A Longitudinal Study to Identify IBS Phenotypes Using Fecal Microbiota and Hydrogen Breath Testing
Phase: PHASE4
Conditions: Irritable Bowel Syndrome
Sponsor: University of Michigan

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A Longitudinal Study to Identify IBS Phenotypes Using Fecal 
Microbiota and Hydrogen Breath Testing 
 
NCT03219528 
 
Date of IRB Approval: May 6, 2023 
 
 
 

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Title 
A Longitudinal Study to Identify IBS Phenotypes Using Fecal Microbiota and Hydrogen 
Breath Testing 
Principal Investigator: 
Allen Lee, MD 
Clinical Lecturer 
University of Michigan 
 
Co-Investigators: 
William Chey, MD 
Shanti Eswaran, MD 
Krishna Rao, MD 
Vincent Young, MD 
 
Funding Mechanism: (e.g., grant or cooperative agreement #) 
Michigan Institute for Clinical and Health Research KL2  
Institution(s):  University of Michigan 
Other Identifying Numbers: (e.g., Institution-assigned number, HUM 
HUM00129427 
 
Protocol Amendments: 
(Any modifications to the protocol should be annotated on the coversheet or in an 
appendix.  The annotation should note the exact words that are changed, the location in 
the protocol the date the modification was approved, and the date it became effective.) 
 
Draft or Version Number: 12.0 
Day Month Year: 16 Feb 2022 

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 TABLE OF CONTENTS 
Page 
Title Page…………………………………………………………………………………………1 
Table of Contents ............................................................................................................ 2 
Signature Page ............................................................................................................... 3 
List of Abbreviations ........................................................................................................ 4 
1 
Background/Scientific Rationale .............................................................................. 5 
2 
Objectives ............................................................................................................... 7 
3 
Expected Risks/Benefits.......................................................................................... 8 
4 
Eligibility ................................................................................................................ 11 
5 
Subject Enrollment ................................................................................................ 12 
6 
Study Design and Procedures ............................................................................... 12 
7 
Data Collection and Management Procedures ...................................................... 18 
8 
Data Analysis ........................................................................................................ 19 
9 
Quality Control and Quality Assurance.................................................................. 19 
10 
Statistical Considerations ...................................................................................... 19 
11 
Regulatory Requirements ...................................................................................... 21 
11.1 Informed Consent ....................................................................................... 21 
11.2 Subject Confidentiality ................................................................................ 21 
11.3 Data Safety Monitoring Plan ....................................................................... 21 
12 
References ............................................................................................................ 25 
Appendices ................................................................................................................... 32 
 
 
 
 
 

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SIGNATURE PAGE 
The signature below constitutes the approval of this protocol and the attachments, and 
provides the necessary assurances that this study 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. 
Site Investigator: * 
 
Signed: 
 
Date: 
 
 
Allen Lee 
 MD 
 
 
 
* The protocol should be signed by the clinical site investigator who is responsible for 
the day to day study implementation at his/her specific clinical site.

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LIST OF ABBREVIATIONS  
IBS, Irritable Bowel Syndrome 
SIBO, Small intestinal bacterial overgrowth 
GHBT, Glucose hydrogen breath tests 
FODMAP, Fermentable Oligosaccharides, Disaccharides, Monosaccharides, and 
Polyols 
IBS-D, Diarrhea-predominant irritable bowel syndrome 
VAS, Visual analog scale 
NRS, Numerical Rating Scale 
PHQ-9, Patient Health Questionnaire  
VSI, Visceral Sensitivity Index  
IBS-QOL, IBS-quality of life 
TpH-1, Tryptophan hydroxylase-1 
SNPs, Single nucleotide polymorphisms 
OTU, operational taxonomic units 
LEfSE, linear discriminant analysis effect size  
AMOVA, analysis of molecular variance  
eCRF, electronic Case Report Form 
PHI, Protected Health Information 
GCP, Good Clinical Practice  
AE, Adverse Event 
 
 
 
 
 
 
 
 
 
 
 
 
 

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1 
BACKGROUND/SCIENTIFIC RATIONALE 
Irritable bowel syndrome (IBS) is a common but incompletely understood condition that leads to 
significant morbidity and substantial health care expenditures (1,2).  IBS is likely a multifactorial 
disorder including disordered motility, immune activation, gut barrier dysfunction, visceral 
hypersensitivity, and altered brain-gut interactions (3–5).   
The role of the gut microbiota is increasingly being recognized for its potential role in the 
pathogenesis of IBS.  Humans are host to a diverse community of microbes collectively known 
as the human microbiota, of which the vast majority live in the gut (6).  The gut microbiota 
normally interacts closely with the intestinal epithelial cells as well as the enteric immune and 
nervous system, which comprises the microbiota-gut-brain axis (7).  There is a symbiotic 
relationship between the microbiota and host with the gut microbiome providing essential 
functions, including maintenance of the gut epithelial barrier and immune function (8).  However, 
alterations in the structure and community function of the fecal microbiota may have a 
significant role in the pathogenesis of human disease, including IBS.  Acute gastroenteritis leads 
to an increased risk for development of post-infectious IBS and is associated with altered gut 
microbiota, increased permeability and immune activation (9–11).  Further evidence of the role 
of gut microbiota in IBS is shown by experiments where transfer of microbiota from patients with 
IBS to germ-free mice leads to rapid GI transit, impaired gut barrier function, and visceral 
hypersensitivity (12,13). 
Several studies have reported an increase in the relative abundance of Firmicutes, a reduction 
in the relative abundance of Bacteroidetes, and decrease in microbial diversity (14–16).  
However, studies are often conflicting and a specific microbial signature has not been 
discovered to date in IBS (17–19).  Incomplete phenotypic characterization, small sample sizes, 
the cross-sectional nature of study design, and probable heterogeneity of the patient 
populations also limited prior studies.  In addition, previous studies have not taken differences in 
diet or stool consistency into consideration despite the fact that these factors heavily influence 
the composition of the gut microbiota (20,21).   
Small intestinal bacterial overgrowth (SIBO) has also been postulated to be an important 
pathophysiologic factor in the pathogenesis of IBS (22–24).  SIBO is defined as presence of 
bacteria in excess of 105 colony-forming units per milliliter on culture of small bowel aspirates 
[34].  Prevalence figures of SIBO in IBS range from 4-64% depending on the method of testing 
(26).  SIBO is characterized by symptoms of diarrhea, abdominal pain, bloating, and gas.  It has 
been proposed that SIBO may lead to immune activation, triggering low-grade inflammation, 
epithelial barrier dysfunction, and visceral hyperalgesia seen in IBS (27).  Meanwhile, antibiotics 
improve symptoms in IBS patients with SIBO while those patients who achieved bacterial 
eradication showed the greatest symptomatic response (23).  However, little is known about the 
specifics of the gut microbiota in SIBO and whether these conditions are separate or 
overlapping entities. 
Small bowel aspirate and culture is an accepted means of quantifying upper intestinal bacteria, 
but this approach is time consuming, invasive, and has a high false-negative rate (28).  As a 
consequence, hydrogen breath tests have largely supplanted intestinal cultures as measures of 
SIBO.  There is some controversy regarding the substrate that is utilized in hydrogen breath 

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tests.  Lactulose has been advocated as a preferred substrate as it is not absorbed in the GI 
tract.  However, simultaneous testing using scintigraphy and lactulose hydrogen breath testing 
(LHBT) demonstrated that abnormal findings with LHBT represent rapid orocecal transit rather 
than SIBO (29).  Glucose is another substrate that is commonly employed in hydrogen breath 
testing.  Although glucose is primarily absorbed in the proximal small bowel and therefore may 
miss distal SIBO, this is still the most common and widely available non-invasive test to 
diagnose SIBO (30). 
Despite our advances in understanding the pathophysiology of IBS, there are still unmet needs 
in terms of treatment options.  Rifaximin is a non-absorbable antibiotic that has been shown to 
improve symptoms in the diarrhea-predominant subtype of IBS (IBS-D) (31,32).  However, 
mechanisms by which rifaximin leads to improvement in IBS-D are still unknown.  In vitro 
studies suggest that rifaximin may prevent changes in barrier dysfunction and reverse mucosal 
inflammation by modulating bacteria in the small intestine (33).  Conversely, prior studies have 
not demonstrated significant effects of rifaximin on fecal microbial composition (34,35).  This is 
likely because rifaximin is insoluble in the colon and has antimicrobial effects primarily in the 
small bowel (36).  Furthermore, rifaximin leads to improvement in symptoms in only 40% of IBS 
patients with a therapeutic gain of 9% over placebo.  Because of these observations, one could 
argue that rifaximin may be effective only in those IBS patients who have concomitant SIBO.  
Indeed, a recent study demonstrated that those IBS patients with SIBO had a significantly 
increased response to rifaximin compared with those IBS patients without SIBO (37). 
Dietary modification has also been advocated as a potential treatment option in IBS-D.  The 
vast majority of IBS patients believe certain foods trigger their IBS symptoms (38).  Fermentable 
oligosaccharides, disaccharides, monosaccharides, and polyols (FODMAP) are short-chain, 
poorly absorbable carbohydrates.  Recently, a diet low in FODMAP has been shown to improve 
symptoms in up to 76% of IBS patients (39,40).  However, two recent randomized controlled 
trials failed to show a low FODMAP diet was superior to traditional IBS dietary advice in 
controlling symptoms (41,42).  Furthermore, improvement in symptoms was reported by only 
50% of patients on low FODMAP diets.  Moreover, the exact mechanism by which a low 
FODMAP diet improves symptoms in IBS is still unknown.  It is postulated that the gut 
microbiota are critical in generation of symptoms in the presence of dietary FODMAPs and lead 
to production of gases, including hydrogen, CO2, and methane.  This may generate symptoms 
in IBS particularly in the setting of visceral hypersensitivity (43).   
Identifying those subjects who are most likely to respond to a low FODMAP diet is clearly 
needed but has been a vexing problem. Hydrogen breath testing has been advocated as a 
potential tool to identify subjects that malabsorb FODMAPs (e.g. lactose, fructose, sorbitol, and 
mannitol) and thereby might have better response to dietary FODMAP restriction.  However, 
questions regarding the reproducibility of breath testing as well as standardization of the test in 
this setting are still unanswered (44).  Furthermore, it was recently shown that a lactulose breath 
test could not discriminate between responders and non-responders to low FODMAP diets (45).  
Interestingly, a recent study in pediatric IBS subjects demonstrated that responders to a low 
FODMAP diet had different fecal microbial composition at baseline compared with non-
responders (46).  As the gut microbiota is postulated to be one of the main mediators in the 
pathogenesis of symptoms in IBS and FODMAP intake, this suggests that alterations in the 
fecal microbiota may predict response to dietary restriction of FODMAP. Genetic variability in 

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tryptophan hydroxylase-1 (TpH-1) and sucrase-isomaltase single nucleotide polymorphisms 
(SNPs) have been demonstrated in IBS patients (47,48).  Unpublished data suggest that 
differences in TpH-1 SNPs may also potentially identify responders vs. non-responders to 
treatments, such as low FODMAP diet, in IBS.  Finally, a recent study suggested that treatment 
with low FODMAP diet may lead to improvement in cytokine profiles (49). 
Taken collectively, our current therapeutic options are quite limited in IBS-D and improve 
symptoms in only ≤ 50% of subjects in the best-case scenarios, which we believe is a 
consequence of multiple pathogenic factors.  Given that IBS is a heterogeneous condition, there 
is a pressing need to stratify IBS patients into distinct subtypes to optimize responses to 
diverse therapies including antibiotics versus dietary measures.  In the investigations included in 
this proposal, we will test the hypotheses that: (i) distinct IBS-D phenotypes with characteristic 
clinical features can be generated by identifying the presence or absence of SIBO and by 
defining fecal microbial populations, and (ii) longitudinal analyses using microbiota-derived 
metrics as well as SIBO status may provide important details into how these treatments improve 
symptoms in IBS and may also provide clues on which patients may respond to different 
therapies.  
2 
OBJECTIVES 
Aim 1:  Randomize IBS-D subjects to receive either treatment with a nonabsorbable 
antibiotic rifaximin vs. low FODMAP dietary intervention.   
a) 200 IBS-D patients will be carefully phenotyped using validated questionnaires (IBS 
symptoms, stool form, psychological function, quality of life) and diet analysis. 
b) IBS-D subjects will undergo baseline testing including glucose hydrogen breath tests 
(GHBT) to define the presence of SIBO.  Fecal microbiota will also be characterized by α- 
and β-diversity and dominant phyla on 16S RNA analyses.  
c) IBS-D subjects will be randomized to receive either rifaximin 550 mg three times daily x 14 
days or low FODMAP diet x 4 weeks under direction of study bionutritionist. 
d) The primary outcome will be changes in mean daily pain or bloating by visual analog scale 
(VAS) after intervention compared with baseline.  Responders to intervention will be 
defined by ≥ 30% reductions in mean daily pain or bloating by VAS compared with 
baseline.  Secondary outcomes will be defined by reduction in IBS Symptom Severity Scale 
by ≥ 50 compared with baseline.  
e) Other secondary outcomes, including GI symptoms, stool form, psychological function, 
quality of life, and diet intake, will be compared to determine if subjects present differently.  
Aim 2: Determine using longitudinal analyses how SIBO status and fecal microbiota 
features associate with response to treatment with rifaximin or a low FODMAP dietary 
intervention. 
a) Fecal microbial analyses will be repeated at 2, 4 and 5 weeks while GHBT will be repeated 
at the end of the intervention.   

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b) The primary aim will be change in fecal microbial diversity after intervention compared with 
baseline.  We hypothesize that low FODMAP diet will result in a 10-15% increase in 
diversity in fecal microbiota compared with baseline. 
c) Secondary outcomes including response to treatment with rifaximin or low FODMAP diet 
will be related to the (i) presence of SIBO on initial testing, (ii) persistence of SIBO after 
intervention, (iii) abnormal α- and β-diversity on initial testing, (iv) changes in α- and β-
diversity as well as in dominant microbial phyla at 2, 4 and 5 weeks, and (v) changes in 
serum cytokine profiles, including TNF-α, IL-1β, IL-6, IL-8, and IL-10.   
d) Other outcomes that will be assessed include differences in tryptophan hydroxylase-1 
(TpH-1) and sucrase-isomaltase single nucleotide polymorphisms (SNPs) in different 
cohorts of IBS-D patients.  
Aim 3: Develop a predictive model to identify subsets of IBS-D patients responsive to 
treatment. 
a) We will develop and validate a predictive model of response to low FODMAP diet using 
data from the subjects randomized to a low FODMAP diet (Aim 1) combined with existing 
data from a separate cohort of IBS-D subjects previously treated with a low FODMAP diet 
(HUM 00053274).  
3 
EXPECTED RISKS/BENEFITS 
The data accumulated in this study will advance our understanding of the 
pathophysiology of IBS, including the role of the gut microbiota and SIBO in the 
pathogenesis of IBS, and may provide important information on which IBS subjects will 
most benefit from therapies including dietary modification and rifaximin.   
There are potential direct benefits for all IBS subjects in this study.  On study entry, 
glucose hydrogen breath tests (GHBT) as well as analysis of fecal microbiota will be 
performed that may provide new insight into causes of symptoms in patients with IBS.  
Furthermore, subjects with IBS will be treated with well accepted treatments for IBS, 
including a nonabsorbable antibiotic rifaximin or diets low in FODMAP.   IBS-D subjects 
treated with low FODMAP diet will be provided education on a low FODMAP diet from a 
trained bionutritionist.  IBS-D subjects will track their symptoms by completing 
questionnaires online including the Bristol Stool Form Scale as well as an 11-point NRS 
on abdominal pain and bloating daily.  IBS-D subjects will be asked to complete these 
questionnaires after their evening meal. They will also be instructed to track their diet for 
24 hours prior to each weekly or biweekly visit using a food questionnaire.  Finally, all 
IBS-D subjects will be seen or receive a phone call by the PI and/or study team every 
week or every two weeks during the study.  At each visit, survey information will be 
obtained using validated questionnaires including IBS-Symptom Severity Scale, 
Gastrointestinal Symptom Rating Scale-IBS, Patient Health Questionnaire (PHQ-9), 
Visceral Sensitivity Index (VSI), and IBS-quality of life (IBS-QOL).  The IBS-Symptom 
Severity Scale is a 10-item questionnaire that tracks severity of IBS symptoms and is a 

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valid way to monitor changes in IBS symptoms over time.  This will take approximately 5 
minutes to complete.  The Gastrointestinal Symptom Rating Scale-IBS is a 15-item 
questionnaires that tracks both upper and lower gastrointestinal symptoms.  This will 
take approximately 8 minutes to complete.   The PHQ-9 is a 9-item questionnaire that is 
a reliable and valid measure of depression severity.  This will take approximately 5 
minutes to complete.  The VSI is a 15-item questionnaire which is a reliable, valid 
measure of GI-specific anxiety.  This will take approximately 8 minutes to complete.  
The IBS-QOL is a 34-item questionnaire that is a reliable, valid quality of life measure 
specific for IBS.  This will take approximately 15 minutes to complete.  This frequent and 
comprehensive assessment provided as part of participating in this study may allow for 
closer monitoring of disease course. 
There are potential risks for subjects, including:  
1) Risk to privacy (rare): There is a potential risk to the IBS-D subject's privacy (<1% 
risk).  While every effort will be made to maintain privacy, it is possible that others 
may learn about information acquired from the medical records. 
 
For Aim 3, there are additional risks to privacy associated with secondary review of 
data from a separate database (HUM 00053274). However, we will follow standard 
procedures to minimize these risks. This includes use of data abstraction forms that 
will not contain any PHI. All subjects will be identified by a study ID number. The list 
linking the subject to his/her study ID number will be retained by the investigators in 
a secure location separate from the source documents. The list linking PHI to study 
ID numbers will be accessed only for verification of data abstraction if any 
inconsistency or incompleteness of study data requires review of a participant’s 
medical record. Once the data has been reviewed, analyzed, and verified, and the 
investigators are satisfied that no further review of the subject’s medical record is 
needed, the list of links to the identifiers for that subject population will be destroyed. 
This will ensure that no links to PHI persist beyond the duration of the study. No PHI 
will be sent outside of the institution. All presentations/publications will deal with data 
in aggregate form. Source documents created for study purposes will identify 
patients by study ID number only and will not contain PHI. 
2) Glucose hydrogen breath testing (likely): IBS-D subjects will be asked to drink 75 g 
of glucose dissolved in 250 ml of sterile water. IBS-D subjects may experience (10-
25%) bloating, abdominal cramping, and/or flatulence after consuming this drink. 
However, symptoms typically resolve quickly and may represent underlying 
physiologic abnormalities causing their symptoms.  Subjects will be allowed to take 
medications, such as simethicone or Tylenol, if they experience significant bloating 
or discomfort. IBS-D subjects may experience dizziness, faint spells, and/or nausea 
after the glucose hydrogen breath test. Subjects should bring a snack with them on 

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days that the hydrogen breath test should be done to help with dizziness and 
lightheadedness.  
3) Risks of Blood Draw: If subjects decide to participate in optional sub-study, 10mL of 
blood will be drawn from a vein at Visits 1 and 5. There could be pain, bruising, or 
lightheadedness. Rarely there could be an infection or fainting. The person drawing 
blood will be trained to do that procedure and use techniques to minimize these 
risks.  
4) Risks of treatment (infrequent): There are theoretical concerns about treatment with 
rifaximin and low FODMAP diet.  Rifaximin is an antibiotic and theoretical concerns exist 
regarding potential for antibiotic resistance.  However, rifaximin is a minimally 
absorbable antibiotic with low risk of clinically relevant bacterial resistance (50,51).  
Randomized controlled trials with rifaximin in IBS have not demonstrated significant 
differences in adverse events between rifaximin and placebo (31,32,52).  General 
antibiotics side effects may occur, such as vomiting, nausea, diarrhea, etc. There are 
also theoretical concerns that prolonged therapy with a low FODMAP diet may have 
detrimental effects on gut microbial composition (53).  However, multiple studies 
examining low FODMAP diet in IBS have demonstrated the safety and tolerability of this 
dietary approach (40–42,45).  The effects of rifaximin in pregnancy and women who are 
breastfeeding are unknown and therefore pregnant women or women who are 
breastfeeding will be excluded from the study.  Finally, women of child bearing age will 
be warned that oral contraception may not be as effective when taken with rifaximin. 
5) General risks (infrequent): This is an interventional study.  IBS-D subjects' condition 
may worsen or improve while undergoing intervention with either rifaximin or a low 
FODMAP diet.  Participation in this study will not prevent normal clinical care of IBS or 
its consequences.  In addition, some prescribed medications may need to be stopped 
temporarily during the study.  This includes medications that may influence pain 
sensation, including narcotic pain medications.  IBS-D subjects will also be asked to 
discontinue antibiotics for at least 3 months and probiotics for at least 1 month prior to 
enrollment.  IBS-D subjects will be asked to avoid Pepto-Bismol for at least 2 weeks 
prior to GHBT.  IBS-D subjects will also be instructed to eat a low carbohydrate diet for 
at least 2 days prior to the GHBT to avoid inaccurate test results.  If IBS-D subjects are 
taking any of these medications, their symptoms may return or worsen when they are 
stopped.  However, the stopping of medications is temporary and is not felt to cause 
any undue hardship.  Finally, there is a risk that some of the questions in the surveys 
may produce emotional distress.  If the IBS-D subject desires, he/she can be referred to 

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12 
a mental health specialist within the UMHS or another provider outside the UMHS 
system. 
6)  Risks for sharing samples (rare):  If subjects decide to participate in an optional sub-
study, saliva samples will be shared with Karolinska Institute and IKMB Kiel to evaluate 
differences in sucrase-isomaltase SNPs. There are potential risks to the IBS-D subject’s 
privacy.  However, all samples will be de-identified and only investigators at UMHS will 
have access to the linkage code.   
4 
ELIGIBILITY CRITERIA 
IBS Patients: 
200 consecutive adult subjects ≥ 18 years of age at screening who meet the following 
inclusion and exclusion criteria will be eligible for enrollment: 
Inclusion criteria: 
• 
Rome IV criteria for IBS-D  
• 
Subjects will be asked to avoid Pepto-Bismol for 2 weeks prior to GHBT. 
• 
Subjects will be instructed to avoid eating or drinking for 8 hours and avoid smoking for 
6 hours prior to the test  
• 
Have colonoscopy or sigmoidoscopy with mucosal biopsy within the past 2 years prior to 
enrollment 
• 
Have an average score of 4 or higher over a 7-day pre-screen taking online symptom 
surveys assessing abdominal pain and bloating 
 
Exclusion criteria:  
• 
Underlying celiac disease 
• 
Inflammatory bowel disease 
• 
Other organic disease that could explain their symptoms.   
• 
History of GI tract surgery, except for appendectomy 
• 
Narcotics, antibiotics taken within 3 months or probiotics taken within one month prior 
to enrollment will not be permitted.  
• 
Previously received formal dietary education, including a low FODMAP diet, for 
treatment of IBS. 
• 
Previously received antibiotics, such as rifaximin, for treatment of IBS or SIBO. 
• 
Women who are pregnant 
• 
Women who are breastfeeding 
 
Additional IBS subjects and Healthy Controls: 
 

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50 consecutive adult subjects ≥ 18 years (25 IBS-D and 25 healthy controls) who are 
undergoing colonoscopy for clinical purposes (e.g. colon cancer screening) 
 
For IBS subjects: 
Inclusion criteria: 
• 
Rome IV criteria for IBS-D  
Exclusion criteria:  
• 
Underlying celiac disease 
• 
Inflammatory bowel disease 
• 
Other organic disease that could explain their symptoms.   
• 
History of GI tract surgery, except for appendectomy 
• 
Narcotics, antibiotics taken within 3 months or probiotics taken within one month prior 
to enrollment will not be permitted.  
• 
Women who are pregnant 
• 
Women who are breastfeeding 
For healthy controls: 
 
 
Exclusion Criteria: 
• 
History of chronic GI disorder, such as irritable bowel syndrome, inflammatory bowel 
disease, and small intestinal bacterial overgrowth 
• 
Narcotics, antibiotics taken within 3 months or probiotics taken within one month prior to 
enrollment will not be permitted 
• 
Women who are pregnant 
• 
Women who are breastfeeding 
5 
SUBJECT ENROLLMENT 
Most patients with irritable bowel syndrome are seen in gastroenterology or general 
medicine clinics.  IBS-D patients will be recruited from gastroenterology and general 
medicine clinics at Taubman Center and in the satellite clinics within the University of 
Michigan Health System. We will also be sending out letters to patients identified from 
DataDirect as eligible for the study. Eligible IBS-D subjects will be contacted and given 
material explaining the nature of the study and introduction to a low FODMAP diet.  
They will be given time to read the materials. If they choose to participate, they will 
undergo a one week pre-screening an 11-point numerical rating scale (NRS) for 
abdominal pain and bloating for 7 days. If the subjects qualify through the pre-screen, 
they will sign the consent document in the presence of the PI and/or Designee.  IBS-D 
subjects will be enrolled on a first come, first served basis.  This approach will ensure 
that IBS-D subjects are representative of the population of patients with IBS in 
Southeast Michigan.   

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14 
Emails to providers in the General Medicine and Gastroenterology clinics in the 
University of Michigan System, public advertisements, telephone calls, and web 
postings on UMClinicalStudies.org will also be utilized to recruit IBS-D subjects. 
 
IBS subjects who do not meet inclusion/exclusion criteria will not be enrolled in the 
study.  All data collected from IBS-D subjects who are screen failures will be saved for 
data analysis. 
 
IBS subjects and healthy controls who are undergoing colonoscopy will be approached 
in the Medical Procedures Unit to determine their interest in participating in the optional 
sub-study and to determine their eligibility. 
6 
STUDY DESIGN AND PROCEDURES 
Specific Aim 1: Randomize IBS-D subjects to receive either a nonabsorbable antibiotic 
rifaximin vs. low FODMAP dietary intervention.  
 
1A.  Research Approach: 
200 IBS-D subjects will be carefully phenotyped using validated questionnaires (IBS symptoms, 
stool form, psychological function, quality of life) and diet analysis.  IBS-D subjects will also 
undergo baseline testing including GHBT as well as fecal microbial analysis.  IBS-D subjects will 
then be randomized to receive either rifaximin or low FODMAP diet intervention at the initial 
screening visit.  Efficacy of therapies will be monitored with validated questionnaires to track 
symptomatic improvement.  The primary outcome will be changes in mean daily pain or bloating 
after intervention compared with baseline.  Responders to intervention will be defined as ≥ 30% 
reduction in mean daily pain or bloating by visual analog scale (VAS) compared with baseline.  
Secondary outcomes will monitor changes in IBS-Symptom Severity Score as well as 
differences in GI symptoms, stool form, psychological function, quality of life, and dietary intake 
before and after intervention. 
 
1B.  Experimental Methods: 
Subject Recruitment:  200 adult subjects ≥ 18 years of age who meet Rome IV criteria for IBS-D 
will be enrolled in the study. Women of childbearing age will undergo a urine pregnancy test to 
exclude the possibility of pregnancy.  Women who are breastfeeding will also be excluded. IBS-
D subjects will be excluded if they have underlying celiac disease, inflammatory bowel disease, 
or other organic disease that could explain their symptoms.  All IBS-D subjects will have prior 
sigmoidoscopy or colonoscopy with mucosal biopsies within the past 2 years to rule out 
microscopic colitis.  IBS-D subjects with a history of GI tract surgery, except for appendectomy, 
will also be excluded from the study.  Antibiotics taken within 3 months prior to enrollment will 
not be permitted (54).  IBS-D subjects on probiotics must discontinue their use at least 1 month 
prior to fecal collection.  IBS medications, including anti-depressants, will be allowed if the dose 
has been stable for at least 1 month before inclusion.  Medications will be carefully tracked to 
follow any potential confounding issues.  IBS-D subjects who have previously received formal 

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15 
dietary education for IBS, including a low FODMAP diet, or previously received antibiotics, 
including rifaximin, for treatment of IBS-D or SIBO will be excluded from the study.   
GI Symptom Assessment:  All IBS-D subjects will complete validated questionnaires to quantify 
severity of IBS symptoms, including IBS Symptom Severity Scale (IBS-SSS), Gastrointestinal 
Symptom Rating Scale-IBS (GSRS-IBS), an 11-point numerical rating scale (NRS) for 
abdominal pain and bloating, and Bristol Stool Form Scale (BSFS) at baseline (55–58). The 
NRS for abdominal pain and bloating and the BSFS will be completed online through the 
REDCap Survey Tool. 
Psychological and Quality of Life Assessment:  IBS-D subjects will also complete validated 
questionnaires to assess for presence of and to measure severity of psychological disorders, GI 
specific anxiety, and impairments in quality of life.  All IBS-D subjects will complete the Patient 
Health Questionnaire (PHQ-9), Visceral Sensitivity Index (VSI), and IBS-quality of life (IBS-
QOL) (59–61).  The PHQ-9 will be reviewed by the study team within 24 hours of completing the 
questionnaire.  If a subject answers affirmative to the question “Thoughts that you would be 
better off dead or of hurting yourself in some way,” they will be provided with the appropriate 
referral to mental health counseling and/or the subject’s PCP will be alerted. 
Dietary Assessment:  All IBS-D subjects will undergo initial consultation with the study team and 
will be instructed on how to monitor and record their dietary intake.  IBS-D subjects will then 
record daily their dietary intake for 3 days prior to fecal collection. 
Fecal Collection and Preparation:  Three fresh stool samples will be collected from all IBS-D 
subjects at baseline.  Each sample will be immediately placed into an OMNIgeneGUT 
collection tube (DNA Genotek, Inc., Ottawa, ON, Canada) which allows for self-collection and 
stabilization of fecal DNA samples for up to 60 days at ambient temperature (62).  Samples will 
then be mailed back or picked up by the study team and stored at -80oC in Vince Young’s Lab 
for long-term storage. If patients consent to the optional portion of the study, they will provide a 
stool sample in 95% ethanol at Visits 1, 3, 4, and 5. We will also collect a one-time stool sample 
and symptom questionnaires from healthy controls. Healthy controls will not have a history of 
gastrointestinal conditions and do not report any abnormal bowel movements or abdominal 
pain.  
Tryptophan hydroxylase-1 (TpH-1) and sucrase-isomaltase single nucleotide polymorphisms 
(SNPs): Buccal swabs will be collected from all IBS-D subjects at baseline.  Each sample will be 
immediately placed into an OrageneDISCOVER (DNA Genotek, Inc., Ottawa, ON, Canada) 
which allows for stabilization of salivary DNA samples for up to 5 years at ambient temperature.  
Samples will then be stored at -80oC for long-term storage. De-identified DNA samples will be 
shared with Karolinska Institute and IKMB Kiel to analyze differences in sucrase-isomaltase 
SNPs.  This portion of the study is optional and subjects have a choice to allow their saliva 
samples to be stored for this sub-study. 
Serum cytokine profiles: Serum cytokines, including TNF-α, IL-1β, IL-6, IL-8, and IL-10 will be 
measured at baseline and at week 5. Whole blood will be collected in a 10 ml SST tube.  SST 
tubes will be allowed to clot at room temperature for 30 minutes.  The serum will then be 
separated from the red blood cells by spinning for 15 min at 1300G at 4oC.  The serum will then 
be aliquoted from each blood collection tube into (3) 2-ml transfer tubes and stored at -80oC for 

 ____________________________________________________________________________________________ 
16 
future analyses. This portion of the study is optional and subjects have a choice to allow their 
blood samples to be stored for this sub-study 
Glucose Hydrogen Breath Test (GHBT): SIBO will be diagnosed using the glucose H2/CH4 
breath test as previously described (63).  Briefly, H2/CH4 breath concentrations will be measured 
by gas chromatography (Quintron Instrument Company, Milwaukee, USA) in parts per million 
(ppm) after administration of an oral loading dose of glucose (75 g in 250 ml of sterile water).  
Breath samples will be collected every 15 minutes for a total of 120 minutes.  The test will be 
considered positive for SIBO when H2 and/or CH4 increase by ≥ 12 ppm above baseline. 
COVID-19 modifications: Patients will be given take home hydrogen test kits either in person or 
through the mail to conduct the test at home. Instructions will be given prior to test with the take 
home test kit. Patients could bring the test result back either in person or through mail and the 
lab will analyze the results. 
Trial Protocol: In order to minimize unequal allocation between arms, IBS-D subjects will be 
randomized using block randomization with random permuted sizes of 2 or 4 in a 1:1 ratio into 
two arms: one group will undergo treatment with rifaximin 550 mg three times daily for 14 days.  
The second group will be placed on a low FODMAP diet (≤ 9 grams per day as previously 
described) for 4 weeks (40,64,65).  IBS-D subjects undergoing dietary intervention will undergo 
consultation with a bionutritionist knowledgeable in low FODMAP diets.  A diet plan will be 
implemented and individualized to subjects based on information from their dietary history.  
Written information regarding allowable vs. non-allowable food items will be provided to IBS-D 
subjects and bionutritionists will be available during the study for further education.  IBS-D 
subjects randomized to the low FODMAP diet group will meet with the GI nutritionist at the 
completion of the 2nd week of dietary intervention to answer any potential questions regarding 
the diet.  IBS-D subjects will complete a 24-hour dietary recall each week or every two weeks 
during the 4-week dietary intervention to ensure compliance. 
Symptom Assessment:  IBS-D subjects will record stool frequency, BSFS, and symptom scores 
for abdominal pain and bloating daily on a diary card.  IBS-D subjects will complete 
questionnaires on a weekly basis during study visits to determine changes in symptoms.  
Responders to intervention arm (both rifaximin and low FODMAP diet) will be defined as ≥ 30% 
reductions in mean daily pain or bloating compared with baseline.  Secondary outcomes, 
including reduction in IBS Symptom Severity Scale by ≥ 50 compared with baseline at the end 
of the interventional arm, will also be monitored.  These are both considered to reflect clinically 
meaningful improvements in IBS (55,57).  IBS-D subjects will be seen on a weekly or biweekly 
basis and diary cards will be collected.  Adherence to medications or dietary intervention will 
also be assessed at that time.   
Specific Aim 2: Determine using longitudinal analyses how SIBO status and fecal 
microbiota-derived metrics associate with response to treatment with rifaximin or a low 
FODMAP dietary intervention. 
2A. Research Approach: 
We will prospectively follow the results of GHBT and fecal microbiota before and after treatment 
with rifaximin and low FODMAP dietary restriction.  We will collect fecal samples throughout the 
study period in IBS-D patients.  All IBS-D subjects will also undergo GHBT before and after 

 ____________________________________________________________________________________________ 
17 
intervention.  The primary aim will be change in fecal microbial diversity after intervention with 
rifaximin or low FODMAP diet compared with baseline.  Secondary outcomes will evaluate 
whether response to treatment with rifaximin or low FODMAP diet is associated with: (i) 
presence of SIBO on initial testing, (ii) persistence of SIBO after intervention, (iii) changes in α- 
and β- diversity as well as in dominant phyla. We will also utilize human intestinal enteroids to 
model how host-microbial interactions may influence response to treatment with rifaximin or low 
FODMAP diet in IBS. 
2B. Experimental Methods: 
Fecal Collection and Preparation: Stool samples will be collected from all IBS-D subjects at 
weeks 2, 4 and 5 as described in Aim 1. Stool samples will be stored for up to 5 years at the 
conclusion of the study for future analyses, including evaluating differences in metabolomics, 
such as histamine, p-hydroxybenzoic acid, azelaic acid, and H2 (45). This portion of the study is 
optional, and subjects have a choice to allow their stool samples to be stored for this sub-study. 
This may provide mechanistic descriptions of how changes in gut microbiota lead to changes 
seen in IBS. If patients consent to this optional portion of the study, they will provide a stool 
sample in 95% ethanol at weeks 0, 2, 4, and 5. Subjects will be compensated a nominal amount 
of $20 for stool samples returned at week 0 and $10 each for weeks 2, 4 and 5 to express our 
gratitude for their participation in the study. We will also provide mailers to mail back samples to 
minimize study burden on participants. 
GHBT: Glucose H2/CH4 test will be repeated at week 5 as described in Aim 1. 
Serum cytokine profiles: Serum cytokines will be repeated at week 5 as described in Aim 1. 
Handling and processing of the blood will be described in Appendix 1.  
Mucosal Samples: If subjects consent to participate in this optional sub-study, mucosal biopsies 
will be taken from eligible subjects who are scheduled to undergo a colonoscopy for clinical 
purposes (e.g. to rule out alternative causes of symptoms or screening for colon cancer). In 
addition to the IBS subjects recruited to participate in the clinical trial, we will recruit an 
additional 25 IBS subjects as well as 25 healthy controls undergoing colonoscopy for clinical 
purposes (e.g. colon cancer screening) to determine how host-microbial interactions may impact 
on IBS vs. healthy controls.  
Mucosal biopsies will be utilized to generate human intestinal enteroids to study host-microbial 
interactions determining response to treatment in IBS. Four mucosal biopsies will be obtained 
from normal appearing mucosa in the ileum, ascending and sigmoid colon (total of 12 mucosal 
biopsies per patient), placed in a sterile cryovial on ice, and immediately stored at -80°C for 
long-term storage. 
Sequencing and Data Analysis: 
(i) MiSeq Illumina Sequencing:  DNA samples will be submitted to the University of Michigan 
Host Microbiome Initiative under the Microbiome Explorers Program and will be processed 
using the MiSeq Illumina sequencing platform.  16S rRNA gene libraries will be constructed 
using primers specific to the V4 region.   
(ii) OTUs and Diversity Measurements:  The open-source software program, Mothur, will be 
used following the steps outlined in the Mothur MiSeq SOP (66,67).  The 16S rRNA gene 
sequencing profiles will be assessed using metrics for α diversity (such as Shannon or inverse-

 ____________________________________________________________________________________________ 
18 
Simpson indices) and β diversity-based distance measures between samples, such as θyc or 
Bray-Curtis (68,69).  Community types will be identified using unsupervised clustering 
algorithms, and individual operational taxonomic units (OTUs) will be identified and compared 
between/within patients (see Biostatistical analysis).   
(iii) Quantitative PCR Targeting the 16S rRNA Gene: qPCR will be used to quantify the total 
number of 16S rRNA gene copies/gram.  Reactions will be performed on a CFX Connect Real-
Time PCR detection system (Bio-Rad Laboratories; Hercules, CA) with universal 16S rRNA 
gene primers as previously described (33). 
Specific Aim 3: Develop a predictive model to identify subsets of IBS-D patients 
responsive to treatment. 
3A. Approach: 
We will develop and validate a predictive model of response to low FODMAP diet using data 
from the subjects randomized to a low FODMAP diet (Aim 1) combined with existing data from a 
separate cohort of IBS-D subjects previously treated with a low FODMAP diet (HUM 00053274).  
3B. Experimental Methods: 
We will use the combined cohort of patients treated with a low FODMAP diet (Aim 1 and HUM 
#00053274), which will be split into a training set from a random sample of 80% of the data 
while the remaining 20% of the data will serve as the test set. Using a random forest model, 5x2 
nested cross-validation (CV) will be performed within the training set for model selection and 
hyperparameter tuning. The candidate models will be compared by testing for overlap of 
confidence intervals for area under the curves (AUCs, two-sided test). Once a final model is 
selected, model performance will be evaluated on the test set.  
 
Study Timeline: 
 
Figure 1 IBS Subject Timeline 
Schedule of Events - Low FODMAP Group 
Visit Number 
Pre-Visit 
Visit 1 
Visit 2 
Visit 3 
Visit 4 
Visit 5 
Visit Description 
Pre-
screening 
(Phone/in 
person) 
Baseline 
(Phone/in 
person) 
Monitoring 
(Phone/in 
person) 
Monitoring 
(Phone/in 
person) 
Monitoring 
(Phone/in 
person) 
Monitoring/ 
Outcomes 
(Phone/in 
person) 
Time point 
Eligibility 
Day 0 
Day 7±3 
days  
Day 14±3 
days 
Day 28±3 
days 
Day 35±3 
days 
7 day pre-screening questionnaire 
X 
 
 
 
 
 
Informed Consent 
X 
 
 
 
 
 
Medical History /Physical 
Examination 
 
X 
X 
X 
X 
X 
Urine pregnancy test (Females of 
childbearing age) 
 
X 
 
 
 
 
Washout  
X 
 
 
 
 
 
Questionnaires 
 
X 
 
X 
 
X 

 ____________________________________________________________________________________________ 
19 
*Female participants capable of bearing children will be required to attend visit 1 in person for the pregnancy test 
*During COVID-19 pandemic, all hydrogen breath tests will be conducted remotely 
Schedule of Events – Rifaximin Group 
*Female participants capable of bearing children will be required to attend visit 1 in person for the pregnancy test 
*During COVID-19 pandemic, all hydrogen breath tests will be conducted remotely 
3-day food diary 
 
X 
 
 
 
 
24-hour dietary recall 
 
 
X 
X 
X 
X 
GI nutrition consultation 
 
X 
 
X 
 
 
Colonoscopy with mucosal 
biopsies (optional) 
X 
 
 
 
 
 
Serum Cytokine Profiles (optional) 
 
X 
 
 
 
X 
Buccal swab collection (optional) 
 
X 
 
 
 
 
Glucose hydrogen breath testing 
 
X 
 
 
 
X 
Stool collection for fecal microbial 
analysis 
 
X (3 
baseline 
samples) 
 
X 
X 
X 
Randomization 
X 
 
 
 
 
 
Dietary Compliance 
 
 
X 
X 
X 
X 
Adverse Events Monitoring 
 
 
X 
X 
X 
X 
Visit Number 
Pre-Visit 
Visit 1 
Visit 2 
Visit 3 
Visit 4 
Visit 5 
Visit Description 
Pre-
screening 
(Phone/in 
person) 
Baseline 
(Phone/in 
person) 
Monitorin
g 
(Phone/in 
person) 
Monitorin
g 
(Phone/in 
person) 
Monitorin
g 
(Phone/in 
person) 
Monitoring/ 
Outcomes 
(Phone/in 
person) 
Time point 
Eligibility 
Day 0 
Day 7±3 
days 
Day 14±3 
days 
Day 28±3 
days 
Day 35±3 
days 
7 day pre-screening 
questionnaire 
X 
 
 
 
 
 
Informed Consent 
X 
 
 
 
 
 
Medical History /Physical 
Examination 
 
X 
X 
X 
X 
X 
Urine Pregnancy Test (Females 
of childbearing age) 
 
X 
 
 
 
 
Washout 
X 
 
 
 
 
 
Questionnaires  
 
X 
 
X 
 
X 
3-day food diary 
 
X 
 
 
 
 
24-hour diet recall 
 
 
X 
X 
X 
X 
Colonoscopy with mucosal 
biopsies (optional) 
X 
 
 
 
 
 
Serum cytokine profiles 
(optional) 
 
X 
 
 
 
X 
Buccal swab collection 
(optional) 
 
X 
 
 
 
 
Glucose hydrogen breath 
testing 
 
X 
 
 
 
X 
Stool collection for fecal 
microbial analysis 
 
X (3 
baseline 
samples) 
 
X 
X 
X 
Randomization 
X 
 
 
 
 
 
Medication Dispensing 
 
X 
 
 
 
 
Medication Compliance 
 
 
X 
X 
 
 
Adverse Events Monitoring 
 
 
X 
X 
X 
X 

 ____________________________________________________________________________________________ 
20 
7 
Data Collection and Management Procedures 
Data Handling and Entry:  All data including questionnaire results and physiologic 
testing will be recorded and reviewed by the study team.  Data will then be entered into 
an electronic case report form (eCRF) that complies with Title 21 of the Code of Federal 
Regulations (21 CFR Part 11).  All passwords will be strictly confidential to protect 
subject’s protected health information (PHI).  Only the Primary Investigator (PI) and 
Study Coordinator will have access to the data and subject identifiers.  Only the PI and 
Study Coordinator will be allowed to enter data onto the password-protected web-based 
server. 
Computer Systems:  Data entry will be entered into REDCAP which is validated and 
conforms to regulatory requirements.  Only the Primary Investigator (PI) and Study 
Coordinator will have access to the data and subject identifiers.  Only the PI and Study 
Coordinator will be allowed to enter data onto the password-protected web-based 
server. 
Phone Visits: All phone visits will be conducted with a University Health System phone 
to protect patient confidentiality. 
SignNow: a secure application that enables users to electronically prepare and send 
University business documents for the purpose of requesting and obtaining digital 
signatures to help protect confidentiality.  
Data Validation:  Validation checks will be programmed within the eCRF system as 
well as supplemental validation performed by review of the downloaded data, will be 
applied to the data in order to ensure accurate, consistent, and reliable data. 
Direct Access to Source Data:  A developed review procedure that complies with 
Good Clinical Practice (GCP) guidelines. 
Source Document/Case Report Form Completion:  Source documents and the 
eCRFs will be completed for each study patient.  It is the Investigator’s responsibility to 
ensure the accuracy, completeness, and timeliness of the data reported in the patient’s 
source document/eCRF.  The source document/eCRF should indicate the patient’s 
participation in the study and should document the dates and details of the study 
procedures, AEs, and patient status. 
Record Retention:  The Investigator will maintain all study records according to 
applicable regulatory requirement(s). 

 ____________________________________________________________________________________________ 
21 
8 
DATA ANALYSIS 
Procedure for Accounting for Missing, Unused, and Spurious Data:  The primary 
outcome measure in our study is changes in mean daily pain or bloating after 
intervention with rifaximin or low FODMAP diet compared with baseline.  We will use 
multiple imputation with 10 imputed datasets to replace missing values on outcome and 
predictor variables [77].  If 10 imputed datasets are not sufficient to ensure stability of 
estimates, we will use 20 imputed datasets.  This allows for maximal use of available 
data while maximizing statistical power.   
9 
QUALITY CONTROL AND QUALITY ASSURANCE 
The PI and Study Coordinator will be responsible for Quality Control and Quality 
Assurance and will be performed in accordance with Standard Operating Procedures at 
the University of Michigan. 
10 
STATISTICAL CONSIDERATIONS 
Aim 1: 
All of the interventional analyses will be analyzed on an intention-to-treat basis.  Each 
IBS-D subject will act as his/her own control for analysis of the effects of either rifaximin 
or low FODMAP diet intervention.  This paired design inherently eliminates a significant 
amount of bias/confounding and affords increased statistical power.  We will power our 
study to detect a ≥30% improvement in mean daily bloating or abdominal pain after 
intervention compared with baseline.  Using available data from a US study on low 
FODMAP diet in IBS patients (42), we conservatively estimate that we will require 14 
IBS-D subjects in each arm to have >90% power to detect a mean difference of 1.7 in 
abdominal bloating scores after intervention via paired t-tests with the expected 
standard deviation (SD) of ±1.8.  Thus, our expected enrollment of 32 IBS-D subjects in 
each arm will be more than sufficient for analyzing this aim (see biostatistical analysis 
for Aim 2), even if the SD is nearly two-fold larger than expected.  
Aim 2: 
We will examine both inter- and intra-individual variability in microbiota-derived metrics.  
We plan to use a paired design to eliminate a significant amount of bias/confounding 
and increase statistical power.  We anticipate that there will be some variability in the 
three samples collected prior to treatment, and various techniques will be employed to 
average these data so they are incorporated as the baseline.  For example, for α 
diversity (Shannon index) a simple average of the three time points will be sufficient to 

 ____________________________________________________________________________________________ 
22 
establish a baseline α diversity.  Similar techniques will be applied to other community 
structure metrics, to create “deltas” between post-treatment time points and the 
baseline. 
These microbe-derived metrics and accompanying clinical variables will be initially 
analyzed through descriptive statistics before/after intervention.  Histograms and 
measures of frequency or central tendency/spread will be used to optimize variable 
constructions.  Subsequently, initial bivariable longitudinal analyses in an inter-individual 
fashion (assessing group-wide trends before/after intervention) that also account for the 
paired nature of the data (intra-individual) will be employed for categorical data 
(McNemar’s test) and continuous data (paired t-test), or their non-parametric 
equivalents for data not normally distributed.  Multiple comparisons will be made for 
individual OTU abundance metrics, with a focus on the most abundant taxa.  However, 
we will avoid a high false discovery rate through use of linear discriminant analysis 
effect size (LEfSe) analysis (70).  Similar to Aim 1, we will power our study using a 
paired t-test based on the primary outcome to detect a change in diversity at the end of 
the intervention compared with baseline.  In addition, we will also examine intermediate 
endpoints or longitudinal trends over time to determine if these are important.  If this is 
the case, we will also use mixed model analyses with repeated measures.   
Although this work is exploratory, we wanted to ensure that we will have sufficient 
power to detect notable differences in microbial and functional characteristics with our 
planned recruitment base.  Based on prior studies with low FODMAP diet in IBS 
patients (42,45,71), we conservatively estimate that 60% of IBS-D subjects will respond 
to treatment.  We further expect that only those IBS-D subjects who respond to a low 
FODMAP diet will show changes in fecal microbial diversity. Given our sample size of 
32 subjects per arm, we will have >90% power to detect a mean difference ≥ 0.4 in θYC 
(a measure of β-diversity) between responders and non-responders. As we are already 
seeing differences in θYC of this magnitude or greater, this suggests we are 
appropriately powered to detect meaningful differences in the microbiota between 
groups even if our standard deviation is 2-fold greater than expected. 
 
 
11 
REGULATORY REQUIREMENTS 
11.1 Informed Consent 1 
 
 
1 The IRB may waive some or all elements of informed consent – consult 
the IRB for consenting requirements. 

 ____________________________________________________________________________________________ 
23 
Informed consent will be signed by subjects after the protocol is explained by either the 
PI, co-I or study coordinator. If an IBS-D subject is a patient of the PI or Co-I, the study 
coordinator will be responsible for obtaining informed consent to avoid potential issues 
of coercion. Before signing, IBS-D subjects will be given ample opportunity to ask 
questions about the protocol. Consent forms could be signed in the Taubman Center or 
in the GI Physiology Laboratory in the Medical Procedures Unit.  Please see Informed 
Consent attached as an appendix. 
 
11.2 Subject Confidentiality  
All study team members will make every effort to protect each IBS-D subject’s 
confidentiality.  Participants will have privacy in a closed exam room to be able to 
provide information to the PI and/or study coordinator regarding medical history, current 
condition, and any other relevant information pertaining to the study.  Research records, 
data, and/or specimens will be kept in a locked office and/or locked cabinet with access 
restricted only to study team members. Study members will also secure electronic data 
on computers by using password protection, installing and regularly updating security 
software, as well as encryption of data. Only the PI and the Study Coordinator will have 
access to the data and IBS-D subject identifiers. Subjects will receive REDCap 
encrypted and secure links to fill out the online questionnaires. Only the PI and the 
Study Coordinator will be allowed to enter data onto the password-protected web-based 
server. Patients will be called using University Health System phones to protect patient 
privacy. 
  
11.3 Data Safety Monitoring Plan 
 
11.3.1. Oversight Responsibilities  
Oversight of the trial is provided by the Principal Investigator (PI), Dr. Lee. Dr. Lee 
assures that informed consent is obtained prior to performing any research procedures, 
that all subjects meet eligibility criteria, and that the study is conducted according to the 
IRB-approved research plan.  
 
11.3.2 Unanticipated Problems, Adverse Events and Serious Adverse Event 
Collection and Reporting2 
 
 
 
2 The Office for Human Research Protections (OHRP) considers unanticipated problems involving risks to subjects or others to 
include, in general, any incident, experience, or outcome that meets all of the following criteria: 

 ____________________________________________________________________________________________ 
24 
There is minimal direct risk to study subjects as all interventions are well accepted 
treatments for IBS. All members of the study team will stay current with training in the 
protection of human research participants and HIPAA. The PI will regularly monitor the 
data in collaboration with the research team, with periodic review by the IRB at the 
University of Michigan. We will minimize risk by using rigorous data security protocols to 
protect protocol deviations (e.g. violation of human subject confidentiality or comprised 
data integrity) and will ensure expedient reporting to the IRB (see below). 
All adverse clinical experiences, whether observed by the investigator or reported 
by the subject, must be recorded, with details about the duration and intensity of each 
episode, any actions taken, and the subject’s outcome. The Principal Investigator must 
evaluate each adverse experience for its relationship to the study and for its 
seriousness. 
The Principal Investigator must appraise all abnormal laboratory results for their 
clinical significance. If any abnormal laboratory result is considered clinically significant, 
the Principal Investigator must provide details about the action taken and about the 
subject’s outcome. 
 
 
All AEs will be assessed by the Investigator(s) and will be recorded in the eCRF, 
including the date of onset and resolution, severity, relationship to study, outcome, and 
action taken. All AEs will be reported to the IRB that meet reporting criteria. Toxicity will 
be graded according to the Common Toxicity Criteria as follows: 
0 = No adverse event or within normal limits 
1 = Mild AE, not requiring treatment 
2 = Moderate AE, resolved with treatment 
3 = Severe AE, resulted in inability to carry on normal activities and required 
professional medical attention 
4 = Life threatening or disabling AE 
 
 
• 
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 subject population being studied; 
• 
related or possibly related to participation in the research (in the guidance document, 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 subjects or others at a greater risk of harm (including physical, psychological, economic, or 
social harm) than was previously known or recognized. 
 
An incident, experience, or outcome that meets the three criteria above generally will warrant consideration of substantive 
changes in order to protect the safety, welfare, or rights of subjects or others.   

 ____________________________________________________________________________________________ 
25 
5 = Fatal AE 
Serious adverse event (SAE) is defined by the FDA as an AE resulting in any of 
the following outcomes: Death, a life-threatening adverse event, inpatient hospitalization 
or prolongation of existing hospitalization, a persistent or significant incapacity or 
substantial disruption of the ability to conduct normal life functions, or a congenital 
anomaly/birth defect. Important medical events that may not result in death, be life-
threatening, or require hospitalization may be considered serious when, based upon 
appropriate medical judgment, they may jeopardize the patient or subject and may 
require medical or surgical intervention to prevent one of the outcomes listed in this 
definition. Once an AE or SAE is known, research team members at the study site will 
ensure that participants receive appropriate care and all actions taken by the PI and/or 
co-investigator after observing the AE or SAE will be documented. 
 
11.3.2. Management of Risks to Subjects 
Expected AEs:  
Expected AEs associated with the interventions being used in the study and study 
procedures include:  
• Abdominal cramping, bloating, flatulence, lightheadedness or dizziness with the 
GHBT 
• Pain, bruising or lightheadedness with the blood draw 
AE Management: 
Subjects will be allowed to take medications, such as simethicone or Tylenol, if they 
experience significant bloating or discomfort with the GHBT. They will also be offered 
juice or other beverages if they experience dizziness or lightheadedness with the GHBT 
or with blood draws. 
 
11.3.3  Entity Responsible for Monitoring 
The Principal Investigator is responsible for complying with local and institutional 
requirements related to the reporting and documenting of SAEs.  These reports must 
include submission of all qualifying SAEs to the IRB and/or Independent Ethics 
Committee (or others).  The guidelines established in the Committees on Human 
Research “Adverse Event and Unanticipated Problems Reporting Policy” will be 
followed.   
 
11.3.3 Data Analysis Plans 

 ____________________________________________________________________________________________ 
26 
Study data are accessible at all times for the PI to review. The PI reviews study 
conduct, including accrual, drop-outs, and protocol deviations, on a monthly basis. The 
PI reviews AEs individually real-time and in aggregate on a quarterly basis. The PI 
reviews serious adverse events (SAEs) in real-time. The PI ensures all protocol 
deviations, AEs, and SAEs are reported to the IRB according to the applicable 
regulatory requirements. 
 
11.3.4 Stopping Rules 
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. 
 
 
 
 
11.3.5 Safety Review Plan 
Study progress and safety will be reviewed monthly (and more frequently if needed). An 
Annual Report will be compiled and will include a list and summary of AEs. In addition, 
the Annual Report 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 Annual Report 
will be forwarded to the IRB. The IRB and other applicable recipients will review 
progress of this study on an annual basis. 

 ____________________________________________________________________________________________ 
27 
12 
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Appendix 1 
Cytokine Profile Collection: 
Visits 2 and 6 (if optional consent is obtained): 
1. 10 ml of blood will be drawn and placed into  2 SST tubes.  
2. Whole blood SST tubes will be transported at room temperature to the lab. 
3. Processing for SST tubes: 
a. SST tubes will be allowed to clot at room temperature for 30 minutes. 
b. Serum will be separated from the red blood cells by spinning for 15 min at 
1300G at 4oC.  
c. The serum will then be aliquoted from each blood collection tube into (3) 
2-ml transfer tubes and then the collection tube will be discarded into the 
red biohazard disposal bucket. 
d. Each collection tube will be labeled with the subject’s unique ID number 
and/or letters. 
e. Each collection tube will then be frozen at -80oC for future analyses.