition: 16S rDNA profiling
Impact of BB-12 on diarrhea/stool frequency
Diary - patient reported outcome
Impact of antibiotics on metabolism of gut microbiota
Fecal SCFA quantification
Impact of BB-12 on metabolism of gut microbiota
Fecal SCFA quantification
Bristol Stool Chart
Diary - patient reported outcome
Gastrointestinal Symptom Rating Scale - IBS version
(GSRS-IBS)
Diary - patient reported outcome
Compliance for antibiotics and probiotic interventions
Daily diary; photo documentation of intervention
bottles; follow-up visits
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6.2.1
Sample Collection and Analyses
6.2.1.1
Sample Collection
Stool samples will be collected into three sterile, screw-capped tubes and frozen immediately. The
participants will be instructed on how to collect the samples and be provided with written, illustrated
instructions. (Refer to Section 6.2.3, “Stool Collection Instructions for Participants.”)
6.2.1.2
Transport
After collection, a member of the research team will be phoned and instructed to retrieve the samples
and transport them to the laboratory. As part of their training, students and the entire research team will
sign an informational sheet documenting their understanding of the risk of handling potentially infectious
materials. The stool will be transported back to the lab and placed in -800 Celsius to freeze for batch
transport to the Institute for Genomic Sciences at the University of Maryland Baltimore (Fraser Lab).
(Refer to Section 6.2.2, “Stool Collection Instructions for Study Personnel.”)
6.2.1.3
Short Chain Fatty Acid (SCFA) Analysis
Primary fecal SCFA produced by anaerobic gut microbiota in the colon (acetate, propionate, butyrate) will
be quantified via a liquid chromatography-tandem mass spectrometry assay. Primary fecal SCFA
produced by anaerobic gut microbiota in the colon (acetate, propionate, butyrate) will be quantified via a
liquid chromatography-tandem mass spectrometry assay. Fecal samples will be homogenized in 50%
aqueous acetonitrile, vortex mixed and centrifuged to yield a supernatant containing SCFAs. SCFA in the
supernatant are then derivatized with 3-nitrophenylhydrazine to enhance sensitivity of detection. Heavy
isotope labeled derivatizing agent (13C6-3nitrophenylhydrazine) is reacted with authentic standards of
SCFAs to generate stable isotope-labeled internal standards for each analyte. SCFA will be separated
using a Waters BEH C18 (2.1 x 100mm, 1.7micron) UPLC column using an acetonitrile/water/formic
acid-based gradient mobile phase where LC-MS/MS analysis will be conducted on either an AB Sciex
5500 QTRAP or a Thermo TSQ QuantumUltra triple quadrupole mass spectrometer operated in negative
ion mode.
6.2.1.4
Microbiome Community Profiling
The gut microbiota will be characterized by sequencing bacterial 16S rDNA gene amplicons from fecal
samples as previously described.(109-113) Raw 16S rDNA reads will be processed using published
protocols.(110-112) Within- (ɑ-diversity) and between- (β-diversity) sample comparisons will then be
performed using QIIME.(113) Microbiota will be compared cross-sectionally and longitudinally, with
emphasis on parameters such as the Bray-Curtis similarity score,(114) Chao1 metric (ɑ-diversity or
richness), the Shannon index (abundance and evenness of species in a community), determined using
standard tools, such as Mothur,(115) QIIME,(113) and statistical R package (www.r-project.org). In
addition, statistically significant correlations between specific microbiota members and clinical outcomes
will be determined using bioinformatics tools, such as eLSA,(116) Metastats,(117) and LEfSe.(118) The
16S rRNA dataset will also be used to simulate the function of the microbiota using PiCRUSt(119) which
uses available genome sequence data to infer functional gene contents based on the 16S rRNA
microbiota composition.
6.2.1.5
Real Time qPCR Analysis of Total Bacterial Number
Reactions will be performed in duplicate using the microbial qPCR DNA kit (Qiagen) and analyzed on the
Fluidigm Biomark platform.
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6.2.2
Stool Collection Instructions for Study Personnel
●
Inform the participants of the following:
1.
Refer to “Stool Collection Instructions for Participants” for detailed instructions.
2.
Use the kit provided to collect sample.
3.
All stool samples must be frozen immediately.
4.
Make sure to use gloves when handling sample.
5.
Write the collection time and date on the label provided.
6.
Call the study personnel after collecting the sample to schedule a pick up.
●
Use the cooler/coolly packs when transporting samples between the participant’s house to
Georgetown University.
●
Confirm participant or a housemate will be available in the time window.
●
Upon receipt of the sample, check the label; if no date/time was marked on the sample, ask the
participant to complete.
●
Fill out Sample Collection (SC) Form, even if the sample was not collected (check “no” on form).
●
Before placing the sample in the freezer, make sure:
1.
Any names on the samples are crossed out
2.
Write correct Study IDs
3.
Make sure all vial caps are tightly closed.
6.2.3
Stool Collection Instructions for Participants
Stool specimens are to be collected 6 times during the study: before the run-in period, after the run-in
period, and on days 7, 14, 21 and 30. At your enrollment visit and throughout the study, you will be
provided with stool collection materials. Each kit will include: disposable gloves, 3 stool vials/containers,
small plastic zipper bags, 1 large plastic zipper bag, labels and a stool collection device (“stool hat”).
Before you begin the collection, please clear a space or shelf in your freezer to store the samples without
contact with food or other items in the freezer. At the time of stool collection:
1. Wash your hands.
2. Put on disposable gloves.
3. Each kit will include labels with your STUDY ID number and the sample date (pre-run-in, post-run-in,
day 7, day 14, day 21 or day 30). If it is not already on the label, please write in your STUDY ID
number, as well as the DATE and TIME the stool was collected. Please label each of the 3 stool vials
and once on the label on the plastic zipper bag. Please do not write your name on the labels.
4. Lift the toilet seat. Align the “stool hat” with the rear of the toilet rim (see Photo A). Replace the seat.
Use the toilet as you normally would, and let the stool collect in the “hat”.
5. For the first 2 vials (without liquid), simply collect stool to the fill line on the vials using the attached
spoon and screw the cap back onto the vial (see Figure 1).
6. Unscrew the cap from the smaller vial that contains liquid, without spilling any of the liquid inside.
Using the spoon attached at the cap of the vial, fill the vial to the “fill line” indicated on the vial (but do
no overfill; see Photo B).
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Photo A.
Photo B.
7. Screw the cap back onto the vial. Make sure the cap is tightly closed. Shake the vial gently until the
stool is thoroughly mixed with the liquid.
1 STOOL (collected in “stool hat”)
Figure 1.
3 VIALS:
VIAL 1 (EMPTY)
VIAL 2 (EMPTY)
VIAL 3 (LIQUID)
Please avoid contact with the liquid preservative in the stool vials.
If any of the liquid comes into contact with skin or eyes, wash thoroughly with water.
Do not ingest/drink the preservative.
8.
Place the vials into the small plastic zipper bag and seal the bag. Then place the small bag into the
large plastic zipper bag. Seal the bag.
9.
Discard remaining stool into the toilet and flush. Discard stool hat.
10. Place the sealed plastic zipper bag into the FREEZER immediately for storage. Avoid contact with
food or other items in the freezer.
11. Discard gloves into the trash and wash your hands.
12. Contact the research staff as soon as possible to arrange a time for stool collection pickup, which
should typically occur within 48 hours.
6.3
Participant Stipends
Participants will receive up to $120 total in compensation for their time. $20 will be paid for each stool
specimen collected for a total of six specimens. All fecal sample tests will be paid for by the study and
participant’s insurance company will not be responsible for tests. As per Georgetown University IRB
protocol and will be elucidated in the Informed Consent, the PI, Georgetown University, Penn State
University and no parties involved in the study will be responsible for additional medical care that
participants may need.
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6.4
Screening Evaluation and Consenting Procedure
Individuals who are interested in participating in this study will contact a member of the research team by
phone, e-mail, or in-person. A member of the research team will establish contact with the individual,
either by phone or in-person, and provide a brief overview of the study and answer any questions. If the
individual would still like to participate, the research coordinator or assistant will ask questions from a
pre-screening form to assess eligibility and exclusion criteria. A visit for enrollment and informed consent
will be arranged at the participant’s home or the Merenstein lab at the Georgetown University Medical
Center, per the participant’s preference.
If the basic screening criteria are met, the potential participant will be offered the opportunity for study
participation and consent will be obtained in-person. Information provided during the consent process
include the purpose of the study, procedures, withdrawal procedures, study termination, risk/discomforts,
benefits, costs, compensation, and alternatives to participation.
If the individual does not qualify after the pre-screening, or decides not to participate, the pre-screening
form will document the reason, and will be filed separately from the participant records, without
personally identifiable information. The research staff will maintain a screening log to track screen fails
and enrollments.
A single informed consent form is initiated prior to an individual’s agreeing to participate in the study and
continuing throughout an individual’s study participation. Extensive discussion of risks and possible
benefits of this study will be provided to the participants. A single consent form describing in detail the
study interventions/products, study procedures, and risks, are given to the participants and written
documentation of informed consent is required prior to starting intervention/administering study product.
The research team will discuss all information outlined in the informed consent document with
prospective participants to ensure that they understand the nature of the research and can voluntarily
decide whether or not to participate.
Research staff will document the following on the Informed Consent Process Documentation form:
a) Were the Informed Consent Form, Health Insurance Portability and Accountability Act (HIPAA)
Authorization and related study documents thoroughly reviewed with the study participant?; b) Did the
study participant have sufficient time to review the documents and ask questions?; c) Was the Informed
Consent and HIPAA Authorization signed prior to the initiation of any study related procedures?; d) Were
copies of the signed documents given to the study participant?
6.5
Enrollment, Baseline, and Randomization
6.5.1
Enrollment
The enrollment/baseline visit will take place at the participants’ homes or an office where comfort and
confidentiality is assured. There is no clinic visit associated with this study. The purpose of this visit is to
have in-person time to collect baseline data, explain all forms, go over the schedule of visits and
intervention, and give the participant the initial compensation. No physical exams or medical
assessments will be conducted as part of the screening or enrollment process.
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6.5.2
Baseline Assessments
Once informed consent is given and it is confirmed that the participant meets all inclusion and exclusion
criteria, baseline data will be collected. These include: Baseline Health Status and Demographic
Information.
Stools will be collected multiple times throughout the study. Research staff will be trained on how to
properly transport stool and how to instruct participants on collecting stool.
During the initial visit, participants will also be given all materials to collect stools, the diary, and study
materials as well as the initial compensation. The participant will commence the 30-day run-in period.
During this 30-day run-in period, the participant will refrain from antibiotics and probiotics (a “What Not to
Eat list will be provided) and provide two baseline stool samples--one pre run-in, and one post run-in
(prior to starting the yogurt and antibiotics).
Following the run-in period and prior to the yogurt start day (day 1), research staff will contact the
participant in order to complete the Post Run-in Health Status form and repeat the Inclusion/Exclusion
form to ensure that the participants are still eligible. Participants who start an exclusionary medication or
experience an onset of an exclusionary, nontransient health condition from the time of enrollment will be
withdrawn from the study.
Should the participant take any antibiotics during this 30-day run-in period, they will start another 30-day
run-in cycle after the antibiotics are completed. The initiation of the yogurt and antibiotic interventions
(day 1) will be delayed until this additional run-in cycle is complete. Participants who take any non-study
probiotics during this 30-day run-in period will document any use on the Post Run-In Health Status form.
Participants who need to delay their day 1 (initiation of the yogurt and antibiotic intervention), e.g. to
complete a full 30-day run-in or due to transient conditions, can be held for a future cohort for up to 90
days from the day the informed consent was signed, without the need to re-consent. After 90 days, the
participant will be re-consented prior to starting the interventions.
Participants who are still eligible based on the completion of the Post Run-in Health Status and post
run-in Inclusion/Exclusion forms will then be randomized to receive the BB-12 yogurt, control yogurt, or
no yogurt for days 1 through 14 to be taken once a day. In addition, they will also receive measuring cups
that denote a level of 4 ounces. The amount of BB-12 supplemented yogurt drink to be supplied will be 4
ounces per day, which is slightly more than 100 grams/day.
6.5.3
Randomization
The group allocation ratio is 28:28:28:28:12: 28 in each of the two BB-12 yogurt groups, 28 in each of the
two control yogurt groups, and 12 in the no yogurt control group. The randomization scheme will be
generated by the study statistician using permuted blocks sizes of 4, 8, and 12 in random order. It will be
impossible for research personnel involved with participants to adjust randomization or discern what
drinks participants were receiving, ensuring true allocation concealment. Participants who receive the
control product will receive all identical interventions (i.e. stool collection, follow-up, etc.) that the BB-12
group receives.
Randomization will be completed after the participant consents to the study and completes the baseline
assessment and run-in. The yogurt or antibiotic interventions may only be initiated after randomization.
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6.6
Blinding
Each participant in the four yogurt groups will be randomly placed into one of the two arms receiving
yogurt, randomly allocated in a 1:1 ratio to either BB-12-supplemented yogurt or an identical yogurt
without the added BB-12 probiotic. Both contain the starter culture YF-L702 (a mixed culture of
Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus). The yogurts are similar in
taste and will have identical nutritional value, such as the same amount of calcium, protein, calories, etc.
The yogurts are packed in identical packaging with four different bin numbers. The participants, PIs,
research staff are all blinded to the treatment allocation.
The study statistician will generate two group-labels (e.g. A, B) where two bin numbers correspond to
one group, and two bin numbers correspond to the other group. . However, Dr. D’Amico will be blinded to
which group received the BB-12 yogurt and which received the control yogurt. The non-yogurt group will
be considered as a separate arm before unblinding.
PSU will send yogurt in four bins with distinct numbers, two of which will be the control yogurt and two
will be the BB-12 yogurt. Only the researchers at Maryland will know which bin numbers correspond to
‘group 1’ and which correspond to ‘group 2’, but will not know which is the active or control. PSU and
Maryland have no contact with participants and will not release this information to the study team until
the analyses are completed and databases are locked.
All study personnel at Georgetown and Dr. D’Amico will remain blinded throughout the study until the
databases are locked. In the unfortunate event that a participant experiences a serious adverse event or
emergency that requires unmasking the treatment groups, Dr. Ranit Mishori, a member of the
Department of Family Medicine who is not affiliated with the study, will discuss the assignment and
follow-up with the participant and their treating physician. Dr. Mishori has done this for our previous
clinical trials.
6.7
Follow-up Visits
6.7.1
Telephone Follow-up
Follow-up phone visits will be completed on Days 7, 14 and 30 (±2 days). It is imperative that all visits be
completed in a timely and regular fashion. Generally, the Follow-Up Form will be the only form completed
during the phone calls, but at times depending on participant’s report, there may be another form or two.
The follow up period includes all the time previous to the day of the interview.  Any incidents that happen
the day of the visit would be captured in the next Follow Up Visit.
There will be a set day that each subsequent visit should be completed for each participant. The
research personnel should call all participant’s phone numbers at least one time per day in order to
collect the data. If more than 3 business days have passed from the set day, the RA should alert the PI
that this week’s Follow-Up Form has not yet been completed. If the RA is not able to contact the
participant, a Missed Visit Report Form should be completed instead and the PI should be alerted once
again.
If a participant is unmasked at any point in time during the study, the PI will be alerted immediately. A
Georgetown University employee in the Department of Family Medicine not associated with the study will
follow up with this participant from this point on. This particular research personnel is not to discuss any
information regarding this occurrence with any other personnel other than the PI.
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6.7.2
Telephone Follow Up Script
In general, the Day 7, 14 and 30 (±2 days) follow up will be administered over the phone, but may be
completed in person. These phone visits are when the primary data will be collected. It is imperative that
all visits be completed in a timely and regular fashion.
1) [If assigned to a yogurt group] Drink Bin Number: Make sure the participant has the correct drink bin
number as you are completing the Follow-Up Form.
a) Ask the participant the number on the drink bottles and this should be confirmed with the
randomization form.
b) If this number is not consistent, the participant should be alerted not consume any more drinks
until further notice and the project coordinator should be immediately alerted after completing this
week’s Follow-Up Form.
c)
Remind the participant when they are supposed to take the yogurt (concurrently or delayed).
2) Daily Assessment Diary: Ask the participant to collect the Daily Assessment Diary for the phone
conversation. A lot of the information that will be collected on the Follow-Up Form has been collected
by the participant on their Daily Assessment Diary. If they have not been using the Daily Assessment
Diary, please positively stress the importance of the Daily Assessment Diary.
3) Explain to participant that we are supposed to fill out this Follow-Up Form today and it should take
about 5 minutes.
4) Administer Follow-Up Form.
●
When entering sick days, missing school or work, etc., this should be generally whole integers.
However, it is possible that a participant missed only 2 hours to go to the doctor. All numbers
under 4 hours should be counted at 0.5 days, and all those above rounded up to the next integer.
●
The participant needs to decide if any activities were missed due to an illness. This is for all
illnesses, even those that we are not collecting data about.
●
For some questions the RA may say, “Did you visit a doctor?”  A doctor is defined as any medical
personnel in this study.  Therefore, the participant may say, “Never, but s/he saw a nurse
practitioner.”  Please count this the same as seeing the doctor.
●
Compliance: Please let the participant know that we would like them to consume the
interventions, diaries, and sample collections as per the schedule, but regardless, we appreciate
their participation.
●
Adverse Events: If the participant believes any AE occurred, it is imperative that the RA
immediately fill out an AE Form, and if there are any SAE, alert the PI and project coordinator
immediately.
●
Confirm the date and time for the next visit or call.
●
At day 7, remind the participant to take a photo of the remaining antibiotics bottles.
●
[If assigned to a yogurt group] At day 14, remind the participant to take a photo of the remaining
yogurt bottle.
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6.7.3
Completion/Final Evaluation
On the final follow-up at day 30, participants will provide the final sample and be queried for changes in
baseline health and other adverse events.
At day 30, the research staff will also collect 24-hour diet recall data. The instrument, the Automated
Self-Administered 24-Hour (ASA24®) Dietary Assessment Tool, will be administered by the research
staff and recorded for one day (https://epi.grants.cancer.gov/asa24/).
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7.
SAFETY ASSESSMENTS
7.1
Specification of Safety Parameters
We will alert the participants of the common reactions to yogurt and by listing common reactions for them
to monitor. We will also have close follow-up and 24-hour phone line accessibility if there is a question of
an allergic or idiosyncratic reaction.
Details of all adverse events reported by participants or observed by research personnel will be recorded
in the adverse event report forms. All adverse events observed will be recorded separately and all will be
reported to the IRB on IRB Adverse Event forms.
In order to prevent the occurrence of adverse events, we will be stringent in enforcing our exclusion
criteria, paying particular attention to the potential for an allergic reaction to strawberry or yogurt on the
part of the participant.
Any participants who develop evidence of allergy or hypersensitivity to any component of the
investigational product will be withdrawn from the clinical study and receive no further doses of either
drink. We will continue to follow these participants for safety for the remainder of the study.
7.2
Methods and Timing for Assessing, Recording, and Analyzing Safety Parameters
Unanticipated problems will be recorded in the data collection system throughout the study.
Details of all expected and unexpected adverse events reported by participants during follow­-up calls,
spontaneously, or observed by research personnel must be recorded in the adverse event report forms.
Participants will be provided with a daily diary with a list of the most likely possible adverse events (see
Table 10), where they can document expected and other unexpected events not already listed. All
non­-serious adverse events will be reported to the IRB during continuing review. All AEs regardless of
severity level will be evaluated by the DSMB during scheduled meetings.
If a participant reports an AE/SAE that does not yet have a Date of Resolution (ongoing) and is
continuous (no more than two days without interruption) across two follow­-up periods, this AE/SAE will
be considered one event. The total duration of the AE/SAE will be collected but only one AE/SAE report
form will be completed for this particular event. If the symptoms resolved for more than two days before
recurrence, these will be reported as separate events.
The study staff will record all reportable events with start dates occurring any time after informed consent
is obtained until 7 (for non-­serious AEs) or 30 days (for SAEs) after the last day of study participation. At
each study visit, the study staff will inquire about the occurrence of AE/SAEs since the last visit. Events
will be followed for outcome information until satisfactory resolution or stabilization. In the event of SAEs,
all clinical and laboratory investigations considered necessary by the investigator will continue until
values have returned to normal. The investigator will provide the IRB with copies of results of
examinations and treatment carried out during the follow­-up of SAEs. These reports will be provided to
the FDA and the NCCIH Program Official.
7.3
Adverse Events and Serious Adverse Events
An adverse event (AE) is any untoward medical occurrence in a participant during participation in the
clinical study or with use of the experimental agent being studied. An adverse finding can include a sign,
symptom, abnormal assessment (laboratory test value, vital signs, electrocardiogram finding, etc.), or
any combination of these regardless of relationship to participation in the study. This includes events not
present at the start of the study, or worsening in grade during the course of the study. An AE can be
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reported anytime after study enrollment; a participant is considered enrolled once the informed consent
form is signed.
A serious adverse event (SAE) is one that meets one or more of the following criteria:
●
Results in death
●
Is life­-threatening (places the participant at immediate risk of death from the event as it occurred)
●
Results in inpatient hospitalization or prolongation of existing hospitalization
●
Results in a persistent or significant disability or incapacity
●
Results in a congenital anomaly or birth defect
An important medical event that may not result in death, be life threatening, or require hospitalization
may be considered an SAE when, based upon appropriate medical judgment, the event may jeopardize
the participant and may require medical or surgical intervention to prevent one of the outcomes listed in
this definition.
Unanticipated Problems (UP): The Office for Human Research Protections (OHRP) considers
unanticipated problems involving risks to participants or others to include, in general, any incident,
experience, or outcome that meets all of the following criteria:
●
Unexpected in terms of nature, severity, or frequency given (a) the research procedures that are
described in the protocol related documents, such as the IRB-approved research protocol and
informed consent document; and (b) the characteristics of the participant population being studied;
●
Related or possibly related to participation in the research (“possibly related” means there is a
reasonable possibility that the incident, experience, or outcome may have been caused by the
procedures involved in the research); and
●
Suggests that the research places participants or others at a greater risk of harm (including physical,
psychological, economic, or social harm) than was previously known or recognized.
7.4
Characteristics of an Adverse Event
7.4.1
Relationship to Study Intervention
To assess relationship of an event to study intervention, the following guidelines are used:
1.
Related (Possibly Related, Probably Related, Definitely Related)
a. The event is known to occur with the study intervention.
b. There is a temporal relationship between the intervention and event onset.
c. The event abates when the intervention is discontinued.
d. The event reappears upon a re­challenge with the intervention.
2.
Not Related (Unlikely Related, Not Related)
a. There is no temporal relationship between the intervention and event onset.
b. An alternate etiology has been established.
7.4.2
Expectedness of SAEs
The Study PIs and Data Safety Monitoring Board (DSMB) will be responsible for determining whether an
SAE is expected or unexpected. An adverse event will be considered unexpected if the nature, severity,
or frequency of the event is not consistent with the risk information previously described for the
intervention.
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Table 10. Possible expected adverse events and grading scale.a
CTCAE Term
Mild
Grade 1
Moderate
Grade 2
Severe
Grade 3
Potentially Life
Threatening Grade 4
Death
Grade 5
Abdominal Pain
(stomach ache)
Mild pain
Moderate pain; limiting
instrumental ADL*
Severe pain; limiting self care
ADL**
-
-
Allergic reaction Systemic intervention not
indicated
Oral intervention
indicated
Bronchospasm;
hospitalization indicated for
clinical sequelae; intravenous
intervention indicated
Life-threatening
consequences; urgent
intervention indicated
Death
Constipation
Occasional or intermittent
symptoms; occasional
use of stool softeners,
laxatives, dietary
modification, or enema
Persistent symptoms
with regular use of
laxatives or enemas;
limiting instrumental
ADL
Obstipation with manual
evacuation indicated; limiting
self care ADL
Life-threatening
consequences; urgent
intervention indicated
Death
Diarrhea
(≥3 loose stools
per day for 2
consecutive
days)
Increase of <4 stools per
day over baseline; mild
increase in ostomy output
compared to baseline
Increase of 4-6 stools
per day over baseline;
moderate ostomy output
compared to baseline;
limiting instrumental
ADL
Increase of >=7 stools per
day over baseline;
hospitalization indicated;
severe increase in ostomy
output compared to baseline;
limiting self care ADL
Life-threatening
consequences; urgent
intervention indicated
Death
Dyspepsia
(upset stomach
or indigestion)
Mild symptoms;
intervention not indicated
Moderate symptoms;
medical intervention
indicated
Severe symptoms; operative
intervention indicated
-
-
Fever
38.0 - 39.0°C
(100.4 - 102.2°F)
> 39 - 40.0°C
(102.3 - 104.0°F)
> 40.0°C (>104.0°F) for ≤ 24
hours
> 40.0°C (>104.0°F) for
> 24 hours
Death
Flatulence
Mild symptoms;
intervention not indicated
Moderate; persistent;
psychosocial sequelae
-
-
-
Lack/loss of
appetite
Loss of appetite without
alteration in eating habits
Oral intake altered
without significant
weight loss or
malnutrition; oral
nutritional supplements
indicated
Associated with significant
weight loss or malnutrition
(e.g., inadequate oral caloric
and/or fluid intake); tube
feeding or TPN indicated
Life-threatening
consequences; urgent
intervention indicated
Death
Loose stools
(not clinically
defined
diarrhea)
Mild symptoms;
intervention not indicated
Moderate symptoms;
limiting instrumental
ADL
Severe symptoms; limiting
self care ADL; hospitalization
indicated
Life-threatening
consequences; urgent
intervention indicated
-
Nausea
Loss of appetite without
alteration in eating habits
Oral intake decreased
without significant
weight loss, dehydration
or malnutrition
Inadequate oral caloric or fluid
intake; tube feeding, TPN, or
hospitalization indicated
-
-
Rash
(Cutaneous
reaction)
Covering <10% body
surface area (BSA) with
or without symptoms
(e.g., pruritus, burning,
tightness)
Covering 10-30% BSA
with or without
symptoms; limiting
instrumental ADL; rash
covering >30% BSA
with or without mild
symptoms
Rash covering >30% BSA
with moderate or severe
symptoms; limiting self care
ADL
-
-
Vomiting
Intervention not indicated Outpatient IV hydration;
medical intervention
indicated
Tube feeding, TPN, or
hospitalization indicated
Life-threatening
consequences
Death
a U.S. Department of Health and Human Services. Common Terminology Criteria for Adverse Events (CTCAE)
Version 5.0. Published: November 27, 2017.
Semi-colon (;) indicates ‘or’ within the description of the grade
Single dash (-) indicates grade is not available
Activities of Daily Living (ADL)
*Instrumental ADL refer to preparing meals, shopping for groceries or clothes, using the telephone, managing
money, etc.
**Self care ADL refer to bathing, dressing and undressing, feeding self, using the toilet, taking medications, and not
bedridden.
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7.4.3
Severity of Event
Adverse events will be graded according to the National Cancer Institutes’ Common Terminology Criteria
for Adverse Events (CTCAE) v5.0:
1. Grade 1 Mild: asymptomatic or mild symptoms; clinical or diagnostic observations only; no
intervention indicated; no to minimal impact on activities of daily living (ADL).
2. Grade 2 Moderate: minimal, local, or non­invasive intervention indicated; moderate impact on ADL.
3. Grade 3­ Severe: severe or medically significant but not immediately life-threatening; hospitalization
or prolongation of hospitalization indicated; disabling; needs major assistance with ADL.
4. Grade 4­ Potentially life­-threatening: life­-threatening consequences; urgent intervention indicated.
5. Grade 5 ­Death: death related to AE.
Please note that a severe AE and an SAE are distinct terms.
7.5
Expected Risks
The expected risks and side effects (listed in Table 10) for each intervention and procedure are as
follows:
●
Cultured yogurt: digestive/gastrointestinal problems such as stomach ache, constipation, diarrhea,
flatulence, fever, lack of appetite, vomiting, and loose, watery bowels, and allergic reaction to any of
the yogurt ingredients, most likely strawberry.
●
Amoxicillin­-clavulanate: nausea and/or vomiting, diarrhea, upset stomach and mild to severe skin
rash. The most serious potential risk is diarrhea caused by a Clostridium difficile infection.
Symptoms of C. difficile infection include stomach pain or tenderness, bloody stools, fever, nausea,
and diarrhea.
●
Fecal sample collection: there are no known risks. However, there may be some discomfort with
fecal sample collection.
These risks are considered to be minimal and are addressed in the protocol and consent form.
It will be explained to the participants that there may also be side effects, other than those listed, that we
cannot predict. Many side effects go away shortly after the treatments are stopped, but in some cases
side effects can be serious, long lasting or permanent. Participants will be informed to discuss any
questions about potential risks with the researchers and their primary care providers. Anyone who
develops evidence of allergy or hypersensitivity to any component of the yogurt products will be
withdrawn from the study and receive no further doses. We will continue to monitor these participants for
safety until the event sufficiently resolves.
7.6
Reporting Procedures
7.6.1
Unanticipated Problem Reporting
Incidents or events that meet the OHRP criteria for unanticipated problems require the creation and
completion of an unanticipated problem report form. OHRP recommends that investigators include the
following information when reporting an adverse event, or any other incident, experience, or outcome as
an unanticipated problem to the IRB:
●
Appropriate identifying information for the research protocol, such as the title, investigator’s name,
and the IRB project number;
●
A detailed description of the adverse event, incident, experience, or outcome;
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●
An explanation of the basis for determining that the adverse event, incident, experience, or
outcome represents an unanticipated problem;
●
A description of any changes to the protocol or other corrective actions that have been taken or are
proposed in response to the unanticipated problem.
To satisfy the requirement for prompt reporting, unanticipated problems will be reported using the
following timeline:
●
Unanticipated problems that are serious adverse events will be reported to the IRB, DSMB, and
NCCIH within 7 days of the investigator becoming aware of the event.
●
Any other unanticipated problem will be reported to the IRB, DSMB, and NCCIH within 14 days of
the investigator becoming aware of the problem.
●
All unanticipated problems should be reported to appropriate institutional officials (as required by
an institution’s written reporting procedures), the supporting agency head (or designee), and OHRP
within one month of the IRB’s receipt of the report of the problem from the investigator.
7.6.1
Adverse Event Reporting
SAEs that are unanticipated, serious, and possibly related to the study intervention will be reported to the
DSMB, IRB, and NCCIH in accordance with requirements.
If any SAEs are reported, the PIs will be alerted immediately, as well as the IRB. If deemed necessary,
members of DSMB will be alerted immediately. The PIs and/or IRB will decide if stopping criteria have
been met. If they believe so, an ad hoc teleconference will be arranged by the DSMB Administrator. No
other participants will be enrolled until after the teleconference, which will occur within 24 hours.
Additionally, SAEs will be reported using the following timeline:
●
Unexpected fatal or life-threatening AEs related to the intervention will be reported to the NCCIH
Program Officer, and DSMB within 3 days of the investigator becoming aware of the event. Other
serious and unexpected AEs related to the intervention will be reported within 7 days.
●
Anticipated or unrelated SAEs will be handled in a less urgent manner but will be reported to the
DSMB, IRB, and other oversight organizations in accordance with their requirements and will be
reported to NCCIH on an annual basis.
●
All other AEs documented during the course of the trial will be reported to NCCIH on an annual basis
by way of inclusion in the annual report and in the annual AE summary which will be provided to
NCCIH and to the DSMB. The DSMB Report will state that all AEs have been reviewed.
7.7
Follow-up for Adverse Events
The following applies only for non-serious adverse events; SAEs will follow the aforementioned policies
for reporting and monitoring of serious adverse events.
For non-serious AE, if the AE has not resolved by the Day 14 follow-up, then depending on the type of
AE (expected or unexpected) the research assistant will either continue to follow-up with the participant
or consider Day 14 as the final Date of Resolution:
●
If the AE reported is listed on the Adverse Event Report Form (Codes 1-12; as listed in Table 10)
as a possible, expected AE, the research assistant will continue to follow-up the participant a
minimum of every 4 days until the AE resolves to record the Date of Resolution.
●
If the AE reported is listed as “Other” (Code 13), or unexpected, and the Principal Investigator
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AND participant considers the AE is not related/or is unlikely related to the intervention, the
research assistant will not continue to follow-up past the Day 14 follow-up and will consider Day
14 as the final Date of Resolution.
7.8
Safety Monitoring
7.8.1
Data Safety Monitoring Board
The Data Safety Monitoring Board (DSMB) will function as an independent oversight committee charged
with overseeing participant safety and providing recommendations to the PIs and Co­-investigators
regarding the continuing conduct of the study. The DSMB will review periodic blinded Safety Reports.
Unblinded but sealed Safety Reports will be available to each DSMB member, should the committee
determine that review of unblinded safety data is warranted.
The DSMB will include independent individuals not involved in this research study and is comprised of
members Nicole Prewitt (community representative), Alexander Krist (physician), Geoffrey Preidis
(probiotics and microbiome physician-­scientist) and Chair, Elizabeth Carter (statistician). No member of
the Committee has collaborated or co­-published with the PIs within the past three years. They are
qualified to review the patient safety data generated by this study because of their unique expertise. The
clinical PI (Merenstein) and research team will provide the DSMB with an up­to­date data set to ensure
safety, effective data collection and progress. During the course of the study, the DSMB will meet
regularly to review adverse events data and approve continuation of the protocol.
7.8.2
Safety Reports
Study progress and safety will be reviewed 25%, 50% and 75% data completion (and more frequently if
needed). Progress reports, including patient recruitment, retention/attrition, and AEs will be provided to
the DSMB. A Safety Report will be compiled and will include a list and summary of AEs. In addition, the
reports will address (1) whether AE rates are consistent with pre­-study assumptions; (2) reason for
dropouts from the study; (3) whether all participants met entry criteria; (4) whether continuation of the
study is justified on the basis that additional data are needed to accomplish the stated aims of the study;
and (5) conditions whereby the study might be terminated prematurely. The report will be sent to the
DSMB and will be forwarded to the IRB and NCCIH. The IRB and other applicable recipients will review
the progress of this study on an annual basis.
The study team will generate Study Reports for the DSMB and will provide information on the following
study parameters: the status of follow­-ups, adverse events and enrollment updates. Study Report tables
will be generated only from aggregate (not by group assignment) baseline and aggregate safety data for
the study population.
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8. INTERVENTION DISCONTINUATION
Study enrollment and/or interventions may be temporarily suspended or terminated if:
●
Any study participant experiences death that the DSMB, PIs or IRB believes may be related to the
study interventions.
●
If a statistically or clinically significant number of SAEs or hospitalizations occur that the DSMB
believes may be related to the treatment.
●
New information from outside studies reveals that it would be unethical to continue enrolling
participants in the study.
●
The IRB decides that the study should be stopped.
●
If statistical differences among the primary outcomes will be determined that require stopping. For
example, if the differences among certain interventions make it unethical to continue or if the effect
size is so small that it is implausible a positive outcome could occur.
A safety review by the DSMB, IRB, and/or NCCIH will be convened to determine if the study
should continue per protocol, proceed with enhanced monitoring, be further investigated, be
discontinued, or be modified and then proceed. The FDA and NCCIH retain the authority to
suspend additional enrollment and study interventions and administration of study product for the
entire study, as applicable.
This study will be stopped prior to its completion if:
(1)
the intervention is associated with adverse effects that call into question the safety of the
intervention;
(2)
difficulty in study recruitment or retention will significantly impact the ability to evaluate the study
endpoints;
(3)
any new information becomes available during the trial that necessitates stopping the trial; or
(4)
other situations occur that might warrant stopping the trial.
Individuals may be withdrawn from the study for any of the following:
●
Adverse events; the clinical PI (DM) will discuss AEs with the participant, and possibly with their
personal physician, as needed, to determine whether the participant should be withdrawn from the
study.
●
If participants are deemed to be non­cooperative (e.g. refuse to participate in follow-­up calls or fail to
complete diaries).
●
If participants are lost to follow­up, i.e. more than 7 days without contact with study personnel during
the follow-up period.
●
Participants will not be withdrawn from the study solely for missed interventions or specimen
collections, but may be withdrawn for non-cooperation or lost to follow-up.
●
Serious adverse events related to the interventions, as determined by the PIs, participant or
participant’s physician.
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9. STATISTICAL CONSIDERATIONS
9.1
General Design Issues
Primary Hypothesis: Antibiotics will result in a reduction in fecal SCFA. BB-12 supplementation will
protect against antibiotic-induced SCFA (with acetate levels being used to represent the production of
SCFA by gut microbiota) reduction and/or be associated with a more rapid return to baseline SCFA levels
(acetate), compared to participants who receive a control yogurt without BB-12.
As reported by Hoverstad et al. (1986) and Clausen (1991), we hypothesize that antibiotic administration
for 7 days will decrease mean fecal acetate levels for each participant, and that upon conclusion of
antibiotic therapy fecal acetate levels will return toward baseline values for each participant. This is
consistent with what we observed in our R61 study.
We will compare the mean fecal acetate levels at days 7 and 30 versus the baseline values for
participants receiving BB-12 or control yogurt. A significantly higher mean level of fecal acetate,
calculated using one-way analysis of variance (Ganes-Howell post hoc test) in participants receiving
BB-12 yogurt compared to the control yogurt during or after antibiotic administration, will be consistent
with our findings in the R61 on the effect of BB-12.
We are moving into the R33 phase because the R61 demonstrated the expected changes described for
each signature, in the direction a priori predicted.
Timing Hypothesis: There is widespread hypothetical belief that timing of probiotic consumption may
impact efficacy. However, to our knowledge this has not been studied while simultaneously providing
antibiotics. We do not believe timing of probiotic and antibiotic consumption will impact SCFA changes.
Secondary Hypothesis: Antibiotics will result in a decrease in the overall number and diversity of
bacterial species present in the fecal microbiota. BB-12 supplementation will protect against
antibiotic-induced shifts in the microbiota and/or will be associated with a quicker return to a baseline
microbiota composition, as compared to the control group. Microbiota composition will be assessed with
16S rDNA profiling and qPCR.
9.2 Sample Size and Randomization
Sample size calculations were informed by our data from the R61 Phase. In our data, we compared
changes in mean SCFA levels between fecal samples from the control group and the active (BB-12
treated) collected at baseline and those collected after seven days administration of antibiotics or placebo
(day 7) as well as subsequent timepoints at day 14, day 21, and day 30. We assume that we will see a
similar baseline and variability at the various time points as our average baseline acetate levels (based
upon our data combining both the control and active group) of 47 ± 20 µM. In the R61 phase, we proposed
to compare differences at day 7 and day 30. Since we observed the most pronounced difference in acetate
at day 30, we calculated the R33 sample size based upon those changes. In our proposed R33 study , we
assume we will see a similar acetate value in the control as we did in the R61 phase which was (decrease
of 25% from average baseline) 35.25 ± 20 µM and a similar acetate level in the active (BB-12) which was
(decrease of only 2% from average baseline) 46 ± 20 µM. Assuming equal sample size per group, SD=20,
and four groups in total [(i) BB-12: concurrent dosing, (ii) control: concurrent dosing, (iii) BB-12: 4 hour
delay, (iv) control: 4 hour delay], a total sample size of 108 participants (27/group) will achieve a power of
80% to detect a difference of 0.05 between the BB-12/concurrent dosing and the BB-12/4 hour delay
groups from the control. In order to balance randomization over time, the number per group has been
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updated to 28/group and 12 in the antibiotic-only group (divisible by 4 treatment groups), for a total
enrollment of 124 participants.
9.2.1
Treatment Assignment Procedures
See Section 6.5.3 on Randomization.
The group allocation ratio is 28:28:28:28:12: 28 BB-12 yogurt concurrent consumption, 28 BB-12 yogurt
delayed consumption, 28 control yogurt concurrent consumption, and 28 control yogurt delayed
consumption, and 12 no yogurt control participants.
The web-based system will integrate the randomization scheme and automatically assign enrolled
participants to the corresponding allocation. All research personnel will be naive to group assignments.
9.3
Definition of Populations
Statistical analysis on the primary outcome will be performed using the intention to treat principle; all
subjects enrolled and randomized will be accounted for in the final analysis in their randomized group.
We will also examine the per protocol group separately and report both the intention-to-treat and per
protocol analyses. The mixed effect model will be used, which is compatible with unbalanced outcome
measurements. The multiple imputation analysis will be performed when the covariates are missing.
9.4
Interim Analyses and Stopping Rules
Since this is a mechanistic study, analyses of primary and secondary outcome data will not be conducted
before the study is completed. The main purpose is to examine the effects of the treatment of BB-12 (on
SCFA and microbiome changes), and thus non-inferiority or bio-equivalence testing is not indicated. There
will be no interim or futility analysis due to mechanistic hypotheses.
9.5
Outcomes
9.5.1
Primary Outcome
The primary outcome is a difference, measured at day 7, in antibiotic-induced changes in fecal SCFA
(with a particular focus on acetate) in participants receiving BB-12-supplemented yogurt versus
non-supplemented control yogurt.
9.5.2
Secondary Outcomes
The secondary outcome is a difference, measured at days 7 and 14, in antibiotic-induced changes in the
number and diversity of bacteria in the gut microbiota.
9.6 Anticipated Results
The most likely outcomes from the study are:
1.
+/- impact of BB-12 on fecal SCFA levels
2.
+/- impact of BB-12 on gut microbiota composition (measured to quantify level of disruption
achieved by antibiotics as a comparator within this study)
3.
+/- impact of BB-12 on AAD (measured, but study not powered to detect clinical difference of
AAD).
4.
4. +/- impact of BB-12 influenced by the timing between AMC dose and BB-12 consumption.
Since BB-12 was found to have a positive effect on the biological signature (acetate levels) as compared
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to the control, the work to be carried out in the R33 will replicate the R61 methods in a second cohort of
patients, with the addition of timing of probiotic administration, as well as expanding the analysis to
characterize the functional microbiome using metatranscriptomic methods. This information will provide a
framework for future research in the characterization of other probiotic strains for AAD.
9.7 Data Analyses
To examine the primary hypothesis that BB-12 supplementation will protect against antibiotic-induced
SCFA reduction, the mixed model will be used to analyze the treatment effect on the antibiotic-induced
SCFA reduction. Statistical analyses will be performed using intent-to-treat analytic strategies. The mixed
effect model will be used, which is compatible with unbalanced outcome measurements. The change of
SCFA from baseline will be considered as the outcome variable of the mixed model. All time points will
be included for the analysis. Covariates including age, sex, BMI and time (a continuous variable) will be
adjusted when necessary. In addition, the time and treatment interaction term will be assessed by the
likelihood ratio test. The random intercept will be used and the AR1 and/or compound symmetry
covariance structure will be considered (and then the likelihood test can help to determine the more
appropriate covariance structure). The final model selection will be performed based on information
criteria such as Akaike information criterion (AIC) or Bayesian information criterion (BIC). Power: with a
sample size of 40 subjects in the treatment arm and 20 subjects in the placebo arm and attrition rate of
5% at day six and 20% at day 30, we would have power of 82% to detect a moderate effect size of
Cohen’s d = 0.63 (assuming the intra-correlation correlation coefficient is 0.3). The α level of 0.05 will be
used for the analysis. Since the effect size of previous study (reduction in acetate levels) is Cohen’s d
=1.23, we should have ample power to detect the treatment effect. (The power calculation is based on
the R software and packages simr, sjstats, and pwr).
9.8 Missing Data
We understand the importance of limiting missing data and as in previous studies the PI and project
coordinator will work very closely to prevent missing data. However, there will be some missing data,
which we anticipate will be missing at random (MAR). Although it is challenging to test the assumption
MAR versus missing not at random (random) due to incomplete information for such test (122), we will
carefully examine the missing patterns and perform sensitivity analysis for the MAR assumption (123).
The multiple imputation will be used to deal with the missing data (e.g. the missing covariate and
baseline outcome variable).
A sensitivity analysis could be used to explore the impact of departures from the MAR assumption
underlying the main analysis. For example, we let the mean of observed data and the mean of the
unobserved data differ from each other, adjusted for other observed variables. Under an MAR
analysis, the difference is assumed to be zero.  We then evaluate the impact of difference on treatment
effect inference under MAR assumption.
To test the secondary hypothesis, we will examine the treatment effects on multiple microbiota
composition scores, for example, Bray-Curtis similarity score, Chao1 metric (α-diversity or richness), the
Shannon index. We will apply mixed models to analyze these metrics and then perform multiple testing
adjustments using FDR and/or FWER correction..
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10. DATA COLLECTION AND QUALITY ASSURANCE
10.1
Data Collection Forms
Blinded research assistants will be collecting participant information in person or over the phone, and will
be responsible for picking up samples from participants. All investigators and research personnel will
take required classes on human subject research and the protection of individually identifiable health
information prior to any contact with the participants.
Participants will be completing questionnaires with protected health information, and the release of
confidential information is a risk. The research staff will take all necessary precautions to minimize this
risk. Participants’ addresses and phone numbers will be collected, as will other private health
information. In accordance with the appropriate HIPAA regulations, all information will be protected and
kept confidential at all times. All contact and study information will be kept on password-protected
computers or online databases, participants will be identified by code, and no information will be released
without proper approvals or permissions.
10.2
Data Management
Data management is a joint effort between the study team, PIs, and the biostatistician. The investigators
are responsible for ensuring the accuracy, completeness, legibility, and timeliness of the data reported.
All source documents and data should be attributable, legible, contemporaneous, original, accurate and
complete (ALCOAC). The investigators will maintain adequate case histories of study participants,
including accurate case report forms (CRFs), and source documentation.
10.2.1 Data Collection
Data collection and accurate documentation are the responsibility of the study staff under the supervision
of the investigator. All source documents and laboratory reports are maintained by the study team, who
will ensure that they are accurate and complete. Unanticipated problems and adverse events must be
reviewed by the investigator or designee.
10.2.2 Data Management System
This study uses a project database within the Remote Electronic Data Capture (REDCap) system hosted
by MedStar Health Research Institute (MHRI), and is FDA 21 CFR Part 11 compliant. The database will
be secured with individual account logins and password protection. Each user must use their unique,
assigned user account and password.
The database incorporates an electronic audit trail to show change(s) to data after original entry
including the date/time and user making the change. REDCap has some built­in validation processes to
ensure data quality, including minimum/maximum ranges, data type, calculations, date, and branching
logic fields.
The data entry system is designed to allow information contained on a single form to be entered over one
or multiple keying sessions. For direct electronic capture, it is recommended to enter any one form in its
entirety rather than breaking up a single form into multiple keying sessions. Once an entry is created, any
later additions or changes to incomplete forms or sessions will require the user to log a ‘change reason’.
The data collection forms were also designed for keying directly into the data entry system without the
need for additional transcription.
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Entries in REDCap may be saved locally on the device and then backed up on the server once the
device is online. In the case of an outage of the REDCap system, a backup paper form will be used to
collect the data, and will be entered into the database at a later time.
The biostatistician will receive only coded information that is entered into the database under study
identification numbers. Electronic communication with outside collaborators will involve only de-identified
information.
10.2.3 Source Documentation
Source documents are original documents, data, and records (e.g., hospital records, clinical and office
charts, laboratory reports, memoranda, diaries or evaluation checklists, or pharmacy dispensing records)
which first records a finding, observation, or other activity during a study.
Since this study will enroll generally healthy participants and data are entered directly into the electronic
database, the case report and data collection forms will serve as the source documents. Any hardcopy
participant study records, such as case report forms, medical records (if applicable), and participant files,
are kept in a locked cabinet inside a locked office. Electronic records, identified by study ID code only,
are stored in the REDCap database or on Georgetown University secure servers, both of which require a
username and password. Only authorized, trained research staff will have access to the data.
Source Document/Form
Abbreviation Key Form
Completed by
Baseline Health Status
BH
X
Research staff
Daily Assessment Diary (Days 1-7, 8-14)
DD
X
Participant
Demographic Information
DI
X
Research staff
Automated Self-Administered 24-Hour (ASA24®) Dietary
Assessment Tool
ASA24
Research staff
Follow-up Form
FU
X
Research staff
Gastrointestinal Symptom Rating Scale - IBS
GS
Research staff
Inclusion/Exclusion
IE
Research staff
Informed Consent Process Documentation
IP
Research staff
Post Run-In Health Status
PR
X
Research staff
Pre-Screening
PS
Research staff
Randomization
RZ
Research staff
Sample Collection
SC
Research staff
Event-driven forms
Adverse Event Report
AE
X
Research staff
Diarrhea Report and Follow-up
DR
Research staff
Missed Contact Report
MC
Research staff
Serious Adverse Event Report
SA
X
Research staff
Withdrawal from Intervention
WI
Research staff
Other source documents may include study progress notes, electronic or paper medical records,
laboratory reports, memoranda, diaries, and other records pertaining to the participant while in the
research study.
10.2.4 Study Records Retention
Study documents will be retained for a minimum of 3 years following the formal discontinuation of clinical
development of the investigational product, unless the funder specifically requests otherwise.
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10.3
Quality Assurance
10.3.1 Training
All study staff will complete training on appropriate data collection and entry procedures, as well as the
study specific databases. Completed trainings will be recorded in the training log, which is on file in the
study regulatory binder. All key personnel (all individuals responsible for the design and conduct of this
study) have completed Human Subjects Research Protection Training.
10.3.2 Quality Control
The PI of the recruitment site, Dr. Merenstein, is responsible for quality management for this study. The
Research Associate has been designated by Dr. Merenstein to be responsible for the implementation of
the quality management plan.
All source documents and case report forms are to be completed in a manner to ensure the data is
attributable, legible, contemporaneous, original, accurate and complete. ALCOAC applies to both paper
and electronic source data, and the records that hold that data. The REDCap system uses an electronic
audit trail and signature capability to ensure each addition or change in the database is attributable to a
unique user, when it was added/changed, the reason for the change, and when the review of the record
is completed.
10.3.3 Metrics
Data validation and quality reports will be generated on a bi-monthly basis. This report will include
validation errors and other discrepancies in the data. The study staff will be responsible for resolving
errors and discrepancies by checking the source documents and making the necessary changes in the
database. Discrepancies should be resolved within 10 business days.
Performance reports will be sent to Dr. Merenstein quarterly. These will report on performance trends, i.e.
enrollment status compared to enrollment goals, rates of missed visits, lost to follow-­ups, and missing
data. The PIs can use these reports to gauge how the study team is performing in the study.
10.3.4 Protocol Deviations
A protocol deviation is any noncompliance with the clinical trial protocol or Good Clinical Practice (GCP)
requirements. The noncompliance may be either on the part of the participant, the investigators, or the
study personnel. As a result of deviations, corrective actions are to be developed by the site and
implemented promptly. It is the responsibility of the study personnel to use continuous vigilance to
identify protocol deviations. All deviations from the protocol must be addressed in study subject source
documents. Protocol deviations must be sent to the local IRB per their guidelines. The PIs/study
personnel will be responsible for knowing and adhering to their IRB requirements.
10.3.5 Monitoring
During the first year of recruitment, an internal source verification and file audit will be conducted on
three randomly selected participant records. Study personnel will not be notified of the three charts in
advance and must be prepared to pull all source documents. The audit is expected to last one day and
will be conducted by Tina Tan, the research associate.
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During the audit, the study databases will be compared with the source documents, and the reviewer will
check that all participants have signed informed consents, verify protocol procedures and records, and
ensure that the research personnel have up­to­date study documentation available. The reviewer will
provide a written report of all findings to the PI within 10 business days of the audit. The PI will send a
written response within 10 business days of receiving the audit report to document the response and
corrective action plans. Significant protocol deviations or issues will be added to the next DSMB report.
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11. PARTICIPANT RIGHTS AND CONFIDENTIALITY
11.1
Institutional Review Board (IRB) Review
Prior to enrollment of subjects into this trial, the approved protocol and the informed consent form as well
as all internal and external advertisements will be reviewed and approved by the Georgetown University
IRB. The Georgetown University IRB operates under the appropriate Federal Regulations, and the
research team have taken required classes on human subject research, ethics and HIPAA.
The responsible official at the Georgetown University IRB will provide the IRB letter of approval of the
protocol prior to the start of this trial. Any amendments to the protocol will be submitted to the IRB and
approval obtained prior to implementation.
11.2
Informed Consent Forms
Informed consent is a process that is initiated prior to an individual’s agreeing to participate in the study
and continuing throughout an individual’s study participation. Extensive discussion of risks and possible
benefits of this therapy will be provided to the participants. Consent forms describing in detail the study
interventions/products, study procedures, and risks are given to the participants and written
documentation of informed consent is required prior to starting interventions or administering study
products. The research team will discuss all information outlined in the informed consent document with
prospective participants to ensure that they understand the nature of the research and can voluntarily
decide whether or not to participate.
After screening, research personnel will meet with the participant and administer the informed consent
process. Dr. Merenstein will review, sign and date each consent form after the participant signs it.
11.3
Participant Confidentiality
Participant confidentiality is strictly held in trust by the investigators, their staff, and the sponsor(s) and
their agents. This confidentiality is extended to cover testing of biological samples in addition to the
clinical information relating to participants. In accordance with the appropriate HIPAA regulations, all
information will be protected and kept confidential at all times. All hardcopy participant study records,
such as case report forms, will be kept in a locked cabinet inside a locked office. All contact and study
information will be kept on password-protected computers or online databases, participants will be
identified by code, and no information will be released without proper approvals or permissions.
The FDA, study monitor, or other authorized representatives of the sponsor may inspect all documents
and records required to be maintained by the investigators. The clinical study site will permit access to
such records.
11.4
Study Discontinuation
The study may be discontinued at any time by the IRB, the NCCIH, the OHRP, the FDA, or other
government agencies as part of their duties to ensure that research participants are protected.
11.5   Ethical Standard
The investigator will ensure that this study is conducted in full conformity with the principles set forth in
The Belmont Report: Ethical Principles and Guidelines for the Protection of Human Subjects of Research
of the US National Commission for the Protection of Human Subjects of Biomedical and Behavioral
Research (April 18, 1979) and codified in 45 CFR Part 46 and the ICH E6 (R2); 83 FR 8882.
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12. COMMITTEES
The Data Safety Monitoring Board (DSMB) will function as an independent oversight committee (see
Section 7.8).
13. PUBLICATION OF RESEARCH FINDINGS
Investigators will make publication decisions jointly, consistent with their respective institutional and NIH
policies. The respective research administration offices of the institutions will be responsible for preparing
and negotiating an agreement for the conduct of the research and disposition of any resulting intellectual
property, consistent with their respective institutional policies and NIH policy.
Efforts will be made to publish our research findings in peer-reviewed, scientific journals in a timely
manner. All final peer-reviewed manuscripts that arise from this proposal will be submitted to PubMed
Central in accordance with the NIH Public Access Policy. Additionally, we will follow any publication
policies outlined in the Notice of Award provided by NCCIH.
This trial is registered at the NIH trials registry (http://ClinicalTrials.gov). Reporting of results of this trial
will be in accordance with the recommendations of the CONSORT statement
(http://www.consort-statement.org) and International Committee of Medical Journal Editors
(http://www.icmje.org/).
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14.
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Pelletier S, Kundrat S, Hasler CM. Effects of a functional foods nutrition education program with cardiac
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Veilleux BG, Rowland I. Simulation of the rat intestinal ecosystem using a two-stage continuous culture
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Clausen MR, Bonnen H, Tvede M, Mortensen PB. Colonic fermentation to short-chain fatty acids is
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Engelbrektson AL, Korzenik JR, Sanders ME, Clement BG, Leyer G, Klaenhammer TR, et al. Analysis of
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Langhendries JP, Detry J, Van Hees J, Lamboray JM, Darimont J, Mozin MJ, et al. Effect of a fermented
infant formula containing viable bifidobacteria on the fecal flora composition and pH of healthy full-term infants. J
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15. SUPPLEMENTS/APPENDICES
APPENDIX 1: Bristol Stool Chart
Heaton, K W & Lewis, S J 1997, 'Stool form scale as a useful guide to intestinal transit time'. Scandinavian
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APPENDIX 2: Summary of BB-12 Safety Articles Using the Identical Strain
A) Langhendries, J.P., et al., Effect of a fermented infant formula containing viable bifidobacteria on the fecal flora
composition and pH of healthy full-term infants. J Ped Gastro Nutr, 1995. 21(2): p. 177-81.
In a double-blind, randomized controlled three-armed study, 54 healthy infants were breast-fed, given a fermented
infant formula containing BB-12, or fed a non-acidified formula during the first two months of life. Growth, tolerance,
acceptability and fecal flora were assessed. No adverse events were reported. The BB-12 formula was found to
be well-tolerated and promoted normal growth during the first two months of life, as compared to the breast-fed
infant group.
B) Haschke, F., et al., Clinical trials prove the safety and efficacy of the probiotic strain Bifidobacterium Bb12 in
follow-up formula and growing-up milks. Monatsschr Kinderheilkd (Suppl 1), 1998. 146: p. S26-S30. Double blind,
randomized, controlled feeding studies with BB-12 in the U.S., China, and Thailand demonstrated normal growth of
healthy children between 4 and 36 months of age and catch-up growth in malnourished children. Feeding the
milk-based formula with BB-12 resulted in protection from rotavirus infection, fewer periods with hard bowel
movements and a lower incidence of diaper rash. No adverse events were reported.
C) Saavedra, J.M., et al., Long-term consumption of infant formulas containing live probiotic bacteria: tolerance and
safety. Am J Clin Nutr, 2004. 79(2): p. 261-7.
In a double-blind, randomized placebo controlled study, 118 healthy infants age 3-24 months consumed formula for
210±127 days. Thirty-nine infants received a standard milk-based formula supplemented with a high dose of BB-12
and TH-4, 39 infants received a low supplementation of BB-12 and TH-4 and 40 infants received an
unsupplemented formula. The supplemented formulas were well accepted and were associated with a significantly
lower frequency of reported colic or irritability (p<0.001) and a lower frequency of antibiotic use (p<0.001) than
unsupplemented formula. Of the 5 infants who developed an intercurrent illness or complaint, 4 had vomiting and
diarrhea (1 in the placebo group and 3 in the high dose group) and 1 (high dose group) had otitis media. Three
infants ended their participation because of an effect perceived by their parents to be related to formula
consumption. Of these three, one infant developed a rash (after consuming the formula for 30 days), but was
diagnosed as being viral by the pediatrician. The second infant had loose stools and the third had loose stools and
vomiting (both in the high dose group). Results showed no significant differences between groups in growth,
health care attention seeking or other health variables, and therefore can be concluded that long-term
consumption of BB-12 and TH-4 supplemented formulas are well tolerated and safe.
D) Bin-Nun, A., et al., Oral probiotics prevent necrotizing enterocolitis in very low birth weight neonates. J Pediatr,
2005. 147(2): p. 192-6. In this study, 145 neonates of less than or equal to 1500g birth weight were given a daily
feeding of a BB-02, BB-12 and TH-3 supplemented probiotic mixture or were given no feed supplements. Using
Bell’s criteria, development of necrotizing enterocolitis (NEC) was assessed. There were no differences in the
incidence of any signs of feeding intolerance such as diarrhea, abdominal distension, or vomiting between the
study and control groups. Three infants in the study group versus eight infants in the control group died (P=.218;
RR 0.38; yielding a relative risk reduction of 62%; 95% CI: 0.38-1.38). The three deaths in the control group were
attributed to NEC, whereas there were no NEC-associated deaths among the treated infants. There were no other
significant differences in the characteristics of those who died between the two groups. No other adverse events
were reported.
E) Weizman, Z. and A. Alsheikh, Safety and tolerance of a probiotic formula in early infancy comparing two
probiotic agents: a pilot study. J Am Coll Nutr, 2006. 25(5): p. 415-9. Fifty-nine full-term infants aged 3-65 days were
randomly assigned for 4 weeks to a standard milk-based formula supplemented with either BB-12, Lactobacillus
reuteri (ATCC 55730) or a probiotics-free formula. The supplemented formulas were well accepted and did not
reveal any adverse effects. A comparison of growth parameters, and variables of feeding, stooling, crying and
irritability did not reveal any significant differences between groups. No adverse events were reported in any of the
participants. Therefore, use of formula supplemented with either BB-12 or Lactobacillus reuteri in early infancy, was
safe, well tolerated and did not adversely affect growth, stooling habits or infant behavior.
F) Saavedra, J.M., et al., Feeding of Bifidobacterium bifidum and Streptococcus thermophilus to infants in hospital
for prevention of diarrhoea and shedding of rotavirus. Lancet, 1994. 344(8929): p. 1046-9.
In a double-blind, placebo-controlled trial, 55 hospitalized infants aged 5-24 months were fed either a formula
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supplemented with probiotics Bifidobacterium bifidum and Streptococcus thermophilus or a formula without
probiotics. Subjects were evaluated for diarrhea and rotavirus shedding for 4447 patient-days during 17 months. All
infants maintained or improved their nutritional status throughout the study. There were no adverse effects judged
to be associated with the feeding of either formulas, and adequate growth was recorded in all subjects. The
probiotic formula was well tolerated by the infants, many of whom were initially malnourished or
immunocompromised.
G) Sheu, B.S., et al., Impact of supplement with Lactobacillus- and Bifidobacterium-containing yogurt on triple
therapy for Helicobacter pylori eradication. Aliment Pharmacol Ther, 2002. 16(9): p. 1669-75.
One hundred and sixty Helicobacter pylori-infected patients were randomized to receive either 1 week of triple
therapy without supplements or triple therapy with Bifidobacterium BB-12 and Lactobacillus acidophilus LA-5 yogurt
(AB-yogurt). In the triple-plus-yogurt group, AB-yogurt was continued for 4 weeks after completion of triple therapy.
Common side-effects, such as vomiting, constipation, diarrhea and metallic taste, were significantly
decreased in the triple-plus-yogurt group, with 15 events reported in the triple-plus-yogurt group and 53 reported
in the triple-only group. In summary, supplementation with AB-Yogurt can improve drug compliance and thus
enhance the intention-to-treat eradication rate of H. pylori after triple therapy. Furthermore, continued
supplementation with AB-Yogurt after triple therapy can restore Bifidobacterium in stools after 4 weeks.
H) Phuapradit, P., et al., Reduction of rotavirus infection in children receiving bifidobacteria- supplemented formula.
J Med Assoc Thai, 1999. 82 Suppl 1: p. S43-8. One hundred and seventy-five children aged 6-36 months were
randomized to receive either a milk based follow-up formula, formula supplemented with BB-12 alone or formula
supplemented with BB-12 and Streptococcus thermophilus for a period of 8 months. There were 81 episodes of
diarrhea during the study period, most of which were caused by bacterial enteropathogens and only 3 were of
rotavirus origin. The caregivers of the participants reported no adverse events.
I) Laake, K.O., et al., Assessment of mucosal inflammation and circulation in response to probiotics in patients
operated with ileal pouch anal anastomosis for ulcerative colitis. Scand J Gastroenterol, 2003. 38(4): p. 409-14. Ten
patients operated with ileal-pouch-anal anastomosis for ulcerative colitis were given a fermented milk product
containing live lactobacilli (La-5) and bifidobacteria (BB-12) daily for 4 weeks. The results suggested that probiotics
primarily act superficially, with change of gross appearance of the mucosa at endoscopy, but without significant
effect on histological picture, mucosal perfusion or faecal calprotectin, during a relatively short period of 4 weeks.
No adverse effects were recorded.
J) Anderson, A.D., et al., Randomised clinical trial of synbiotic therapy in elective surgical patients. Gut, 2004.
53(2): p. 241-5. In a double blind, randomised placebo controlled study, 137 patients for elective laparotomy were
enrolled 2 weeks prior to receive a combination of pre- and probiotics (synbiotic) Lactobacillus acidophilus La5,
Bifidobacterium lactis BB-12, Streptococcus thermophilus, and Lactobacillus bulgaricus, together with the prebiotic
oligofructose or a treatment of placebo capsules and sucrose powder. Seventy-two patients were in the synbiotic
group and 65 patients in the placebo group. There were no differences in the incidence of septic morbidity
between the placebo and synbiotic groups. The most common sites of infection were the urinary tract,
respiratory tract and surgical wound.
K) Anderson, A.D., et al., Randomized clinical trial of multimodal optimization and standard perioperative surgical
care. Br J Surg, 2003. 90(12): p. 1497-504. Twenty-five patients undergoing colonic resection were randomized into
two groups receiving a ten-point optimization program or standard care. Optimized care included pre- and probiotic
treatments for 7–14 days before surgery. The prebiotic used was oligofructose and the probiotic capsules contained
Lactobacillus acidophilus La5, Lactobacillus bulgaricus, Bifidobacterium lactis BB-12 and Streptococcus
thermophilus. Five patients in the conventional group reported 6 instances of postoperative complications: urinary
retention (one patient), atrial fibrillation (two), respiratory depression related to PCA (two) and ileus (one); compared
to four patients reporting of 5 instances of complication in the optimization group: ineffective epidural (two patients),
ileus (one), urinary tract infection (one) and wound infection (one). No adverse events inconsistent with
post-operative recovery were reported. One patient in the control group died from a perioperative myocardial
infarction on the first postoperative day. In conclusion, optimization of surgical care significantly improved patients’
physical and psychological function in the early postoperative period and facilitated early hospital discharge.
L) Mohan, R., et al., Effects of Bifidobacterium lactis Bb12 supplementation on intestinal microbiota of preterm
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infants: a double-blind, placebo-controlled, randomized study. J Clin Microbiol, 2006. 44(11): p. 4025-31. A double
blind, placebo-controlled, randomized clinical trial was performed on 69 preterm infants born with a gestational age
of <37 weeks. The infants were randomized into two groups to receive a probiotic formula supplemented with
BB-12 or a placebo formula from the first day after birth for 21 days. No adverse effects were observed in any of
the infants.
M) Pitkala, K.H., et al., Fermented cereal with specific bifidobacteria normalizes bowel movements in elderly
nursing home residents. A randomized, controlled trial. J Nutr Health Aging, 2007. 11(4): p. 305-11. In a
randomized, double blind, placebo-controlled trial, 209 elderly nursing home residents were randomized to a daily
fermented oat drink with either 1) Bifidobacterium longum strains, or 2) BB-12, or 3) without viable bacteria
(placebo) for 7 months. No adverse events were reported. The fermented oat drinks were well taken and well
tolerated by subjects, with compliance at 85%.
N) Saarela, M., et al., Tetracycline susceptibility of the ingested Lactobacillus acidophilus LaCH-5 and
Bifidobacterium animalis subsp. lactis Bb-12 strains during antibiotic/probiotic intervention. Int J Antimicrob Agents,
2007. 29(3): p. 271-80. In this study, 10 patients suffering from respiratory tract infections consumed doxycycline
(tetracycline) for 10 days together with probiotic capsules containing Lactobacillus acidophilus La-5 and
Bifidobacterium Bb-12. As a control group, 10 volunteers consumed only probiotic capsules for 14 days. No
adverse events were reported.
O) Matsumoto, M., et al., Effect of Bifidobacterium lactis LKM 512 yogurt on fecal microflora in middle to old aged
persons. Microbial Ecology in Health and Disease, 2000. 12: p.77-80. In this study, eleven long-term inpatient
volunteers aged 50-93 years, consumed a yogurt product containing Bifidobacterium lactis LKM 512 (BB-12),
Lactobacillus bulgaricus LKM 1759 and Streptococcus thermophilus LKM 1742 daily for 2 weeks. The study lasted
7 weeks with the following schedule: pre-administration (1 week), probiotic-supplemented yogurt administration (2
weeks), post-administration period (2 weeks) and administration of ordinary yogurt without probiotics (placebo
yogurt) (2 weeks). No adverse events were reported.
P) Laake, K.O., et al., Influence of Fermented Milk on Clinical State, Fecal Bacterial Counts and Biochemical
Characteristics in Patients with Ileal-Pouch-Anal-Anastomosis. Microbial Ecology in Health and Disease, 1999. 11:
p. 211-17. In a double-blind, randomized, controlled study, 16 patients with restorative proctocolectomy for
ulcerative colitis randomized to two groups: 8 patients ingested 500 ml of a fermented milk product containing live
LA-5 and BB-12 (in its regular form) and 8 patients ingested 500 ml of a heat-treated form of the same product for
one week. No adverse effects were recorded.
Q) Matsumoto, M., et al., Impact of LKM512 yogurt on improvement of intestinal environment of the elderly. FEMS
Immunology and Medical Microbiology, 2001. 31: p. 181-86. In a placebo controlled study, 6 elderly volunteers of
average age 78 years, consumed BB-12 yogurt then consumed a placebo yogurt without BB-12 supplementation.
Fecal samples were tested before and after administration of both products as well as at Week 1 and Week 2
during administration. It was observed that the intestinal environment of the volunteers improved after the ingestion
of BB-12 yogurt. No adverse effects were reported.
R) Jain, P.K., et al., Influence of synbiotic containing Lactobacillus acidophilus La5, Bifidobacterium lactis Bb 12,
Streptococcus thermophilus, Lactobacillus bulgaricus and oligofructose on gut barrier function and sepsis in
critically ill patients: a randomised controlled trial. Clin Nutr, 2004. 23(4): p. 467-75.
The aim of this study was to determine whether the oral administration of a synbiotic preparation could alter gut
barrier function in critically ill patients and thus reduce sepsis. Ninety patients admitted to an intensive care unit
were randomised to either synbiotic or placebo preparations (45 into each group). The synbiotic preparation
consisted of Lactobacillus acidophilus La5, Bifidobacterium lactis BB-12, Streptococcus thermophilus and
Lactobacillus bulgaricus (probiotics), in dose of one capsule three times a day, with oligofructose (prebiotic),
administered twice a day. The median duration of intake of study medication was 10 days in both groups. Fifty-nine
patients developed one or more septic complications during the study; 33/45 (73%) in the synbiotic group and
26/45 (58%) in the control group. This did not represent a statistically significant difference (χ2 2.41, P=0.12).
The mortality rate was similar in both groups; 49% (22/45) in the synbiotic group and 45% (20/45) in the control
group (χ2 0.17, P=0.672).
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S) Laake, K.O., et al., Assessment of mucosal inflammation and blood flow in response to four weeks' intervention
with probiotics in patients operated with a J-configurated ileal-pouch-anal-anastomosis (IPAA). Scand J
Gastroenterol, 2004. 39(12): p. 1228-35. Forty-one patients with ulcerative colitis and ten patients with familial
adenomatous polyposis operated with ileal-pouch-anal-anastomosis were given 500 ml of a fermented milk product
(Cultura) containing BB-12 and LA-5 daily for 4 weeks. The aim of the study was to determine, in conjunction with
an earlier study, if mucosal perfusion in the distal part of the ileal pouch is reduced in patients on probiotic
intervention. All patients completed the treatment period and reported intake of Cultura according to the study
protocol with no report of adverse effects.
T) Laake, K.O., et al., Outcome of four weeks' intervention with probiotics on symptoms and endoscopic
appearance after surgical reconstruction with a J-configurated ileal-pouch-anal-anastomosis in ulcerative colitis.
Scand J Gastroenterol, 2005. 40(1): p. 43-51. In a previous double-blind, randomized, controlled study, clinical
improvement of symptoms was demonstrated in patients with ulcerative colitis (UC) operated on with
ileal-pouch-anal-anastomosis (IPAA), during intervention with live probiotic microbes Lactobacilli and
Bifidobacteriae. The aim of the present study was to confirm the previous results in a larger population, including
clinical symptoms, fecal flora and endoscopic evaluation, and to compare the results in UC/IPAA patients with those
of patients with familial adenomatous polyposis (FAP) with IPAA and UC patients with ileorectal anastomosis (IRA).
Five hundred millilitres of a fermented milk product (Cultura) containing live lactobacilli (La-5) and bifidobacteriae
(BB-12) was given daily for 4 weeks to 51 UC patients and 10 patients with FAP, operated on with IPAA, and six UC
patients operated on for IRA. No adverse events related to consumption of product were reported.
U) Gatt, M., et al., Randomized clinical trial of multimodal optimization of surgical care in patients undergoing major
colonic resection. Br J Surg, 2005. 92(11): p. 1354-62. Thirty-nine patients undergoing major elective colonic
resection were randomized to receive a ten-point multimodal optimization package or conventional perioperative
care. The optimized program included the supplementation of Trevis (Lactobacillus acidophilus La5, Lactobacillus
bulgaricus, Bifidobacterium lactis BB-12 and Streptococcus thermophilus) daily 7-14 days before surgery.
Twenty-four patients developed complications, nine in the optimized group and 15 in the control group, and five
patients required readmission within 30 days of surgery (one and four, respectively). There was one death, which
occurred after a perioperative myocardial infarct in a patient randomized to multimodal optimization. There were no
differences in morbidity and mortality between the optimization and conventional group.
V) Wildt, S., et al., Probiotic treatment of collagenous colitis: a randomized, double-blind, placebo-controlled trial
with Lactobacillus acidophilus and Bifidobacterium animalis subsp. Lactis. Inflamm Bowel Dis, 2006. 12(5): p.
395-401. In a randomized, double blind, placebo-controlled trial, 29 patients with collagenous colitis and diarrhea
were divided into two groups; 21 patients consumed Lactobacillus acidophilus LA-5 and Bifidobacterium animalis
subsp. lactis BB-12 capsules (AB-Cap-10) and 8 patients consumed a placebo for 12 weeks. The overall tolerance
of AB-Cap-10 and placebo was good, with only minor adverse events. In no case were side effects the cause of
withdrawal from study. Only the gastrointestinal symptoms were considered possibly related to AB-Cap-10. In the
probiotic group, worsening of diarrhea (n=1), abdominal pain and constipation (n=2), stomach burn (n=1), nausea
(n=1), and flatulence (n=1) were reported. In the placebo group, 4 patients complained of nausea.
W) Hol, J., et al., The acquisition of tolerance toward cow’s milk through probiotic supplementation: a randomised,
controlled trial. J. Allergy Cli. Immunol., 2008, 121(6): p. 1448-1455. In a randomized, double-blind, placebo
controlled trial, 119 infants between the of ages 1.4 to 6 months with cow’s milk allergy were assigned to two
groups to receive extensively hydrolyzed formula or formula with Bifidobacterium lactis BB-12 and L. casei CRL 431
for twelve months. The tolerance to cow’s milk was similar at 6 and 12 months. Probiotic intake was confirmed
because probiotics were isolated from the feces more often in the treated infants than the control infants. Parents
were asked to document whether their infants showed any symptoms as a result of the study formula. The study
formula with or without the probiotic supplementation was well tolerated. They did not note any difference
between the groups.
X) Kajander, et al., Clinical trial: multispecies probiotic supplementation alleviates the symptoms of irritable bowel
syndrome and stabilizes intestinal microbiota. Aliment Pharmacol Ther. 2008, 27: p.48-57.
In a randomized, double-blind, placebo-controlled study, irritable bowel syndrome (IBS) patients were randomized
to receive once daily either 1.2dL of a probiotic milk-based drink containing Bifidobactium lactis (BB-12), L.
rhamnosus GG (LGG), L. rhamnosus Lc705, and P. shermanii JS or 1.2dL of a placebo drink devoid of probiotics
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but similar otherwise to the probiotic drink. The purpose of the study was to evaluate the probiotic drink compared
to the placebo on the health-related quality of life and IBS symptoms of each patient. At 20 weeks, the IBS
symptom score of subjects in the probiotic group decreased significantly, with a 37% reduction compared
to baseline, while the placebo-controlled group had a 9% reduction in IBS symptoms. Significant beneficial
effects were also seen on the quality of life of patients in the probiotic group.
Y) Meng, et al., Effect of a lactose-free milk formula supplemented with bifidobacteria and streptococci on the
recovery from acute diarrhea. Asia Pac J Clin Nutr. 2008, 17(1): p.30-34. In a double-blind, randomized,
placebo-controlled study, 212 infants between the ages of 6 and 36 months were studied to determine the effect of
a lactose-free formula, supplemented with S. thermophilus and B. Lactis BB-12 at concentrations of 108 and 109
cfu/gram of powder, on the duration of the infants’ episodes of acute diarrhea. 71 infants were randomized to the
control group, a milk-based lactose-free formula, 71 infants were randomized to receive the same formula but
supplemented with 108 CFU/g BB-12, and 70 infants were randomized to receive 109 CFU/g BB-12 in their formula.
The duration of the diarrhea episodes was not significantly different among the three groups.
Z) Smerud, et al., Effect of a probiotic milk product on gastrointestinal and respiratory infections in children
attending day-care. Microbial Ecology in Health and Disease. 2008, pp 1-6. In a randomized, double-blind, placebo
controlled trial 240 children between the ages of 12-36 months were studied to determine if three probiotic strains
(L. rhamnosus GG, L. acidophilus La-5, Bifidobactium lactis BB-12) given daily during their first year of day-care
could prevent infections versus a placebo product. The children were asked to drink 150ml of investigational
product every day. The product had > 10 e 8 cfu / ml of LGG and BB-12 and > 10 e 7 cfu/ ml of La-5. The probiotic
reduced the number of days with gastrointestinal symptoms (1.7 days for the probiotic product compared
to 3.0 days for the placebo, i.e. 43% lower mean of the probiotic product, p=0.02) No significant difference
between treatments was seen with respect to respiratory symptoms.
AA) Rautava, et al., Specific probiotics in reducing the risk of acute infections in infancy – a randomised,
double-blind, placebo-controlled study. Brit J Nutr. 2009, 101: p.1722—1726. In this study, 81 infants before the age
of 2 months were randomized to receive formula supplemented with Bifidobacterium lactis (BB-12) and L.
rhamnosus GG (LGG) or normal formula daily until the age of 12 months to determine whether probiotics can be
effective in reducing the risk of infections in infancy. The study probiotics reduced the risk of early acute otitis
media and need for antibiotic treatment during the first 7 months of life, and significantly reduced the
incidence of recurrent respiratory infections during the first 12 months of life. No serious adverse effects
resulting from probiotic supplementation were detected during the study.
AB) Dotterud, et al., Probiotics in pregnant women to prevent allergic disease: a randomized, double-blind trial. Brit
J Dermatol. 2010, 163: p.616–623. The goal of this study was to determine whether a probiotic supplement given to
pregnant women during the last 4 weeks of pregnancy up until 3 months after birth would reduce the incidence of
allergic disease and allergic sensitization in their children at 2 years of age compared with a placebo. The pregnant
women were randomized to receive daily either 250mL probiotic low fat fermented milk, containing L. acidophilus
(La-5), Bifidobacterium lactis (BB-12), and L. rhamnosus GG (LGG) or 250mL placebo skimmed fermented milk.
The cumulative incidence of atopic disease was significantly reduced in children born to mothers treated
with a probiotic supplement for 4 months. However, there was no reduction in the incidence of asthma or
allergic rhinoconjunctivitis in the probiotic group.
AC) Merenstein, et al., The study to investigate the potential benefits of probiotics in yogurt, a patient-oriented,
double-blind, cluster-randomised, placebo-controlled, clinical trial. Eur J Clin Nutr. 2010, 64: p. 685-691. In this
study, 182 subjects between the ages of 1-3 years were studied to determine if daily consumption of a
probiotic-supplemented yogurt-based beverage containing Bifidobacterium lactis BB-12 can reduce daycare
absences. The children were asked to drink 4 oz. of active or control drink for 90 consecutive days. The active drink
had >1010 CFU/serving of BB-12. The active drink did not decrease absences due to illness in daycare/school
for healthy children. The strain of probiotic studied did not show any positive impact on absences.
AD) Simren, et al., Clinical trial: the effects of a fermented milk containing three probiotic bacteria in patients with
irritable bowel syndrome—a randomized, double-blind, controlled study. Aliment Pharmacol Ther. 2010, 31:
p.218-277 This study aimed to assess the effect of milk fermented with L. bulgaricus and St. thermophiles and
containing L. acidophilus (La-5), B. Lactis BB-12, and L. paracasei F19 versus acidified milk without the bacteria on
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the symptoms of patients with irritable bowel syndrome (IBS). 74 IBS patients were randomized to receive daily
400mL of either the active or control drink for 8 weeks. In both groups, the severity of IBS symptoms was gradually
reduced over the 8-week period. A significant positive effect of probiotic milk could not be detected on the
symptoms of IBS patients compared to the control treatment.
AE) Merenstein et al., Study to investigate the potential of probiotics in children attending school. Eur J Clin Nutr.
2011, 65: p.447-453. In a randomized, double-blinded, placebo-controlled trial, 172 subjects between the ages of
2-4 years were studied to determine if once-daily consumption of a probiotic-supplemented yogurt-based beverage
containing Bifidobacterium lactis (BB-12) can reduce daycare absences. 91 children were randomized to receive
the active drink and 81 children were randomized into the control group. The active drink had >1010 CFU / serving
of BB-12. The probiotic-containing yogurt-based beverage did not decrease absences due to illness in
daycare/school for healthy children.
AF) Taipale, et al., Bifidobacterium animalis subsp. lactis BB-12 in reducing the risk of infections in infancy. Brit J
Nutr. 2011, 105: p.409-416. In this double-blinded, placebo-controlled study, 109 newborn 1-month-old infants were
randomized to receive either a B.lactis BB-12 tablet or the control placebo tablet in order to investigate the impact
of BB-12 supplementation on the risk of acute infectious diseases. The tablets were administered twice daily until
the age of 8 months. The study demonstrated that BB-12 supplementation significantly reduced the
incidence of respiratory infections during the first 8 months of life in healthy breastfed infants.
AG) Wildt, S., et al., A randomised double-blind placebo-controlled trial with Lactobacillus acidophilus La-5 and
Bifidobacterium animalis subsp. lactis BB-12 for maintenance of remission in ulcerative colitis. J Crohns Colitis.
2011, 5: p.115-121. Thirty-two patients with ulcerative colitis (UC) were randomized to receive either Probio-Tec
AB-25, a probiotic capsule containing L. acidophilus (La-5) and Bifidobactium lactis (BB-12), or a placebo capsule
in order to study the clinical effect of probiotic treatment in maintaining remission in patients with UC. The study
drug was administered for 52 weeks. Of the 20 patients randomized to the experimental group, 5 patients (25%)
maintained remission after one year of treatment, while only 1 patient out of the 12 in the placebo group maintained
remission. ProbioTec AB-25 demonstrated no effect on maintenance of remission in patients with UC.
AH) Jacobs, S.E., et al., Probiotic Effects on Late-onset Sepsis in Very Preterm Infants: A Randomized Controlled
Trial. Pediatrics. 2013, 132: p.1055-1062. In this double-blind, placebo-controlled, randomized trial, very preterm
infants defined as born <32 weeks’ gestation and weighing <1500g were supplemented soon after birth with either
probiotics containing S. thermophilus, B. Lactis BB-12, and B. infantis BB-02 or placebo to determine the effect of
the daily administration of probiotics on the incidence of definite late-onset sepsis. 1099 infants were enrolled with
548 randomized to the probiotic group and 551 to the control group. The study found no significant effect of the
probiotic combination on definite late-onset sepsis in very preterm infants.
AI) Smith, T.J., et al., Effect of Lactobacillus rhamnosus LGG and Bifidobacterium animalis ssp. lactis BB-12 on
health-related quality of life in college students affected by upper respiratory infections. Brit J Nutr. 2013, 109:
1999—2007. The aim of this study was to determine the effect of probiotics on the health-related quality of life in
college students suffering an upper respiratory infection (URI). In this double-blinded, placebo- controlled trial, 231
healthy students at Framingham State University were randomized to receive a daily dose (5g) of either
strawberry-flavored probiotic powder or strawberry-flavored placebo powder. Each powder package contained 109
CFU of LGG and BB-12. The duration of URIs in the probiotic group was significantly shorter than the
duration of the URIs of the control group. Also, the probiotic group had URIs of significantly lower severity
compared to the placebo group.
AJ) Fox M, et al., Can probiotic yogurt prevent diarrhoea in children on antibiotics? A double-blind, randomised,
placebo- controlled study. BMJ Open. 2015; 5(1): e006474. In this study, 72 children between the ages of 1-12
years prescribed broad-spectrum oral antibiotics were studied to examine the efficacy of yogurt containing
Lactobacillus rhamnosus GG (LGG), Bifidobacterium lactis (Bb-12), and Lactobacillus acidophilus (La-5) in
reducing the rate of antibiotic-associated diarrhea (AAD). Seventy children, thirty-six of whom received the
pasteurized placebo yogurt and thirty-four of whom received the probiotic study yogurt, completed the trial, which
consisted of consuming two 100g tubs of yogurt/day for the entire duration of their antibiotic treatment. The
probiotic group had significantly fewer instances of adverse events. Children in the probiotic group had
fewer instances of diarrhea and no cases of severe diarrhea, compared to the placebo group.
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