Probiotic Treatment: The Role of the Gut Microbiome in Childhood Abdominal Pain and Anxiety
Phase: PHASE2
Conditions: Anxiety, Abdominal Pain
Sponsor: Nancy Zucker

 
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Research Summary Template 
 
1. Protocol Title: 
The Probiotic Study: Using Bacteria to Calm your Mind  
 
2.  Purpose of the Study:  
We seek to 1) conduct the first open-label trial of probiotics in young children, 2) examine the effects 
of probiotic treatment on the reduction of anxiety and abdominal pain, 3) examine changes in stress-
reactivity as a potential mediator of treatment effects, 4) assess the feasibility and efficacy of this 
treatment, and 5) elucidate the pre- and post-treatment composition of enteric microbiota in the 
middle-lower GI tract as a potential mediator of treatment effects.   
 
3.  Background & Significance:  
Approximately 10% of children presenting to primary care facilities complain about abdominal pain, 
the most frequent symptom in gastrointestinal (GI) diseases (Peery et al., 2012; Ramchandani, 
Hotopf, Sandhu, & Stein, 2005). Annual costs for the diagnosis and treatment of GI diseases in the 
US have been estimated at $142 billion in direct and indirect costs (Peery et al., 2012). In addition to 
the direct costs related to GI disease is the added burden of comorbid psychiatric illness. When 
present, an anxiety disorder can significantly prolong the course and increase the burden of GI 
disease, which in turn can increase symptoms of anxiety (Shelby et al., 2013). 
 
In the US, 28.8% of the population will receive a diagnosis of an anxiety disorder during their 
lifetime and 5-8% of 5-year olds meet diagnostic criteria (Kessler, Berglund, Demler, Jin, Merikangas, 
& Walters, 2005). Given the high rates of comorbidity and synergistic vulnerability imposed by GI 
disease and anxiety disorders, understanding and treating core processes that contribute vulnerability 
to both disorders may significantly alter the developmental course and outcome. This transdiagnostic 
focus is consistent with recent emphases of the NIMH, part of the Research Domain Criteria 
(RDoC) initiative (Cuthbert & Insel, 2013). 
 
One barrier to treatment development is that the biological mechanisms that contribute to enhanced 
abdominal pain and/or anxious experience in children are unknown. Some studies show that 
interventions that directly alter gut microbiome composition (e.g., as in the use of probiotics), not 
only alter pain behavior, but also decrease anxious behavior in animal models (Collins, Verdu, 
Denou, & Bercik, 2009). In adult human studies, the gut microbiota is altered in individuals with 
irritable bowel syndrome, and symptoms may be relieved by probiotic consumption (Jeffery, 
O'Toole, Ohman, Claesson, Deane, Quigley, & Simren, 2012; Rajilic-Stojanovic, Biagi, Heilig, 
Kajander, Kekkonen, Tims, & de Vos, 2011; Saulnier et al., 2011; Whorwell et al., 2006).  
 
Stress is one unifying biological mechanism that influences anxiety and GI pain. Heightened stress 
reactivity is a transdiagnostic process along the anxiety spectrum that is also associated with GI 
disease. Heightened stress reactivity is defined as heightened reactivity (e.g., increased autonomic 
reactivity and subjective perceived threat) to innocuous stimuli perceived as threatening, stronger 
physiological reactivity to 
noxious stimuli, and a slower 
return to baseline following stress 
provocation.(Chida, Sudo, 
Sonoda, Hiramoto, & Kubo, 
2007). Elevated stress reactivity is 
also a marker of HPA axis 
dysregulation.(Kryski, Smith, 
Sheikh, Singh, & Hayden, 2013). 
Due to evidence that shows 
stimulation of the HPA axis, the 
Hyper-
sensitivity to 
perceived or 
actual stress
Heightened 
physiological 
reactivity
Enhanced 
perception of 
physiological 
arousal 
Stronger 
“feelings” of 
emotion and 
pain
Figure 1. Red borders signify heightened stress reactivity and blue lines signal heightened visceral sensitivity. 
Probiotics are proposed to affect stress reactivity via changes in HPA-axis function and affect visceral 
hypersensitivity via changes in inflammation. 
Probiotics 
influence via 
changes in HPA-
axis function
Probiotics 
influence via 
changes in 
inflammation

 
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psychosocial stress induction task, Trier Social Skills Task is a justifiable mean of measuring stress 
reactivity (See Design and Procedures)(Gunnar, Talge, & Herrera, 2009a). Given the nature of the 
autonomic response associated with stress reactivity, heart rate variability and cortisol changes 
will be collected pre and post to evaluate the effectiveness of the probiotic treatment.  
 
Additionally, HPA axis dysregulation has important implications for the development of the gut 
microbiome as will be discussed. A potentially related cross-diagnostic process is visceral 
hypersensitivity, subjective sensitivity to changes in visceral organs (e.g., an individual who readily 
senses changes in gut motility) (Azpiroz, Bouin, Camilleri, Mayer, Poitras, Serra, & Spiller, 2007; 
Bradesi et al., 2004; Mayer, Bradesi, Chang, Spiegel, Bueller, & Naliboff, 2008; Theodorou, 2013) 
In fact, such sensitivity to visceral change can become a subsequent cue for intensified reactivity 
in a vicious cycle (Figure 1). Given the early onset and chronic course of anxiety and GI 
disorders, an intervention that can impact transdiagnostic processes of stress reactivity and 
visceral hypersensitivity would be transformative. In the following section, we discuss how 
understanding communication between the gut and brain and the influence of the gut 
microbiome on such communication may provide key intervention mechanisms.  
 
Therefore, alternations in the gut microbiome may specifically impact the core processes relevant for 
anxiety and GI disorders. Further, such changes may be evidenced behaviorally and physiological via 
improvement in stress reactivity (more sensitive cortisol dynamics in response to a lab-based 
stressor).  Study of proposed biological and behavioral mechanisms of new treatments for mental 
disorders is consistent with the recent strategic plan of the NIMH emphasizing more mechanistically-
informed intervention development.  
 
4.  Design & Procedures:  
In this open-label trial, self-report measures and a laboratory task will be collected from children or 
their primary caregivers prior to and following a 30-day probiotic administration (See Figure 2).  
 
Figure 2: Study design and timeline. Acronyms are explained in the subsequent paragraphs.  

 
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Inclusion (Phone Screening): Parents of study participants will provide verbal consent over the 
phone prior to answering our screening questions about their children’s abdominal pain and anxiety 
(see Telephone Consent for Screening). Abdominal pain will be assessed with questions taken from 
the ROME III questionnaire, and anxiety will be assessed with the Beck Youth Inventory (BYI). 
Participants below cutoff for both parameters will meet the exclusion criteria whereas those above 
cutoff will be given the probiotic treatment (Green Group in Figure 2). Participants who have 
immune disorders will automatically be excluded from this study.  
 
1st Lab Visit (Introduction to the Study): Parents and children (dyads) will be invited to come to 
our lab for an introductory session and provide written informed consent. The researcher will explain 
specific details about the study as well as how to collect stool samples and complete the daily Pain 
Diaries. After dyads consent to participate, they are given the EasySampler Stool Collection Kit and 
Pain Diaries to be collected by the researcher in the next visit in one week. Children will also fill out 
the State Scale of the State-Trait Anxiety Inventory (STAI) to obtain a measure of current anxiety. 
 
The researcher will specifically explain stool sample collection procedures as follows: 
i. 
Please follow the instructions provided in the EasySampler® Stool Collection Kit to ensure 
an easy and hygienic stool collection and proper labeling.  
ii. 
Collect samples either the day before or the day of a lab session. The closer the stool 
collection is to the delivery time the better.  
iii. 
Place the tubes containing the samples in your freezer as soon as possible after collection.  
iv. 
Bring the tubes on your appointment day.   
v. 
You’re done! 
 
 
 

 
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The EasySampler® Stool Collection Kit manual instructs to: 
 
1. Follow instructions provided in the package 
2. Handle and label collection containers accordingly 
3. Use disposable gloves for stool collection  
4. Tape EasySampler (paper device) onto the toilet rim as shown below: 
 
5. Collect stool sample accordingly 
6. Remove disposable gloves and flush EasySampler  
 
For more information, refer to EasySampler® Stool Collection Kit manual and/or visit: 
https://www.alpco.com/store/easysampler-stool-collection-kit.html. 
 
 
 
1-Week Assessment Period (Light Blue in Diagram). 
Participants will complete a 1-week worth of a daily pain diary prior to treatment (or the control 
period) and for another week post-treatment. One -day prior to the 2nd lab visit, parents will have 
their children take a stool sample to bring with to their laboratory visit.  
 
2nd Lab Visit (Pre-Treatment): One week later, dyads will return to the laboratory and will provide 
their stool samples and completed Pain Diaries. Stool samples will be stored in a -20°C freezer at the 
Duke Center for Developmental Epidemiology, in Suite 24-E, for less than 24 hours before being 
transported to Seed Laboratory located at Edwin L. Jones Building, Room 437, Research Drive, 
DUMC, Durham, NC 27710. Subsequently,  dyads will fill out a full battery of diagnostic and self-
report measures (See Table 1). Then, each child will participate in the Trier Social Skills Task (TSST). 
The Trier Social Skills Task has been designed to induce psychosocial stress in humans, and has been 
used in as early as 7 year-old children (Gunnar, Talge, & Herrera, 2009b). This task has shown 
evidence of psychosocial stress induction in humans via stimulation of the Hypothalamic-Pituitary-
Adrenal (HPA) axis as measured by changes in cortisol dynamics. This task is divided in several 
sections, including a speech preparation period, followed by the presentation of the speech and 
subsequent arithmetic problem solving. Pre and post resting periods precede and follow the task 
respectively. Physiological responses such as heart rate and changes in cortisol are collected 
throughout the whole experiment.  The lab task lasts approximately 20 minutes, however, 
completion of questionnaires, salivary sampling, and the lab task combined will take about an hour 
and a half (Buske-Kirschbaum et al., 1997; Gunnar, Wewerka, Frenn, Long, & Griggs, 2009; Jansen 
et al., 2000;Krishnaveni et al., 2014).   
 
There will be two between-subject and within-subject TSST randomized versions, such that all 
individuals participate in both versions: one version at pretest and the other at posttest. Each version 
contains a different story lead. For more information on the story leads, refer to the TSST protocol. 
Both versions are followed by an arithmetic problem (see TSST protocol). Saliva samples will be 
collected 20 and 1 minute before TSST, 9 minutes before public speaking (i.e. at the end of 
preparation period), immediately after math problem, and at 20 minute intervals after TSST for 60 
minutes. 

 
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Treatment: Participants will be given a 30-day 
supply of an over-the-counter Probiotic 
Nutritional Supplement, Culterelle®, taken as 
directed per dosing instructions for children, 1 
capsule/packet per day (see Figure 3 for product 
labeling). This administration of probiotics will be 
supervised by Dr. Richard Chung, MD. One of our 
staff members will give weekly calls to the parents, 
every seventh day of treatment, for four weeks, to 
assess tolerability address questions or concerns, 
and encourage compliance (See Tolerability 
Monitoring Script).  
 
Caregivers and children will continue to complete 
an abbreviated daily pain diary that is completed at 
the end of each day and that assesses abdominal 
pain, anxiety, and other somatic symptoms. 
 
3rd Lab Visit (Post-Treatment): After the 30-day 
treatment period, children will again be asked to 
collect a stool sample, and the questionnaires, and 
laboratory task will be repeated. Dyads will be 
given another week of daily pain diaries to 
complete.  
 
 
Follow-up: Participants will 
complete one week of PDs. At 
the time of completion, they will 
turn in their PD and will receive 
their final compensation.  
 
5.  Selection of Subjects. We 
will recontact participants who 
are now between the ages of 9 
years old and 13 years old who 
were part of a longitudinal study 
of preschool anxiety with 
protocol number  Pro00008503 
(See eligibility criteria below). To 
be eligible, participants need to 
meet the criteria for functional 
abdominal pain as specified by 
the Rome III criteria and 
depicted in Table 2. They also 
have to be above cutoff (i.e. 
TS>= 55) for BYI which can be 
determined by finding the 
corresponding raw score, gender, 
and age on Appendix A in the 
BYI manual:  
 
TABLE 1: List of diagnostic and self-
report measures 
Parent about Child (PaC) 
Daily Pain Diary 
MASC  
Visceral Sensitivity Index  
Anxiety Sensitivity Scale 
BBC Sensory Scale Questionnaire 
Response to Stress Questionnaire 
Child about Child (CaC) 
MASC 
Visceral Sensitivity Index 
Hyperarousal Scale for Children 
Anxiety Sensitivity Scale 
MFQ 
Response to stress questionnaire 
STAI  (state anxiety sub-scale) 
Emotion Awareness Questionnaire 
Parent about Self (PaS) 
Visceral Sensitivity Index 
Anxiety Sensitivity Scale 
BBC Sensory Scale 
MFQ  
Difficulties and Emotional Regulation Scale 
Figure 3 

 
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 
TS >= 55 represent mildly elevated to extremely elevated anxiety; therefore, eligible.  
 
TS <55 not eligible 
Participants with immune disorders, and taking immunosuppressive medication (other than inhaled 
corticosteroids), will be automatically excluded from this study. Participants who have taken 
probiotics and antibiotics in the past month will also be excluded. Those with medical causes of 
abdominal pain (e.g. inflammatory bowel disease), and a mental disorder, other than an anxiety 
disorder, will be excluded. Those who have taken anxiolytics in the past year will be excluded as well.  
 
 
 
 
Eligibility Criteria for the study “Preschool Anxiety Disorders in Primary Care,” Pro00008503 
  
Inclusion/exclusion criteria for the screened sample (n=4,410) 
Screening inclusion criteria will be (1) a child who is between 24 and 71 months old, (2) the presence 
of a parent/legal guardian at the clinic visit who speaks English well enough to consent to participate 
in the screening, (3) the child’s attendance at the Duke pediatric clinic during a screening period. 
Exclusion criteria will be (1) lack of a parent with adequate fluency in English to complete the 
interview, and (2) the index child being known to have mental retardation (IQ < 70), autism, or other 
pervasive developmental disorders. Parents of children younger than 3 years of age will be asked to 
complete the Modified Checklist for Autism in Toddlers (M-CHAT), while parents of children 3 
years of age and older will be asked to complete the Social Communication Questionnaire (SCQ).  
Any positive questionnaire would be followed up with a phone interview that used items from the 
Autism Diagnostic Interview (ADI) to determine whether the behavior is truly atypical. Exclusion for 
autism or other PDD will be based on the results of the screen and telephone follow-up; (3) sibling 
already enrolled in the study. If the parent brings more than one child between the ages of 24-71 
months old to see the pediatrician, the recruiter will consult pre-computed random selection tables to 
select which child to enroll in the study.  
Inclusion criteria for screen-stratified in-home interviewed sample (n=929) 
Children who score 4 or more on the narrow band anxious/depressed syndrome scale of the CBCL1 
½ -5 will be recruited to participate in the study (N=706). We will randomly select 7.9% of children 
who score less than 4 on the anxious/depressed scale (N=223). 
Inclusion criteria for nested case-control sample (n=500) 
We will run the diagnostic algorithms on data from the in-home assessment to determine whether 
the child meets diagnostic criteria for any DSM-IV anxiety disorder (SAD, GAD, simple phobia, 
social phobia, PTSD, and/or selective mutism), and attempt to recruit all children with an anxiety 
disorder. We expect to recruit 250 children who meet these criteria, 240 from the screen positive 
group and 10 from the screen negative group. We will also randomly select 7.9% of children who do 
not have an anxiety disorder (N=37 from the screen highs, and 223 from the screen lows). We will 
ask the parent who completed the in-home assessment to complete the laboratory assessment with 
his/her child. 
 
6.  Subject Recruitment & Compensation:  
Table 2. Scenario #1 
Criteria for inclusion 
A. In the last 2 months, how often did your child have pain or an 
uncomfortable feeling in the upper abdomen above the belly 
button?  
If ≥ 8 (or   If ≥ 1 plus 
impairment ≥ 25% of the time 
)  
B. In the last 2 months, how often did your child have pain or an 
uncomfortable feeling in the area around or below the belly 
button? 
If ≥ 8 (or   If ≥ 1 plus 
impairment ≥ 25% of the time 
) 

 
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We will recruit 40 children who are between 9-13 years old, both female and male, above cut-off for 
anxiety and abdominal pain. Potential participants will receive a letter/e-mail from Helen Egger, 
principal investigator for the pre-school anxiety study (Pro00008503), in which they will find a phone 
number they can call to opt in or opt out of being re-contacted for future studies. Those who call 
expressing interest in the study, will be screened to determine their eligibility. The contact 
information of those who opt out will be deleted immediately.  
 
We will conduct a DEDUCE search using the terms “anxiety” and “abdominal pain” within the case 
load of Dr. Gary Maslow. Potential participants that meet these search criteria and are verified by Dr. 
Maslow as being appropriate for the study will receive a letter and/or email from Dr. Maslow 
informing the parent that their child may be eligible. Interested parents will notify the investigators 
by phone or email and a telephone screening call will be scheduled. 
 
Lastly, we will start recruitment efforts at the Duke Children's Primary Care Southpoint, where our 
research assistant, Adam Kiridly, will introduce our study to parents who agree to learn more about 
it. Parents who show interest will be asked to complete a short contact form for phone screening.  
 
 Those who score below cut-off for anxiety and abdominal pain will be excluded. Each child-parent 
dyad will receive a compensation of $190.00 USD. The compensation amount will be broken down 
as follows: 
 
Compensation Breakdown 
1st Lab Visit (DPD) 
$20.00 
1Wk PD 
10.00 
2nd Lab Visit (Questionnaires and Physiological Measures) 
40.00 
Treatment (Probiotics) 
30.00 
Bonus 
10.00 
3rd Lab Visit  
30.00 
1WK PD 
10.00 
Gift Card/Bonus 
40.00 
Total 
$190.00 
 
7. Consent Process – see Section 14 of the e-IRB submission form and complete the questions in 
that section.  – consent from parent and assent from child 
 
Samuel Marsan, the project coordinator, will be conducting the consent process with prospective 
participants at the Duke Center for Eating Disorders, which is physically located at the Duke Center 
of Developmental Epidemiology. The consent process is expected to take about 10-20 minutes 
unless parents and children need more time to ask questions and think about participation. 
Participants who are below the age of 12 years of age will provide verbal assent; whereas, those who 
are 12 years of age or older will sign the consent form should they decide to participate in the study. 
Parents or legal guardians are also required to sign the consent form if they decide to participate in 
the study.  
 
In order to protect the privacy of prospective participants, the consent process will take place in a 
laboratory at the center. The laboratory is accessible to Duke staff only. Study documents will be 
stored under three locks in the project coordinator’s office. After consent is signed, participants will 
be given the PI contact information in case they have more questions after the study. The project 
coordinator has been trained in the consent process, and will follow good clinical practice guidelines 

 
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to minimize the possibility of coercion or undue influence. Participants who do not read, are blind, 
or who do not read/understand English will be excluded from the study.     
 
8.  Subject’s Capacity to Give Legally Effective Consent:  
Children who are below 12 years of age will provide assent and those who are 12 years or older will 
sign the consent form.  
 
9.  Study Interventions:  
We will use Culturelle®, a probiotic composed of the micro-organisms Lactobacillus GG. This 
formulation has been used for the treatment of gastrointestinal inflammation in numerous clinical 
trials. Dosing will follow the manufacturer’s recommendation for children (1 capsule/packet per 
day), and will be monitored by Dr. Chung. Weekly side effects and clinical changes will be monitored 
by both study therapist and caregiver using the Children’s Global Assessment Scale and the Clinical 
Global Impression Scale (Severity, Improvement, and Efficacy) 
 
10. Risk/Benefit Assessment:  
Physical risks -There are no known physical risks to the child or parent associated with this 
proposed study. However, as we are recruiting children with gastrointestinal symptoms, there is the 
possibility that children’s gastrointestinal problem will worsen due to the natural course of their 
disorder. The following steps will be taken with children whose abdominal symptoms worsen during 
the course of the study. 
1. They will be referred for additional medical management. 
2. Any medical management they receive will be documented. 
3. They will be allowed to continue in the study so that they continue to receive psychological 
services. 
 
Psychological risks –Assessment Risk: The parents may experience some psychological discomfort 
talking to the interviewer about some aspects of their lives or their children’s lives. Children might 
find the observational assessments frustrating, disappointing, scary or boring. To address this risk, we 
will inform and reiterate that participants are free to refrain from answering any question that makes 
them uncomfortable.  
 
Intervention – There are no known risks from taking Probiotics, a dietary supplement that attempts 
to increase the quantity of naturally-occurring healthy gut bacteria. The active cultures found in 
Culterelle® have been added to many food products available on grocery shelves (e.g., yogurt). Some 
of the side effects of taking this supplement may include mild bloating, gas or digestive gurgling 
when you first begin taking Culturelle. This is normal due to the changing environment in the 
digestive system and should go away within a short period of time. 
 
Social risks - There are no known social risks.  
 
Legal risks -There is a risk that study data might be subpoenaed for legal purposes. We will obtain a 
Certificate of Confidentiality from the FDA which includes the protections the certificate affords as 
well as the limitations and exceptions of the protection. In addition, except under circumstances 
covered under the mandated child abuse reporting laws, and/or situations in which the child and/or 
a caregiver is judged clinically to be a danger to themselves or others, no information about the child 
or family will be shared with any individual or agency without prior written consent. 
 
Other risks - Participants will be informed of the federally mandated reporting laws for child abuse 
and neglect, verbally and in the written consent form. Specifically, the consent form will read: "This 
protection, however, does not prohibit the investigator from voluntarily reporting information about 
suspected or known sexual or physical abuse of a child or a subject’s threatened violence to self or 
others. If the researchers learn that you or someone with whom you are involved is in serious danger 

 
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or harm, they may inform the appropriate agencies". Therefore, a serious, though rare, risk to 
families is disruption of the home based upon the severity of the abuse or neglect disclosed. 
 
Potential Benefits. The project has the potential for direct benefit to the participants in the form of 
decreased abdominal pain, anxiety, and impressed capacity to cope with stress. In light of these 
potential benefits, we deem that the magnitude and low likelihood of risk is small relative to the 
magnitude of potential benefit.  
 
11. Costs to the Subject: none 
Costs to participants are their time and effort. There are no expenses associated with this study. 
 
12. Data Analysis & Statistical Considerations:  
Statistical Methods 
 
In this pilot study, we have established stress reactivity, anxiety and abdominal pain as our primary 
endpoints. A post-hoc one-tailed power analysis will be conducted to determine the probability in 
which a probiotic treatment can significantly reduce anxiety and abdominal pain. Tests of hypotheses 
will be based on standard binomial models for proportions and Student’s t-test or standard 
ANCOVA regression models as appropriate for continuous measures; ordinal data not meeting 
assumptions of normality will be analyzed using nonparametric Wilcoxon Mann-Whitney procedures 
or, in some cases, generalized linear regression (Poisson, logistic) models. Subjects who are below 
cutoff in our measures of anxiety and abdominal pain will be excluded from the study  
 
Cortisol. Several steps will be taken to increase the accuracy with which children’s baseline cortisol 
levels were indexed. All visits will begin between 1200 h and 1530 h to minimize the effects of 
diurnal variation on cortisol samples.(Gunnar et al., 2009b) Children will refrain from eating or 
drinking for a half hour prior to the visit to minimize the influence of food/drink on cortisol assays. 
Children will first play quietly with the experimenter for 30 minutes followed by a baseline salivary 
cortisol sample and the stress task described above. Following the stress task, the child and 
experimenter will resume quiet play while the remaining saliva samples are obtained. Children will 
chew on a 2-inch absorbent cotton dental roll until wet with saliva, which will be expunged into a 
micro tube and frozen until assayed in duplicate using an expanded range, high sensitivity, salivary 
cortisol enzyme immunoassay kit (Salimetrics, PA, USA). Standard curve and concentration of 
unknown samples will be generated according to manufacturer’s instructions using a 4- parameter 
sigmoid minus curve fit.  
 
Heart rate. We will measure heart rate continuously starting at 20 minutes before and ending at 60 
minutes after TSST. We will use Garmin vivosmart HR, a wrist heart rate monitor , to collect this 
data. Heart rate variability will be analyzed pre and post treatment.    
 
Data Extraction of Enteric Gut Microbiome. We will utilize next generation sequencing of stool 
ribosomal DNA to elucidate the enteric microbiota of children with anxiety and abdominal pain. We 
have prior experience with all of the described methods herin.(LaTuga, Ellis, Cotton, Goldberg, 
Wynn, Jackson, & Seed, 2011) Total DNA will be isolated from stool using a bead lysis protocol 
(Zymo Research). The V3 region of 16s rDNA will be amplified by polymerase chain reaction (PCR) 
using multiplexing primer pairs. Pooled amplicons will be sequenced on the Ion Torrent PGM 
platform in the Duke Genome Analysis core facility of the Institute for Genome Sciences and Policy. 
The sequences will be de-multiplexed and analyzed through the Qiime and Mothur pipelines to make 
taxonomic assignments and determine alpha and beta diversity.(Caporaso et al., 2010; Schloss et al., 
2009) 
 
Data Analysis of Enteric Gut Microbiome. Intra- and inter-individual statistical comparisons will 
be performed using the Unifrac metric combined with Kuskal Wallis testing and principle coordinate 

 
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analysis. Analyses will be performed to identify potential correlations between specific taxa and 
clinical pain and anxiety symptoms.  
 
Statistical Considerations. Because of limited sample size and the iterative nature of intervention 
development, criteria for success at the pilot phase are based primarily on clinical rather than 
statistical criteria. We have established goals for compliance, completion, and reduction in physical 
and psychological distress aimed at establishing the feasibility and efficacy of the intervention. We 
carefully considered statistical constraints of pilot investigations by framing outcomes based on 
clinical but not statistical significance.  
 
 
 
 
13. Data & Safety Monitoring:  
 
DATA AND SAFETY MONITORING PLAN:  
Data will be collected in the form of electronic forms collected via Qualtrics and the Duke owned 
and approved Redcap® database management system. Ensuring the integrity and confidentiality of 
the data is an essential part of safeguarding the rights of subjects. The Center for Developmental 
Epidemiology, from whose offices the study will be conducted, has instituted a series of checks at 
several levels of data handling, to maintain its quality and confidentiality. (1) Interviewers, many of 
whom have years’ experience in survey work, will be given extra training in the importance of 
confidentiality in psychiatric interviewing. Like all study personnel, they will promise in writing to 
maintain that confidentiality. (2) After collection, coding, and review for accuracy by the project 
coordinator, data are entered into computer files at our offices at The Center for Developmental 
Epidemiology. The protocol, in both its physical (paper and pencil) and electronic (entered data 
forms), will be identified by number only. From this point it will not be possible to trace it back to an 
individual except by linking with a separate computer file that includes both the identification 
number and the individual’s identity.  
In accordance with federal regulations the PI will monitor for, review, and promptly report to the 
IRB, appropriate institutional officials, sponsor, coordinating center and the appropriate regulatory 
agency head all unanticipated problems involving risks to subjects or others that occur in the course 
of a subject’s participation in a research study (45 CFR 46.103(b)(5)(i) and 21 CFR 56.108(b)(1)), and 
all reportable AEs will be submitted per the DUHS IRB policies; available at: 
(http://irb.duhs.duke.edu/modules/irb_pols/index.php?id=2) 
 
ADVERSE EVENTS MONITORING: 
In accordance with federal regulations the PI will monitor for, review, and promptly report to the 
IRB, appropriate institutional officials, sponsor, coordinating center and the appropriate regulatory 
agency head all unanticipated problems involving risks to subjects or others that occur in the course 
of a subject’s participation in a research study (45 CFR 46.103(b)(5)(i) and 21 CFR 56.108(b)(1)), and 
all reportable AEs will be submitted per the DUHS IRB policies; available at: 
(http://irb.duhs.duke.edu/modules/irb_pols/index.php?id=2) 
 
All adverse events (AEs), whether observed by the Investigator, elicited from the participant or 
volunteered by the participant, and whether ascribed to the drug or not, will be recorded.  This will 
include the following: a brief description of the event, the date of onset, the date of resolution, the 
duration and type of the event, the severity, contributing factors and any action taken with respect to 
the study drug. 
 
For each adverse event, the relationship to the study drug will be recorded as one of the choices on 
the following scale: 

 
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DEFINITE 
Causal relationship is certain (i.e., the temporal relationship between drug exposure 
and the adverse event onset/course is reasonable, there is a clinically compatible response to de-
challenge, other causes have been eliminated and the event must be definitive pharmacologically or 
phenomenologically using a satisfactory re-challenge procedure if necessary). 
PROBABLE 
High degree of certainty for causal relationship (i.e., the temporal relationship 
between drug exposure and the adverse event onset/course is reasonable, there is a clinically 
compatible response to de-challenge [re-challenge is not required] and other causes have been 
eliminated or are unlikely). 
POSSIBLE 
Causal relationship is uncertain (i.e., the temporal relationship between drug 
exposure and the adverse event onset/course is reasonable or unknown, de-challenge/re-challenge 
information is either unknown or equivocal and while other potential causes may or may not exist, a 
causal relationship to the study drug does not appear probable). 
UNLIKELY 
Not reasonably related, although a causal relationship cannot be ruled out (i.e., while 
the temporal relationship between drug exposure and the adverse event onset/course does not 
preclude causality, there is a clear alternate cause that is more likely to have caused the adverse event 
than the study drug). 
NOT RELATED 
No possible relationship (i.e., the temporal relationship between drug 
exposure and the adverse event onset/course is unreasonable or incompatible, or a causal 
relationship to study drug is implausible). 
 
The severity of each adverse event must be recorded as one of the choices on the following scale: 
MILD  
No limitation of usual activities 
MODERATE Some limitation of usual activities 
SEVERE 
 
Inability to carry out usual activities 
 
The expectedness of an AE must be indicated when reporting adverse events.  An unexpected 
adverse event is any adverse experience for which the specificity or severity of the event is not 
consistent with the current labeling.   
 
A serious adverse drug event (SAE) is defined as any adverse event that occurs during the study that 
results in any of the following outcomes: death, a life-threatening adverse event (i.e., the participant 
was at immediate risk of death from the event as it occurred; does not include an event, that had it 
occurred in a more severe form, might have caused death), inpatient hospitalization or prolongation 
of existing hospitalization (hospitalizations scheduled before enrollment for an elective procedure or 
treatment of a pre-existing condition which has not worsened during participation in the study will 
not be considered a serious adverse event), a persistent or significant disability/incapacity (substantial 
disruption of one's ability to conduct normal life functions), a congenital anomaly/birth defect, a 
medically important event or required medical intervention to avoid one of the above outcomes.  In 
addition to the above procedures for AEs, all SAEs will be reported to the IRB within 24 hours of 
recording.  All serious adverse event information will be followed until resolution or an appropriate 
end point is reached. This may involve contacting other clinicians responsible for the participant’s 
care to obtain information on diagnoses, investigations performed and treatment given 
 
Fatal or life-threatening, unexpected adverse events will be reported to the FDA by telephone, 
facsimile, or in writing as soon as possible, but no later than 7 calendar days after first knowledge by 
the Sponsor-investigator. Serious, unexpected adverse events that are not fatal or life-threatening will 
be reported to the FDA by telephone, facsimile, or in writing as soon as possible, but no later than 
15 calendar days after first knowledge by the sponsor-investigator. 
 
14. Privacy, Data Storage & Confidentiality –  
Data stored is password protected and kept in a special computer volume to which only our (a) 
network administrator and (b) senior data manager have access rights. Participant information on the 
computer files is identified only by a study-specific identification number. Only approved research 

 
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12 
staff has access to the data. All faculty and staff have taken and passed Duke’s required training 
modules on HIPAA regulations for protecting participants’ privacy, and the Center for 
Developmental Epidemiology, where the study records are held, observes all approved security rules 
to protect the data. (3) Protocols and tapes will be kept in locked file cabinets in a locked storage 
room in a research facility that is locked at night. (4) Written reports and papers will not identify 
subjects or make identification possible, without the written consent of the person concerned. Our 
data storage plans have been developed to be fully compliant with HIPPA safeguards. Electronic 
communication between the research team and participants will follow Duke Medicine Electronic 
Communication policy guidelines, including encryption of Sensitive Electronic information (SEI) by 
prepending the Subject line of the message with the string: 
(secure).      
 
 
 
 
 
 
 
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